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K P Heirwegh

Publications and source records attributed to K P Heirwegh.

At least 19 recordsLinked to original sources
PubMed1996

Thermodynamic studies of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast microsomes.

An approach to the quantitative spectral analysis of substrate binding and inactivation of cytochrome P-450 in microsomes is described. The method is based on the application of the principal component analysis technique on the Soret-region spectra measured at different temperatures at various concentrations of substrate. This approach allowed us to study the thermodynamic parameters of substrate binding and spin transitions in human cytochrome P-450 3A4 expressed in yeast (Saccharomyces cerevisiae) microsomes. These parameters are discussed in comparison with the values reported earlier by Ristau et al. [(1979) Acta Biol. Med. Ger. 38, 177-185] for rabbit liver cytochrome P-450 2B4 in solution with benzphetamine as a substrate. Our analysis shows the substrate-free states of 2B4 and 3A4 to be very similar. However, substrate binding seems to perturb haem-protein interactions in 3A4 in contrast with 2B4, where the effect of substrate binding on the thermodynamic parameters of spin transitions was insignificant. The implication of the results for the mechanism of substrate-induced spin shift is discussed.

Animals↗
PubMed1994

Multiphasic modelling of ligand/acceptor interactions. The hydrophobicity-dependent binding of relatively small amphiphilic substances to acceptor proteins and the nature and facedness of acceptor sites.

The modelling of multiphasic ligand/acceptor equilibrium binding systems proceeds at three logically distinct levels: (1) A suitable response quantity, e.g. the amount of acceptor-bound ligand nEL, is expressed as a function of the ligand concentrations [Li] (L = A,B,...) in the compartment i that contains the acceptor sites. One thus obtains a response function nEL = f1([Li]). In general, the equilibrium constants KL contained in such mathematical models are physically ill-defined. (2) Each local concentration [Li] is further expressed as a function of [Laq], the corresponding concentration in the aqueous phase, leading to nEL = f2([Laq]). In this way, the constants KL are transformed into effective constants K'L which (i) can be assessed experimentally and (ii) depend on ligand hydrophobicity in a way that is characteristic of the binding site. Formulation of the functions f1 and F2 only requires knowledge of the reactions in which the acceptor sites participate directly. (3) For each ligand, the experimentally accessible total ligand concentration Lt is expressed as a function of [Laq], leading to concentration balance equations Lt = Lt([Laq]). The latter transformation takes account of any reactions, distinct from ligand/acceptor interaction, in which the ligands are involved, e.g. binding to additional protein sites. As a result of steps 2 and 3, each binding system is described by a set of simultaneous equations dependent on the auxiliary variable [Laq]: (i) the response function f2([Laq]) and (ii) a concentration balance for each ligand Lt = Lt([Laq]). The formulae are rendered more conscise and their discussion and application to data fitting are simplified by introducing, for each ligand L, a function FL characterising the distribution of unbound monomeric ligand over the various partition compartments. When the acceptor acts on unbound ligand, the formulae are further expressed in terms of a new auxiliary variable i.e. the total concentration of unbound monomeric ligand microL. In contrast to data analysis as a function of local concentrations, analysis in terms of total ligand concentrations avoids losing sight of alternate hypotheses about the nature of the binding sites. The present formulation has also permitted clarification of several consequences of the multiphasic nature of the binding systems that, as yet, have been poorly recognised.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1993

The substrate-depletion error on the multiple Michaelis-Menten equation with and without an added term linearly dependent on the substrate concentration.

Saturation curves for substrate interaction with an acceptor (enzyme, binding or carrier protein) are often analysed on the assumption that the amount of acceptor-bound substrate is negligible compared to its total amount. Analytical criteria permitting one to decide whether the assumption is justified or not for systems described by a single Michaelis-Menten equation have been derived previously. For more complicated systems, error formulae often cannot be obtained in closed form, and, if obtainable, are unwieldy. How this more complicated problem can be tackled is shown for mixtures of acceptor sites described by a sum of several Michaelis-Menten terms, without or with an additional term for 'non-specific' uptake or binding. In particular, it is shown that the maximum error, for which simple analytical expressions are obtained, provides a valid criterion for assessing whether substrate depletion is negligible or not.

