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Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland (B.S.Z., S.T.S., J.D.C., S.E.M.); and Department of Pharmacology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania (J.M.K., Y.H., M.K.)
(Received November 6, 2007; Accepted May 15, 2008)
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Abstract |
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). Endogenous long-chain ceramide levels have been shown to undergo sphingomyelinase-mediated regulation in response to external stimuli such as cytokines, hypoxia, heat, ionizing radiation, or antitumor drugs (Ogretmen and Hannun, 2004; Ogretmen, 2006). Together with the cell signaling function of ceramide, these data support a therapeutic role for ceramide.
Exogenous ceramide is cytotoxic to various tumor cell lines (Shabbits and Mayer, 2003b; Stover and Kester, 2003). Long-chain (C16) ceramide has been shown to increase the life span of murine tumor models by 20% (Shabbits and Mayer, 2003a). Short-chain (C6) ceramide has also been shown to be effective in vivo, reducing murine tumor size by 6-fold (Stover et al., 2005). The therapeutic mechanism by which short-chain C6-ceramide induces tumor regression is thought to involve caspase 3/7 activation and inhibition of the Akt prosurvival pathway, resulting in DNA fragmentation and apoptosis (Stover and Kester, 2003).
Researchers have shown that ceramide, in addition to its chemotherapeutic activity, can act synergistically with existing antineoplastic agents such as paclitaxel and tamoxifen (Lucci et al., 1999; Mehta et al., 2000). This synergy may result from reversion of multidrug resistance associated with altered ceramide metabolism. In addition to cancer therapy, ceramide may have the potential to treat a variety of other conditions, such as human immunodeficiency virus infection (Finnegan et al., 2004) and brain ischemia (Furuya et al., 2001; Zimmermann et al., 2001).
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; Stover and Kester, 2003; Stover et al., 2005) and emulsions (Desai et al., 2007). Short-chain C6-ceramide (Fig. 1), delivered as a polyethylene glycol (PEG)-coated liposomal formulation, has been shown to be efficacious in a mouse xenograft model of human breast adenocarcinoma (Stover et al., 2005). This liposomal formulation may be expected to take advantage of passive targeting properties related to its nanoscale size and reticuloendothelial system (RES) evading PEG coating, as has been shown for other similar liposomal formulations (Gabizon and Martin, 1997). Indeed, the pharmacokinetics of the radiolabeled [3H]C6-ceramide liposomes were also investigated, and shown to follow two-compartmental first-order kinetics with sustained drug levels in the tumor tissue (Stover et al., 2005). This previous study, however, did not address the loss of liposomal drug loading in vivo, because the analytical methods did not differentiate between incorporated and free ceramide. From a drug delivery perspective, it is more informative to monitor both drug as well as liposomes to gain information on the formulation's integrity/stability, as this may affect the potential therapeutic efficacy and toxicity of the drug. To better characterize the pharmacokinetics and tissue distribution of liposomal C6-ceramide, in the present study we examined the plasma and tissue profiles of a [14C]C6-ceramide and [3H]DSPC labeled C6-ceramide nanoliposome system. The rapid, liposome-independent tissue distribution of the C6-ceramide observed in these studies may have many advantages over longer-chain liposomal ceramide formulations with greater stability but less efficient delivery. |
Materials and Methods |
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Liposomal Formulation. PEGylated ceramide liposomes and control liposomes (non-C6-ceramide) were prepared as described previously, with a minor modification being the addition of radiolabeled [14C]ceramide and [3H]DSPC (Stover et al., 2005). Briefly, lipids were solubilized in chloroform, combined in a specific molar ratio DSPC/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxyPEG(2000)]/N-octanoyl-sphingosine-1-[succinyl(methoxyPEG(750))]/C6-ceramide (5:2.3:1:1:4 M ratio), dried under nitrogen, and hydrated with sterile isotonic 0.9% NaCl solution. Control liposomes (non-C6-ceramide) contained a molar ratio of lipids equivalent to that of the ceramide liposomal formulation. Liposomes were prepared by sonication and extrusion of the solution through 100-nm polycarbonate membranes (Stover et al., 2005). Ceramide loading in liposomes was 14% (w/w). The specific activities of [14C]C6-ceramide and [3H]DSPC were 97.4 and 194.8 µCi/g of liposomes, respectively. The specific activity of [3H]DSPC in the control liposomes was 343 µCi/g. The liposomes were kept at 4°C in saline at a concentration of 25 mg/ml before use.
