Hepatobiliary disposition of rhodamine 123 in isolated perfused rat livers.

The disposition of rhodamine 123 (RH-123), a known marker of P-glycoprotein, and its liver-generated glucuronide metabolite (RH-Glu), a marker of Mrp2, was studied in an isolated perfused rat liver model. Livers were perfused with a buffer containing 0.1 microg ml(-1) RH-123 for 30 or 60 min or for 30 min followed by 90 min of drug-free perfusion, and the concentrations of the drug and its metabolites were determined in the perfusate, bile, and the liver tissue. The outlet perfusate concentrations of RH-123 and RH-Glu reached an apparent plateau during the continuous infusion of the drug, with a very extensive extraction ratio of approximately 96% for the parent drug. However, the biliary excretion rates of both RH-123 and generated RH-Glu continued to rise almost linearly during the entire 60 min of drug infusion. This was associated with a linear increase in the amount of RH-123 recovered in the liver between 30 and 60 min of drug infusion, resulting in a significant (>50% of the administered dose) recovery of the marker in the liver both after 30 and 60 min of perfusion. Additionally, the washout experiments showed that the declines in the biliary excretion rates of RH-123 and RH-Glu were parallel to that of RH-123 concentration in the liver in the absence of drug input. The hepatobiliary disposition of RH-123 in rats is unique because of its substantial and time-dependent accumulation in the liver, resulting in a lack of steady-state in its biliary excretion despite apparent steady-state in the perfusate.

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: plasma and tissue disposition.

Plasma and tissue disposition of a macromolecular prodrug of methylprednisolone (MP), dextran (70 kDa)-methylprednisolone succinate (DMP), was studied in rats. Single 5-mg/kg doses of DMP or unconjugated MP were administered into the tail veins of different groups of rats (n = 4/group/time point). Blood (cardiac puncture) and tissues (liver, spleen, kidney, heart, lung, thymus, and brain) were collected at various times after DMP (0-96 h) or MP (0-2 h) injections. Concentrations of DMP and MP in samples were analyzed by size-exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (HPLC), respectively. Conjugation of MP with 70-kDa dextran resulted in 22-, 300-, and 30-fold decreases in the steady-state volume of distribution, clearance, and terminal plasma rate constant of the steroid, respectively. As for tissue distribution, the conjugate delivered the steroid primarily to the spleen and liver as indicated by 19- and 3-fold increases, respectively, in the tissue/plasma area under the curve (AUC) ratios of the steroid. On the other hand, the tissue/plasma AUC ratios of the prodrug in other organs were negligible. Active MP was released from DMP slowly in the spleen and liver, and AUCs of the regenerated MP in these tissues were 55- and 4.8-fold, respectively, higher than those after the administration of the parent drug. In contrast, no parent drug was detected in the plasma of DMP-injected rats. These results indicate that DMP may be useful for the targeted delivery of MP to the spleen and liver where the active drug is slowly released.

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: dose-dependent pharmacokinetics in rats.

The dose-dependency in the pharmacokinetics of a macromolecular prodrug of methylprednisolone (MP), dextran-methylprednisolone succinate (DMP), was investigated in rats. Single doses (MP equivalent) of 2.5, 5.0, 10, 20, and 30 mg/kg of DMP were administered intravenously to rats (n=5/group), and serial blood samples (0-96 h) and spleen and liver tissues (96 h) were collected. The concentrations of DMP in plasma and spleen were analyzed using a size-exclusion chromatographic method. The concentrations of DMP in the liver samples were determined by an indirect method after sequential hydrolysis by dextranase and esterase enzymes, followed by HPLC analysis of MP. The kinetics of DMP were analyzed by non-compartmental methods. The systemic clearance of DMP decreased approximately 5-fold (from 42.1+/-11.0 to 7.72+/-1.84 ml/h per kg) when the dose was increased from 2.5 to 30 mg/kg. The nonlinearity in the clearance of DMP could be adequately described by a Michaelis-Menten type elimination with a maximum velocity of elimination of 1.72 mg/h per kg and a constant of 24.9 microg/ml. Additionally, the percent of the dose of DMP found at 96 h in the liver and spleen, where the prodrug is sequestered and gradually eliminated, significantly decreased with an increase in the dose. It is concluded that the clearance of DMP in rats is modestly dose-dependent in the dosage range of 2.5-30 mg/kg.