Binding Sites↗
PubMed1992

Modelling of interaction of basic lipophilic ligands with cytochrome P-450 reconstituted in liposomes. Determination of membrane partition coefficients of S-(-)-nicotine and N,N-diethylaniline from spectral binding studies and fluorescence quenching.

The spectral interaction of N,N-diethylaniline and S-(-)-nicotine with cytochrome P450IIB4 reconstituted into large unilamellar vesicles could properly be described by a model for interaction of basic lipid-soluble ligands with membrane-bound acceptor sites in which linear partitioning of non-ionized ligand in the membrane is postulated. Apparent spectral dissociation constants Ksapp for type I binding of N,N-diethylaniline and for type II binding of S-(-)-nicotine increased linearly with increasing lipid volume fraction alpha L of the proteoliposomes. From plots of Ksapp vs. alpha L, the membrane partition coefficient of each ligand was calculated. The apparent affinity of cytochrome P450IIB4 for the ligands increased as the pH was raised from 6.0 to 8.5. However, effective dissociation constants were virtually independent of the pH, indicating that only the uncharged form of the basic ligands interact with cytochrome P450IIB4. For each compound, the apparent quenching rate constants kqapp derived from the Stern-Volmer plots for dynamic quenching of the fluorescence intensity of 8-(2-anthryl)octanoic acid in liposomes, decreased with increasing liposomal concentration. Plots of (kqapp)-1 vs. alpha L of the liposomes yielded the overall bimolecular quenching rate constant kq of each quencher. The kq value for S-(-)-nicotine was about three orders of magnitude less than that for N,N-diethylaniline. The values of the partition coefficient of N,N-diethylaniline, obtained from the binding studies and the fluorescence quenching measurements, were identical (on average, Kp amounted to 383). Analysis of the quenching data of N,N-diethylaniline with Scatchard plots likewise revealed that the association of the compound with liposomal membranes is a pure partition process.

Animals↗
PubMed1992

Testing and characterizing enzymes and membrane-bound carrier proteins acting on amphipathic ligands in the presence of bilayer membrane material and soluble binding protein. Application to the uptake of oleate into isolated cells.

1. A multiphasic modelling approach [Heirwegh, Meuwissen, Vermeir & De Smedt (1988) Biochem. J. 254, 101-108] is applied to systems containing poorly water-soluble amphipathic reactants, membrane material, soluble binding protein and acceptor protein (enzyme or membrane-bound carrier protein). 2. The field of application is constrained by the assumptions (i) that the amount of acceptor-bound substrate is small compared with the total amount and (ii) that all preceding chemical reactions and steps of mass transport are rapid compared with the chemical change monitored. 3. Initial-rate formulae for systems in which an acceptor interacts with unbound or protein-bound ligand are given. The saturation curves are near-hyperbolic or sigmoidal, depending both (i) on the form of ligand (unbound or protein-bound) acted upon by the acceptor and (ii) on whether the assays are performed at constant concentration of soluble binding protein Cp or at constant substrate/binding-site molar ratio RS. 4. Several diagnostic features permit unequivocal distinction between acceptor action on unbound or protein-bound substrate. In the former case, saturation curves, run at the same constant concentration of one of several binding proteins of increasing binding affinity, will show progressively increasing inhibition, the shape changing from near-hyperbolic at Km' less than K1' to sigmoidal at Km' greater than K1'.Km' is the effective Michaelis constant of the acceptor and K1' the effective dissociation constant of the binding sites of the soluble protein (for the sites with the higher binding affinity, if several classes of binding site are present on the protein). Alternatively, the maximum velocity obtained at constant RS less than or equal to 1 should increase hyperbolically with RS/(1-RS) for a binding protein with a single class of binding site. The formula that applies when the binding protein contains two classes of independent binding site is also available. When the acceptor acts on protein-bound ligand, the maximum velocity obtained at constant binding-protein concentration, Cp, increases hyperbolically with Cp. 5. Application of these and additional criteria to initial-rate data on the uptake of oleate into isolated cells supports a mechanism of carrier-mediated uptake of the unbound ligand and allows one to clarify some observations that hitherto had been poorly explained. 6. The influence of soluble binding protein on the reaction and substrate specificities of ligand/acceptor interaction is also discussed. 7. In its present state, data treatment for 'double binding-protein systems' generally requires separate determination of the binding parameters of the soluble binding protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Carrier Proteins↗
PubMed1992

Interaction of 7-n-alkoxycoumarins with cytochrome P-450(2) and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients.