Particle Size Measurements. A Zetasizer Nano ZS instrument (Malvern Instruments, Southborough, MA) with a backscattering detector was used for measuring the hydrodynamic size (diameter) in batch mode at 25°C in a low-volume quartz cuvette. Hydrodynamic size is reported as the volume-weighted average diameter and its percentage of the size populations present. Stock solutions of control and ceramide liposomes were diluted in phosphate-buffered saline to give a final concentration of 1 mg/ml. A minimum of seven measurements were taken for each sample.
Zeta Potential Measurements. The Zetasizer Nano ZS instrument was used to measure the zeta potential of control and ceramide liposomes. Samples of liposomes were diluted in 10 mM NaCl to give a 1 mg/ml final concentration. Samples were loaded into a folded capillary flow cell and a voltage of 100 V was applied. A minimum of three measurements were taken for each sample.
Pharmacokinetic and Tissue Distribution Studies. Ten-week-old double jugular catheterized female Sprague-Dawley (SD) rats (approximate weight of 200 g) were purchased from Charles River Laboratories, Inc. (Raleigh, NC). The animals were fed standard rat food (Purina) and chlorinated tap water ad libitum. The animal rooms were maintained on a 12-h light/dark cycle, with a temperature range between 20 and 22°C and 50% relative humidity.
Ceramide liposomes (50 mg of liposomes/2 ml of saline/kg) or control liposomes were administered via the left jugular vein catheter (four to five rats per treatment group). A nonliposomal C6-ceramide control group was not included because of the insolubility of ceramide. Blood samples (200 µl) were collected via the right jugular catheters at each time point (15 and 30 min and 1, 2, 4, 8, 24, and 48 h postdose) and placed in lithium heparinized tubes. The blood was centrifuged immediately after collection, and the resulting plasma was used for pharmacokinetic (PK) analysis. For tissue distribution studies, rats were euthanized by CO2 asphyxiation at 2 min and 1, 2, and 48 h postdose, and the following tissues were recovered: brain, liver, lungs, kidneys, heart, and spleen.
NCI-Frederick is accredited by Association for Assessment and Accreditation of Laboratory Animal Care International and follows the U.S. Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1996).
Plasma and Tissue Analysis. For plasma samples 80 µl of plasma was mixed with 1 ml of BTS-450, followed by addition of 15 ml of Ready Organic liquid scintillation cocktail. For tissue samples, 70 to 100 mg of tissue was mixed with 1 ml of BTS-450 and incubated at 50°C for 12 h to solubilize the tissue completely. Subsequently, the samples were cooled to room temperature, treated with 200 µl of a 30% (w/w) H2O2 solution, and allowed to set for 1 h. Samples were then diluted with 15 ml of Ready Organic liquid scintillation cocktail.
[14C]C6-ceramide and [3H]DSPC radioactivities were determined by scintillation counting using dual-label mode (Beckman Coulter LS6500). The counting efficiencies were 50 and 80% for 3H and 14C, respectively. It should be noted that radioactivity measurements in tissues, blood, and plasma did not differentiate between intact tracer and metabolites derived therefrom.
Analysis of Pharmacokinetic Parameters. WinNonLin software (Pharsight, Mountain View, CA) was used for noncompartmental analysis of pharmacokinetic data. Pharmacokinetic parameters were determined as follows: the area under the time concentration curve (AUC) was calculated using the linear trapezoidal rule with extrapolation to time infinity; clearance (CL) was calculated from dose/AUC; apparent volume of distribution (Vd) was calculated from dose/C0 (concentration at time 0 calculated from extrapolation of the plasma time curve); and plasma half-life (t1/2) was calculated from 0.693/slope of the terminal elimination phase.