Stereospecific pharmacokinetics and pharmacodynamics of beta-adrenergic blockers in humans.

The beta-blockers comprise a group of drugs that are mostly used to treat cardiovascular disorders such as hypertension, cardiac arrhythmia, or ischemic heart disease. Each of these drugs possesses at least one chiral center, and an inherent high degree of enantioselectivity in binding to the beta-adrenergic receptor. For beta-blockers with a single chiral center, the (-) enantiomer possesses much greater affinity for binding to the beta-adrenergic receptors than antipode. The enantiomers of some of these drugs possess other effects, such as antagonism at alpha-adrenergic receptors or Class III antiarrhythmic activity. However, these effects generally display a lower level of stereoselectivity than the beta-blocking activity. Except for timolol, all of these drugs used systemically are administered clinically as the racemate. As a class, the beta blockers are quite diverse from a pharmacokinetic perspective, as they display a high range of values in plasma protein binding, percent of drug eliminated by metabolism or unchanged in the urine, and in hepatic extraction ratio. With respect to plasma concentrations attained after oral or intravenous dosing, in most cases the enantiomers of the beta-blockers show only a modest degree of stereoselectivity. However, the relative magnitude of the concentrations of the enantiomers in plasma is not constant in all situations and varies from drug to drug. Further, various factors related to the drug (e.g., dosing rate or enantiomer-enantiomer interaction) or the patient (e.g., racial background, cardiovascular function, or the patient metabolic phenotype) may affect the stereospecific pharmacokinetics and pharmacodynamics of beta-blockers. An understanding of the stereospecific pharmacokinetics and pharmacodynamics of beta-blockers may help clinicians to interpret and predict differences among patients in pharmacologic responses to these drugs.

Dextrans for targeted and sustained delivery of therapeutic and imaging agents.

Dextrans are glucose polymers which have been used for more than 50 years as plasma volume expanders. Recently, however, dextrans have been investigated for delivery of drugs, proteins/enzymes, and imaging agents. These highly water soluble polymers are available commercially as different molecular weights (M(W)) with a relatively narrow M(W) distribution. Additionally, dextrans contain a large number of hydroxyl groups which can be easily conjugated to drugs and proteins by either direct attachment or through a linker. In terms of pharmacokinetics, the intact polymer is not absorbed to a significant degree after oral administration. Therefore, most of the applications of dextrans as macromolecular carriers are through injectable routes. However, a few studies have reported the potential of dextrans for site (colon)-specific delivery of drugs via the oral route. After the systemic administration, the pharmacokinetics of the conjugates of dextran with therapeutic/imaging agents are significantly affected by the kinetics of the dextran carrier. Animal and human studies have shown that both the distribution and elimination of dextrans are dependent on the M(W) and charge of these polymers. Pharmacodynamically, conjugation with dextrans has resulted in prolongation of the effect, alteration of toxicity profile, and a reduction in the immunogenicity of drugs and/or proteins. A substantial number of studies on dextran conjugates of therapeutic/imaging agents have reported favorable alteration of pharmacokinetics and pharmacodynamics of these agents. However, most of these studies have been carried out in animals, with only a few being extended to humans. Future studies should concentrate on barriers for the clinical use of dextrans as macromolecular carriers for delivery of drugs, proteins, and imaging agents.

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: in vitro immunosuppressive effects on rat blood and spleen lymphocytes.

The in vitro immunosuppressive activity of a conjugate of methylprednisolone (MP) with dextran 70 kDa (DEX-MPS) was tested using the lymphocyte proliferation assay after stimulation of lymphocytes with concanavalin A (Con-A). Blood and spleen lymphocytes, isolated from drug-free male Sprague-Dawley rats, were used in the assay. First, the optimum concentration of Con-A for stimulation of lymphocytes was determined. The inhibition of the lymphocyte proliferation was then tested in the presence of 0.25,0.5, 1.0,2.5,5.0,10,20, and 50 nM concentrations (MP equivalent) of DEX-MPS or free MP. The maximum stimulation of lymphocytes with Con-A was observed at mitogen concentrations of 2.5 and 10 microg/ml for the spleen and blood lymphocytes, respectively. For free MP, sigmoidal relationships were observed between the effect (% inhibition of lymphocyte proliferation) and the logarithm of MP concentration. Additionally, the maximum inhibitory effect (I(max)) and MP concentration producing half of I(max) (IC(50)) were, respectively, 98% and 1.38 nM for the blood and 86% and 3.1 nM for the spleen lymphocytes. For MP conjugated to dextran, the response-log concentration curves were substantially shifted to the right with IC(50) values of 40 and 52 nM for the blood and spleen lymphocytes, respectively. It is concluded that compared with free MP, the steroid attached to dextran has minimal immunosuppressive activity. Therefore, to be effective in vivo, DEX-MPS should release MP in the body.