A study was made of the binding of 7-ethoxy-, 7-n-propoxy- and 7-n-pentoxy-coumarin to cytochrome P-450(2) reconstituted into large unilamellar liposomes composed of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants Ksapp. increased linearly with increasing proteoliposomal concentration. When both cytochrome P-450(2) and NADPH:cytochrome P-450 reductase were reconstituted into liposomes, the apparent Michaelis constants Kmapp. for O-dealkylation of 7-methoxy-, 7-ethoxy- and 7-n-propoxy-coumarin showed a similar dependence on the proteoliposomal concentration. The results were in accordance with models for kinetic or equilibrium processes in biphasic systems containing membrane-bound catalytic or acceptor sites, in which a linear solute partition in the bilayer membrane is postulated. The methyl, ethyl and n-propyl ether were readily dealkylated. However, the O-dealkylation rate of 7-n-butoxycoumarin was low and became very small for longer alkyl ethers. Both the effective dissociation constants and effective Michaelis constants decreased with elongation of the alkyl side chain of the coumarins. From plots of the apparent dissociation constants and apparent Michaelis constants against the lipid volume fraction of the proteoliposomes, the membrane partition coefficients for several homologues were calculated. When protein-free liposomes were added to 7-n-alkoxycoumarin solutions, the fluorescence intensity of the coumarins decreased and eventually became negligible in the presence of an excess of liposomal material. On the assumption that the overall fluorescence can be ascribed exclusively to the fraction of 7-n-alkoxycoumarin molecules present in the aqueous phase, partition coefficients for liposomal accumulation of the test compounds could be determined directly. For several coumarin ethers, comparable values were derived for the membrane partition coefficients from binding, kinetic and fluorescence intensity measurements. The change in free energy per methylene group of the 7-n-alkoxycoumarins for partitioning between n-octanol and buffer was significantly different from the value for liposome partitioning.

Alkylation↗
PubMed1992

Analysis of membrane-bound acceptors. A correction function for non-specific accumulation of poorly water-soluble hydrophobic or amphipathic ligands based on the ligand partition concept.

Non-specific ligand accumulation into membrane material, which may contribute considerably to the experimental signal obtained in binding studies with labelled amphipathic and hydrophobic ligands, may be accounted for by linear partition of the ligands into the membrane phase. For application to binding data obtained at a single membrane-lipid concentration, a fitting procedure is proposed which allows one to correct for non-specific ligand partition. If the assumption is met that the amount of acceptor-bound ligand is small compared to the total amount present in the system, one can validly interpret the data in terms of total ligand concentrations. The apparent dissociation constants Kd(app) thus obtained should be corrected for the often large effects of the size of the partition compartment(s), by performing assays at several membrane-lipid concentrations. The importance of the latter correction is stressed and an approach for obtaining the characteristic effective dissociation constants Kd' is indicated. The procedure also yields estimates of the ligand/membrane partition coefficients.

Binding Sites↗
PubMed1991

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.

Bilirubin↗
PubMed1989

Chromatographic analysis and structure determination of biliverdins and bilirubins.

Recent applications of thin-layer chromatographic (TLC) and high-performance liquid chromatographic (HPLC) procedures has revealed an unexpected wide variety of naturally occurring unconjugated and conjugated bilirubins. Biliverdins seems to occur only in unconjugated forms, mainly as the IX alpha isomer. Several synthetic biliverdins and bilirubins present interesting models for biochemical and metabolic studies. Owing to recent recognition of the astounding heterogeneity of natural bilirubins and to the various artifactual changes that bile pigments can undergo, considerable confusion has existed, and still exists, with regard to the nomenclature of the bile pigments and their derivatives. To set a background for further discussion, the present review starts with a brief discussion of nomenclature and of the various characteristic forms of lability of the bile pigments. TLC and HPLC procedures for preparation and analysis of unconjugated biliverdins and bilirubins and their methyl ester and sugar ester conjugates, as well as procedures for analysis of bilirubin-protein conjugates, are then discussed. Since, in view of the lability and pronounced heterogeneity of bile pigments, it is important to assess the composition and nature of chromatographically isolated pigments, the review is concluded by a brief evaluation of various structural tests.