In Vitro Blood Interaction Studies. In vitro blood interaction studies were conducted using the blood from untreated female SD rats. All Eppendorf collection tubes were washed with heparin solution (70 units/ml) before addition of blood. Blood samples were incubated at 37°C for 5 min before mixing with ceramide liposomes to mimic in vivo temperature conditions. Eppendorf tubes containing the ceramide liposome-blood mixture (liposome concentrations of 34.7, 85.4, 112.9, and 166.7 µg/ml) were incubated at 37°C for 10 min in a rotary shaker (Barnstead Thermolyte Labquake Shaker, rotation at 8 rpm) to ensure complete mixing. After incubation, a 10-µl sample of the ceramide liposome-blood mixture was removed for radioactivity measurement. Plasma was separated from the remaining ceramide liposome-blood mixture by centrifugation (VWR Galaxy mini microcentrifuge, 2000g, 5 min), and 10 µl of the resulting plasma was used for radioactivity measurement. Plasma and blood samples were prepared by adding 1 ml of BTS-450 to a 10-µl sample, followed by addition of 200 µl of a 30% (w/w) H2O2 solution and 15 ml of Ready Organic liquid scintillation cocktail. Radioactivity was measured by scintillation counting using dual-label mode (Beckman Coulter LS6500). The fraction of ceramide and liposomes in plasma (Fp) was calculated according to eq. 1 (Weiss et al., 2006). The estimated hematocrit value (Hc) was 0.46, according to the literature (Sharp and LaRegina, 1998; Webb et al., 1998). Cp and Cb refer to concentration in plasma and in blood, respectively. All measurements were conducted in triplicate, and the mean values and S.D. are reported.
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Results |
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Pharmacokinetic and Tissue Distribution Studies. Radiolabeled, ceramide-loaded liposomes and control liposomes were administered to SD rats via jugular vein catheters. The resulting plasma concentration profiles are reported as the percentage of injected dose per milliliter of plasma for each treatment group, calculated from the amount of radioactive [14C]C6-ceramide and [3H]DSPC administered. Figure 2 shows the percentage of injected dose of [3H]DSPC (ceramide liposomes and control liposomes) and [14C]C6-ceramide per milliliter of plasma versus time profile, on the y1- and y2-axis, respectively. Pharmacokinetic parameters (C0, AUC, CL, t1/2, and Vd) for the ceramide, ceramide liposomes, and control liposomes are summarized in Table 1.
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Plasma profiles (Fig. 2, y1-axis) and calculated PK parameters (Table 1) for the lipid component of the ceramide liposomal and control liposomal formulations were similar, indicating that ceramide incorporation did not alter liposome pharmacokinetics. In contrast, the PK parameters and plasma profile for ceramide and liposomes were quite divergent. The calculated Vd values for ceramide and liposomes was 1020 and 63 ml/kg, respectively. This low Vd for liposomes is consistent with the systemic volume reported for rats (57.5–69.9 ml/kg) (Sharp and LaRegina, 1998), suggesting that liposomes were confined to the vasculature. The Vd of ceramide, however, was approximately 20-fold greater than the systemic volume and 2-fold greater than the total body water, implying that ceramide undergoes high tissue distribution.
Tissue distributions of [14C]C6-ceramide and [3H]DSPC were evaluated in the following organs: brain, heart, kidney, liver, lung, and spleen. The percentages of injected radioactive dose per organ for [3H]DSPC ceramide liposomes, [3H]DSPC control liposomes, and [14C]C6-ceramide are shown in Table 2. As with their plasma concentration profiles, tissue distribution of lipid tracer in the ceramide liposome and the control liposome groups was also comparable, demonstrating again that ceramide incorporation did not alter liposome pharmacokinetics. The [14C]C6 ceramide tissue profile mimicked the plasma profile. Approximately 23% of the injected [14C]C6 ceramide dose was accounted for in the liver at 2 min. In comparison with [14C]C6 ceramide, tissue accumulation of [3H]DSPC was delayed, with peak levels in liver at 1 h corresponding to 20% of the injected dose in both the ceramide liposome and control liposome groups.