High-performance size-exclusion chromatographic analysis of dextran-methylprednisolone hemisuccinate in rat plasma.

A size exclusion chromatographic method is presented for the measurement of the concentrations of a macromolecular prodrug of methylprednisolone (MP), dextran-methylprednisolone succinate (DEX-MPS), in rat plasma. After precipitation of the plasma (100 microl) proteins with perchloric acid, the samples are injected into a size exclusion column with a mobile phase of water:acetonitrile:glacial acetic acid (75:25:0.2) and a flow-rate of 1 ml/min. The DEX-MPS conjugate, detected at 250 nm, elutes at a retention time of approximately 6.5 min, free of endogenous peaks. Excellent linear relationships (r2=0.997) were found between the detector response and the concentrations of DEX-MPS in the range of 2-100 microg/ml (MP equivalent), with intra- and inter-run C.V.s of <6% and error values of <5%. The application of the assay was also demonstrated by measurement of the plasma concentrations of DEX-MPS after single 5 or 10 mg/kg doses of the conjugate administered intravenously to rats.

Kinetics of hydrolysis of dextran-methylprednisolone succinate, a macromolecular prodrug of methylprednisolone, in rat blood and liver lysosomes.

A macromolecular prodrug of methylprednisolone (MP) was synthesized by conjugating MP with dextran with a M(W) of 70000 through a succinic acid linker. It has been shown previously that the dextran-MP conjugate (DMP) releases MP directly or indirectly through formation of methylprednisolone succinate (MPS) which is further hydrolyzed to MP. To investigate the suitability of DMP conjugate as a prodrug of MP for systemic administration, the kinetics of hydrolysis of the conjugate was studied in vitro in rat blood and liver lysosomes. In blood, the hydrolysis of MPS to MP was approximately ten-fold faster than that in buffer. However, the hydrolysis rate constants of DMP conjugate to MP or MPS in blood were not different from those in buffer. Overall, the hydrolysis of DMP in the rat blood occurred with a half life of approximately 25 h. Hydrolysis of MPS to MP also occurred in the liver lysosomal fraction, but not in the control samples lacking lysosomes. However, the rate constants for the hydrolysis of DMP conjugate to MP and MPS in the lysosomal fraction were not significantly different from those in the control samples. These data suggest that the slow hydrolysis of DMP conjugate to MP or MPS in both rat blood and liver lysosomes occurs mostly, if not completely, via chemical hydrolysis. However, the conversion of MPS to MP is apparently enzymatic. The data may have significant implications for systemic administration of the prodrug.

Simultaneous analysis of methylprednisolone, methylprednisolone succinate, and endogenous corticosterone in rat plasma.

A reversed-phase HPLC method is reported for simultaneous quantitation of methylprednisolone (MP), MP succinate (MPS), and endogenous corticosterone (CST) in plasma of rats. Additionally, the 11-keto metabolite of MP (methylprednisone, MPN) is resolved from the other analytes. After addition of internal standard (triamcinolone acetonide: IS) and an initial clean up step, the analytes of interest are extracted into methylene chloride. The steroids are then resolved on a reversed-phase polymer column using a mobile phase of 0.1 M acetate buffer (pH 5.7): acetonitrile (77:23) which is pumped at a flow rate of 1.5 ml min-1. Sample detection was accomplished using an UV detector at a wavelength of 250 nm. All the five components (MPS, MP, MPN, CST and IS) were baseline resolved from each other and other components of plasma. Linear relationships were found between the steroids: IS peak area ratios and plasma concentrations in the range of 0.1-4 mircog ml-1 for MP and MPS and 0.1-1.0 microg ml-1 for MPN and CST. The assay is accurate as intra- and inter-run error values were < +/- 8% for all the components. Further, the intra- and inter-run CVs of the assay were < 16% at all the concentrations and for all the components. The application of the assay was demonstrated after the injection of a single 5 mg kg-1 (MP equivalent) dose of MPS or a macromolecular prodrug of MP to rats.