Animals↗
PubMed1989

Modelling of chemical reactions catalysed by membrane-bound enzymes. Determination and significance of the kinetic constants.

A model of multiphasic systems, based on the assumption of zero-order partition of substrates and products into the membranes, is applied to reversible mono-substrate and bi-substrate reactions catalysed by membrane-bound enzymes. Apart from replacement of single-phase kinetic constants by apparent kinetic constants, the derived kinetic expressions are formally identical with those for corresponding single-phase systems. The model confers to the apparent kinetic constants an experimentally verifiable meaning. For full characterization of membrane-kinetic systems, experiments at various concentrations of enzyme-embedding phospholipid are required. Extrapolation to zero phospholipid concentration of each Km app then yields the corresponding true kinetic constant characteristic of the membrane-bound enzyme and also provides a technique for determination of the membrane-partition constants. The procedure implies that the phospholipid content should be assayed for full characterization of membrane-bound enzymes. If, for practical reasons, the assays have to be limited to a single enzyme concentration, correction of the apparent kinetic constants is still possible provided the phospholipid concentration and the partition constants of the reactants are known. The model has permitted prediction of a number of previous observations reflecting the multiphasic nature of the systems. The assumptions, underlying the model, and their implications are examined as well as some commonly used experimental designs for determination of the type of enzymic site.

Binding Sites↗
PubMed1989

Thyroid hormones and the hepatic handling of bilirubin. I. Effects of hypothyroidism and hyperthyroidism on the hepatic transport of bilirubin mono- and diconjugates in the Wistar rat.

The effects of thyroidectomy and of thyroid hormone administration on the hepatic transport of endogenous bilirubin were investigated in the Wistar R/APfd rat. Hypothyroidism resulted in an enhanced hepatic bilirubin UDP-glucuronosyltransferase activity and in a decreased p-nitrophenol transferase activity. It caused a cholestatic condition with a 50% decrease in bile flow and bile salt excretion, and an increased proportion of conjugated bilirubin in serum. The biliary output of unconjugated and monoconjugated bilirubins decreased in parallel by about 65%, whereas the excretion rate of the diconjugate dropped by only 47%, resulting in an increased di- to monoconjugate ratio in bile. Hyperthyroidism was characterized by a decreased bilirubin and an increased p-nitrophenol transferase activity, and by an augmented bilirubin output in bile. The output of unconjugated and monoconjugated bilirubins increased in parallel by about 50 or 100%, whereas the excretion of the diconjugate increased by only 20 to 50%, depending on the dose of thyroxine administered; this resulted in a decreased di- to monoconjugate ratio in bile. A linear positive relationship was found between bilirubin UDP-glucuronosyltransferase activity and the ratio of bilirubin di- to monoconjugates present in bile or formed by in vitro incubation of liver homogenates at low concentration of bilirubin (10 to 15 microM), indicating that bile pigment composition is mainly determined by the conjugation activity in the liver. The inverse relationship observed between hepatic beta-glucuronidase activity and the ratio of di- to monoconjugates in bile warrants further investigation to analyze whether this enzyme activity also plays a possible role in the changes in bile pigment composition in hypo- and hyperthyroid rats.

4-Nitrophenylphosphatase↗
PubMed1989

Similarities in maximal biliary bilirubin output in the normal rat after administration of unconjugated bilirubin or bilirubin diglucuronide.