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: immunosuppressive effects after in vivo administration to rats.

PURPOSE: To study the immunosuppressive activities of a macromolecular prodrug of methylprednisolone (MP), dextran-methylprednisolone succinate (DEX-MPS), in rats. METHODS: Single 5 mg/kg (MP equivalent) doses of MP or DEX-MPS were administered intravenously to rats, and blood and spleen samples were collected over 96 h. The immunosuppressive activity was determined by the effects of the free or dextran-conjugated drug on the mitogen-stimulated spleen lymphocyte proliferation. Additionally, the number of lymphocytes in the spleen cell suspensions was estimated. Further, the plasma and spleen concentrations of the conjugated and free MP were determined using size-exclusion and reversed-phase chromatographic methods, respectively. RESULTS: Both MP and DEX-MPS injections resulted in the inhibition of the spleen lymphocyte proliferation. However, the maximal effect of DEX-MPS was significantly (P < 0.003) more intense (approximately 100% inhibition) and delayed (24 h) relative to that of MP (approximately 50% inhibition at 2 h). The DEX-MPS injection also resulted in a significantly (P < 0.0001) higher decline in the estimated number of spleen lymphocytes (approximately 80% at 24 h), compared with the MP injection (approximately 30% at 2 hr). Whereas the plasma and spleen concentrations of MP could not be measured at > or = 2 h after the drug injection, relatively high concentrations of DEX-MPS persisted in plasma and spleen for 24 h and 96 h, respectively. CONCLUSION: Dextran-methylprednisolone conjugate can effectively deliver the corticosteroid to its site of action for immunosuppression, resulting in more intense and sustained effects when compared with the free drug administration.

Preparation and release of ibuprofen from polyacrylamide gels.

The conditions of preparation of polyacrylamide (polyAC) gels, the incorporation of ibuprofen (IB), and the kinetics of IB release under various conditions have been evaluated. Transparent, opaque, or elastic gels were prepared depending on the concentration of acrylamide (AC) and the cross-linking agent, N,N'-methylenebisacrylamide (BIS). Release studies in media below pH 5.0 resulted in opaque gels. The kinetics of IB release was a function of the AC, BIS, and the pH of the medium, but the optimum composition, in terms of gel integrity and release characteristics, was 7% AC cross-linked with BIS at a 50:1 ratio. Modulation of the release rate was possible with the incorporation of 10% of certain polymers. The amount of IB that could be incorporated per gram of transparent gel was a function of the amount of polymer initiator N,N,N',N'-tetramethylene diamine (TEMED) used per gram of gel. More than 200 mg of IB could be incorporated per gram of transparent gel by using 100 microliters of TEMED. The release of IB obeyed matrix/swelling-controlled kinetics and 70-80% of the IB was released from gels containing 10 to 40 mg IB per gram of gel in 5 hr at pH 7.4 and 37 degrees C.

Simultaneous analysis of dextran-methylprednisolone succinate, methylprednisolone succinate, and methylprednisolone by size-exclusion chromatography.

An analytical HPLC method is reported for simultaneous measurement of low (1.0-100 microg ml(-1)) concentrations of dextran-methylprednisolone succinate (DEX-MPS) and its degradation products methylprednisolone hemisuccinate (MPS) and methylprednisolone (MP). The analytes were detected at 250 nm after resolution using a size exclusion column with a mobile phase of KH2PO4 (10 mM): acetonitrile (3:1) and a flow rate of 1 ml min(-1). The resolution of MP and MPS peaks was substantially affected by the pH of the mobile phase; while MP and MPS co-eluted at pH 3.4, they were baseline-resolved at pH > or = 5. Linear relationships (r > or = 0.997) were found between the detector response and the concentrations of the analytes (1.0-100 microg ml(-1) for MP and MPS and 2.5-100 microg ml(-1) for DEX-MPS). Intra- and inter-run error (< 13%) and precision (CV of < or = 6%) data indicated that the assay could accurately and precisely quantitate all three components in the examined concentration range. The application of the assay to determination of degree of substitution, purity, and stability of DEX-MPS was also demonstrated.

Bioequivalence of chiral drugs. Stereospecific versus non-stereospecific methods.

Guidelines for bioequivalence of non-racemic pharmaceuticals are abundant in the literature. However, few guidelines exist for the bioequivalence of racemic drugs which consist of 2 or more stereoisomers. The aim of this article is to address the question of whether the bioequivalence of racemic drugs should be based on the measurement of the individual enantiomers or that of the total drug. Several pharmacokinetic-pharmacodynamic cases are examined to test the validity of extrapolating the bioequivalence of racemic drugs to that of their individual enantiomers after administration of the racemate; simulation and experimental data are presented to support these cases. It is shown that for drugs which exhibit non-linear pharmacokinetics, the results of bioequivalence studies based on the total drug may differ from those based on the individual enantiomers. Similar discrepancies can be shown for a racemic drug with linear pharmacokinetics whose enantiomers substantially differ from each other in their pharmacokinetic parameters. Therefore, it is suggested that stereospecific assays be used for these drugs. Additionally, it is recommended that for racemic drugs which undergo chiral inversion, and for most products with modified release characteristics, the bioequivalence be assessed using stereospecific assays. Conversely, for racemic drugs with linear pharmacokinetics and minimal to modest stereoselectivity in their kinetic parameters, and for those with non-stereoselective pharmacodynamics, the use of stereospecific analytical methods are not warranted. Finally, the limited, controversial literature in favour of or against the use of stereospecific assays in bioequivalence of chiral drugs are reviewed and a preliminary guideline is proposed.

Kinetics of hepatic accumulation of dextrans in isolated perfused rat livers.

The role of various processes (uptake, release, metabolism, and excretion) in the hepatic accumulation of dextrans was investigated in isolated perfused rat livers (IPRLs). Single-pass IPRLs were infused with fluorescein-dextran (FD) with a molecular weight (MW) of 70,000 (FD-70) for 15, 30, 45, or 60 min, and inlet and outlet samples and livers were collected. In addition, two groups of livers were infused with FD-70 for 60 min, followed by 30 or 60 min of drug-free perfusion. The concentrations of the macromolecule in the samples were measured by a size exclusion chromatographic method. Similar, but limited, experiments were also conducted for FDs with MWs of 4,000 (FD-4) and 150,000 (FD-150). In addition, the metabolism of all three FDs were investigated using liver homogenates. Because of low hepatic extraction, the concentrations of dextrans in the inlet and outlet perfusates were almost the same during the entire perfusion. However, liver concentrations increased almost linearly during the infusion of FD-70 (0-60 min) and declined slowly thereafter (60-120 min). The apparent hepatic extraction ratio (Eapp) values, estimated directly from the concentrations of FDs in the liver, were MW dependent; Eapp of FD-4 (0.124% +/- 0.015%) was significantly (p < 0.05) less than that for FD-70 (0.677% +/- 0.193%) or FD-150 (0.711% +/- 0.022%). The metabolism and biliary excretion of all FDs were negligible during the perfusion time. The mean residence time of FD-70 in the liver, estimated by nonlinear regression analysis of experimental data, was 248 min. These studies define the role of various processes involved in the slow (but substantial) and MW dependent hepatic accumulation of dextrans.

Reversal of stereoselectivity in the hepatic availability of verapamil in isolated perfused rat livers. Role of protein binding.

The effects of serum proteins on the stereoselective kinetics of the high clearance drug verapamil (VER) and its metabolite, norverapamil (NOR), were studied in isolated perfused rat livers (IPRLs). Livers were perfused, in a recirculating manner, with a solution containing human serum albumin (HSA), bovine serum albumin (BSA), or no serum albumin (N = 5 for each group). After presystemic administration of a single dose of racemic VER (2 mg), the concentrations of VER and NOR enantiomers in the perfusate were measured over 90 min. In addition, the fraction of the enantiomers bound to the plasma of perfusate was determined. Perfusate concentrations of both VER and NOR were stereoselective in all of the perfusates studied. However, the direction of stereoselectivity in the concentrations of VER enantiomers in the BSA perfusate (S-VER > R-VER) was opposite that in the HSA and albumin-free perfusates (R-VER > S-VER); this was associated with an opposite stereoselectivity in the concentrations of NOR in the BSA perfusate was higher than that in the HSA and albumin-free perfusates, an observation in agreement with the higher stereoselectivity in the binding of NOR to BSA. These data, along with other kinetic parameters such as apparent hepatic availability and intrinsic clearance, suggest that the apparent stereoselectivity in the presystemic elimination of VER by IPRLs is significantly influenced by the stereoselectivity in the protein binding of the drug.