The rate-limiting step in the overall plasma-to-bile transport of a saturating load of bilirubin is still a matter of controversy. We reassessed the apparent maximal biliary bilirubin excretion following i.v. infusion of unconjugated bilirubin and--for the first time--of highly purified bilirubin diglucuronide in the rat. The bilirubin diglucuronide preparation could be kept in a stable form at -20 degrees C for at least 2 months after addition of 3 mM sodium ascorbate. The biliary bilirubin excretion rates in animals with and without bile depletion in order to induce different flow rates were comparable after infusion of unconjugated bilirubin and of bilirubin diglucuronide. No significant hydrolysis of bilirubin diglucuronide seemed to occur during the hepatic transport of the pigment. Injection of bilirubin diglucuronide into rats which were already being infused with saturating doses of unconjugated bilirubin did not result in increased biliary bilirubin excretion. In contrast, a reversible inhibition of bilirubin output and bile acid-dependent bile flow was observed. If unconjugated and diglucuronidated bilirubin follow the same intracellular routes, the present results would suggest that conjugation did not restrict maximal biliary excretion. However, if exogenously administered diglucuronide utilizes a separate pathway, as was recently proposed, the biliary secretion of this exogenous conjugate might be restricted, presumably due to a toxic effect of the high local concentration of diglucuronide. The pathways utilized by the unconjugated pigment, on the other hand, could be primarily determined by the conjugating capacity.

Animals↗
PubMed1989

Subcellular localization of UDP-glucuronyltransferase by differential centrifugation. Changes produced by pretreatment of rats with secretin, glucagon, vasoactive intestinal polypeptide and phenobarbitone.

Subcellular fractionation of liver homogenates from treated rats was carried out in order to study the mechanism of action of the gastrointestinal polypeptides on glucoronidation. Rats were treated for 90 min with an intravenous infusion of secretin (0.4 cU/h/100 g body weight), glucagon (100 micrograms/h/100 g body weight) and vasoactive intestinal polypeptide (VIP) (300 ng/h/100 g body weight); controls were sham-treated rats. For comparison, another group of animals was treated with a daily injection of phenobarbitone (10 mg/kg), a well-established enzyme inducer. Treatment with the different polypeptides produced minor changes in the subcellular localization of the enzyme. The bulk of activity was always recovered in the microsomal fraction, as identified by both differential centrifugation and the enrichment in specific activity of glucose-6-phosphatase, esterase and NADPH-cytochrome c reductase. Secretin produced a specific increase of bilirubin glucuronidation, more evident in all nuclear fractions. Glucagon increased both bilirubin and p-nitrophenol glucuronidation in all subcellular fractions. VIP had a selective action on p-nitrophenol conjugation of similar extent in nuclear and microsomal fractions. The type of changes observed is suggestive of physicochemical modifications occurring into the cell, perhaps at the membrane environment of different organelles, able to modify the overall conjugation of different substrates by the cell.

Animals↗
PubMed1988

Liposomes as carriers of poorly water-soluble substrates: linear modelling of membrane systems with catalytic or binding sites of different facedness. Significance of experimental membrane partition coefficients and of kinetic and equilibrium parameters.

1. A multiphasic modelling approach to systems containing membrane-bound receptors or catalytic sites and a liposomal preparation as a substrate carrier is described. Kinetic expressions are derived for a single-substrate enzymic reaction operating at constant liposome concentration or at a fixed substrate/liposome concentration ratio. 2. The assumption that accumulation of exchangeable components into the phospholipid bilayers can be described by linear bulk-phase partition leads to simple relationships between the initial reaction rate and (a) two kinetic coefficients (V and K'm), (b) the partition coefficients of the solutes for the lipid compartments of the membrane (Pms) and liposomal preparations (P1s) and (c) the total concentrations of substrate, membrane lipid and liposomal lipid. K'm is called the effective Michaelis constant. 3. For correct estimation of the coefficients V, K', Pms and P1s extrapolation to zero lipid concentration is required. 4. The distinction is introduced between hydrophilic and hydrophobic aqueous-faced sites, lipid-faced sites and mixed sites, i.e. sites overlapping an aqueous and a lipid region. For hydrophilic aqueous-faced sites K'm is equal to the true Km and for the other types of site to Km/Ps. For lipid-faced and for mixed sites Ps corresponds to the membrane partition coefficient Pms. For binding of homologous compounds to a hydrophobic aqueous-faced binding pocket Ps is the incremental site partition coefficient Pbss, which takes into account the energetic contribution to the binding process due to the hydrophobic tail of the ligands. 5. K'm accounts for any effects due to the facedness and nature of the enzymic sites. The dependence of the systems on the size of the lipidic partition compartment(s) is expressed exclusively by a distribution function F.6. When enzyme assays are performed with a series of chemically different substrates containing the same catalytically sensitive group, independence of K'm from partition indicates a hydrophilic aqueous-faced binding site. For the low-molecular-mass members of the homologous series a linear increase in -log (K'm) with the logarithm of the partition coefficient will be observed with any of the other site types considered 7. Equilibrium relationships for binding of a ligand to a membrane-bound receptor are also derived. 8. The significance of experimental membrane partition coefficients is discussed.

Binding Sites↗
PubMed1988

Novel method for high-performance liquid chromatography of azo derivatives of conjugated and unconjugated bilirubin.

A method for the separation and quantitation of ethyl anthranilate or p-iodoaniline azo derivatives of bile pigments was developed using reversed-phase high-performance liquid chromatography. A convenient separation was achieved in 15 min, permitting the quantitation of the unconjugated azo-dipyrrole (alpha o) and its glucuronide (delta), xyloside (alpha 2) and glucoside (alpha 3) conjugates. The pathological beta- and gamma-azo pigments, derived from bilirubin glucuronide isomers that occur in cholestatic bile or plasma, are also detected in this system. The results of this method as applied to bile from 25 healthy dogs were in excellent agreement with the values obtained by reversed-phase chromatography of bilirubin and its mono- and dimethyl esters produced from the corresponding conjugates by alkaline methanolysis. This system permits the sensitive and convenient determination of bilirubin and its conjugation pattern in biological fluids.

Animals↗
PubMed1988

Principles of a competitive binding assay for the study of the interactions of poorly water-soluble ligands with their soluble binding partners. Application to bilirubin with the use of Sephadex G-10 as a competitive adsorbent.

1. A generally applicable method is described for obtaining experimental data on the interactions between a poorly water-soluble ligand and soluble binding factors, with the use of chemically inert solid adsorbent. The equilibrium distribution of the ligand between the liquid phase containing the soluble binders and the adsorbent must be measured and knowledge of the binding isotherm of the adsorbent is required. Procedures are given for the calculation of the binding parameters. 2. The method has been applied to quantify the interactions of bilirubin with serum and liver cytosol from the rat, Sephadex G-10 serving as the competing adsorbent. Reversible adsorption keeps the concentration of the free ligand low, thereby preventing formation of colloidal bilirubin. The sensitivity of the procedure accommodates the rather high binding affinities by which bilirubin generally interacts with its specific binding proteins. 3. The binding activities of serum and liver cytosol are of comparable magnitude. Binding of bilirubin by rat serum can be described by two independent binding sites, the affinities of which differ by two orders of magnitude. Only the site with the higher affinity appears to be of physiological importance. The major bilirubin-binding sites of rat liver cytosol seem to contribute equally to the overall binding activity of this preparation, provided that GSH is present.

Adsorption↗
PubMed1988

Transplantation of isolated hepatocytes into the pancreas.

In previous research into hepatocyte transplantation (HTX) the spleen was the preferred acceptor organ for isolated donor hepatocytes. In this study the pancreas was tested as an acceptor organ for HTX. HTX into the pancreas or spleen was performed by injection of 10(7) isolated hepatocytes into the parenchyma of these organs. Intrapancreatic hepatocytes showed good viability 3 months after syngenic HTX as assessed by histological and immunocytochemical parameters. Definite proof of sustained metabolic activity of normal hepatocytes, 3 months after transplantation into the pancreas of congenitally jaundiced rats, was obtained by demonstration of bilirubin conjugates in bile of the recipients: 4.0% of total biliary bilirubin was conjugated. Intrasplenic HTX, however, was more effective and resulted in a conjugated fraction of 17.7% of total biliary bilirubin (p less than 0.001). Reduction of total plasma bilirubin was significant with both methods, but more pronounced in intra-splenic HTX. Bile drainage from the hepatocellular transplant via the pancreatic excretory system into the gut was not observed: conjugated bilirubins were not found in pancreatic juice of HTX-treated jaundiced rats. Intrapancreatic HTX did not adversely affect the host rat; evidence of pancreatitis or diabetes was not found. It is concluded that the pancreas is a suitable acceptor organ for HTX. However, intrapancreatic HTX appears to be less effective than intrasplenic HTX in the treatment of enzyme deficiency disease.

Animals↗