Enantioselective distribution of verapamil and norverapamil into human and rat erythrocytes: the role of plasma protein binding.

In this in vitro study, the distribution of the enantiomers of verapamil (VER) and its active metabolite, norverapamil (NOR), into the red blood cells (RBCs) of humans and rats was investigated using a chiral liquid chromatographic assay. When plasma was replaced with buffer, the distribution of VER and NOR enantiomers into both human and rat RBCs was substantial (RBC:blood concentration ratios, 1.39-1.79), non-stereoselective, concentration (125-1000 ng mL-1) linear, and species independent. However, in the presence of plasma, the RBC distribution of VER and NOR was stereoselective, with opposite stereoselectivity for human (S > R) and rat (R > S) blood. Additionally, the presence of plasma caused a reduction in the extent of RBC distribution for both VER and NOR enantiomers and in some cases resulted in nonlinearity in the RBC distribution of the enantiomers. Plasma protein binding studies revealed opposite stereoselectivity in the free fractions in human (S > R) and rat (R > S) plasma for both VER and NOR. These data suggest that the stereoselective protein binding is responsible for the apparent stereoselectivity in the RBC distribution of VER and NOR. The data are also in agreement with the opposite stereoselectivity in the plasma concentrations of VER observed in vivo in rats and humans.

Pharmacokinetics of atenolol in clinically normal cats.

OBJECTIVES: To determine the pharmacokinetics of atenolol (AT) after i.v. and oral administrations in cats, to assess duration of beta-blocking effect, and to determine whether AT can be effectively used once per day. ANIMALS: 9 clinically normal cats. PROCEDURE: Single doses of 1 (i.v.) or 3 (oral) mg of AT/kg of body weight were administered to each cat on different occasions, and serial blood samples were collected. Plasma concentrations of AT were subsequently determined, using high-performance liquid chromatography. The plasma concentration data were analyzed, using noncompartmental analysis. An isoproterenol challenge test was used to determine the beta-blocking effect of AT on heart rate after 3 consecutive days of oral treatment (3 mg/kg, once a day). RESULTS: After i.v. administration, mean +/- SD apparent volume of distribution at steady state and systemic clearance values were 1,088 +/- 148 ml/kg and 259 +/- 72 ml/h/kg, respectively. Bioavailability was 90 +/- 9% after oral administration. The half-life values were 3.44 +/- 0.5 and 3.66 +/- 0.39 hours after i.v. and oral administrations, respectively. Compared with baseline values prior to AT administration, heart rates at 6 and 12 hours after administration of AT were significantly reduced. CONCLUSIONS: AT has high oral bioavailability in cats, resulting in small interindividual variability in its kinetics in this species. The drug has beta-blocking effect in cats, as indicated by the attenuated heart rate response to isoproterenol; this effect persists for at least 12 hours in clinically normal cats.

Effects of storage and homogenization methods on the hepatic recovery of dextrans determined by size-exclusion chromatography.

The effects of storage and homogenization methods on the analytical recovery of dextran macromolecules from rat livers were investigated using a high-performance size-exclusion chromatographic (HPSEC) method. Livers were collected from rats dosed with fluorescein-labeled dextrans with molecular weights of 150 or 70 kD. Subsequently, the livers were subjected to different methods to study the effects of the following parameters on the hepatic recovery of dextrans: storage method (freezing the livers before homogenization or freezing the homogenates); contents of the homogenization buffer (addition of 1% Triton X-100); and sample type (HPSEC analysis of the whole homogenate or the supernatant after centrifugation). It is shown that in the absence of Triton in the homogenization buffer, the hepatic recovery of dextrans is substantially affected by all the factors studied. However, in the presence of 1% Triton in the buffer, the hepatic recoveries were maximal and independent of the storage method or sample type. These studies suggest that for optimal recovery of dextran macromolecules from the liver, a sample preparation method capable of disrupting the subcellular membranes should be used.