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Tissue distribution data were also analyzed on a per gram of tissue basis to determine uptake selectivity relative to plasma. Tissue [14C]C6-ceramide concentrations indicated greater preferential distribution toward lung and liver, followed by heart and spleen (Table 3). In contrast, [3H]DSPC (Table 3) accumulated in liver and spleen only and displayed slower tissue distribution kinetics than that of ceramide.
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In Vitro Red Blood Cell Interaction Studies. In vitro studies were conducted to measure the extent of red blood cell (RBC) partitioning of the ceramide and liposome components of the ceramide liposomes. After equilibration of ceramide liposome-spiked whole blood samples at 37°C, [14C]C6 ceramide and [3H]DSPC concentrations in blood and plasma were measured. Control experiments without plasma were conducted to ensure that ceramide did not absorb to plastic and confound results. Table 4 displays the percentages of [14C]C6 ceramide and [3H]DSPC in the plasma fraction at various ceramide concentrations. These data suggest that [14C]C6 ceramide equilibrates between the plasma and blood compartments (Fp = 53–46% at 35–177 µg of ceramide/ml), whereas the majority of the [3H]DSPC ceramide liposome remains in the plasma fraction (Fp = 91–78%, at 35–177 µg of ceramide/ml).
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Discussion |
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). The chemotherapeutic utility of ceramide, however, is limited by its extremely low solubility. To overcome this limitation, liposomes consisting of one (Stover and Kester, 2003) or more (Shabbits and Mayer, 2003a) phospholipid bilayers with enclosed aqueous phases have been used to deliver incorporated ceramide to tumors in vivo.
Liposomal C6-ceramide has been shown to cause concentration-dependent cytotoxicity in mammary adenocarcinoma cells, with IC50 values of 5 µM (Stover et al., 2005). Efficacy of the liposomal C6-ceramide has also been demonstrated in a murine xenograft tumor model of human breast adenocarcinoma (Stover et al., 2005). Most intriguingly, cytotoxicity was not observed in normal epithelial cell lines at concentrations up to 50 µM (Stover et al., 2005), suggesting a neoplasm-selective mechanism. This neoplastic selective mechanism has also been observed in melanoma versus melanocytes (Tran et al., 2008) and is the basis for the untargeted, interbilayer-dependent nanoliposomal ceramide exchange delivery system used in these studies. In the present study we sought to further characterize the pharmacokinetics and tissue distribution of liposomal C6-ceramide in rats. The in vivo integrity (stability) of liposomes is an important characteristic for liposomal formulations. The drug/lipid ratio in plasma, relative to initial loading, can be used to determine the integrity of liposomes in vivo. The assessment of the integrity of liposomes is crucial, because this may affect the distribution, metabolism, and excretion profile, and in turn the therapeutic efficacy and toxicity of the liposomal drug. For example, premature release of drug from the liposomes may result in acute toxicity. To monitor the in vivo integrity of liposomes, dual radiolabeled ceramide liposomes with [14C]C6-ceramide and [3H]DSPC were used in this study.
Nanotechnology drug delivery systems, such as nanoscale liposomes, have several advantages for cancer therapy (Gabizon and Martin, 1997). In particular, the prolonged circulation half-life and size-dependent distribution mechanisms of nanotechnology drug delivery systems allow for passive tumor accumulation, i.e., the enhanced permeation and retention effect, whereby the tumor's leaky vasculature and poor lymphatic drainage result in selective tumor uptake (Iyer et al., 2006). Longer circulation times can be achieved by shielding liposomes with a PEG coating to create "stealth liposomes," thus minimizing RES uptake and enabling passive tumor targeting. As expected, PEGylated C6-ceramide liposomes and control liposomes exhibited a long circulation t1/2 of 14 h (Table 1). Also as expected, the Vd of the [3H]DSPC tracer (63 ml/kg) indicated that liposomes were confined to the vasculature and did not exhibit high tissue distribution. PK parameters (C0, AUC, CL, and Vd) were similar for both ceramide liposome and control liposome formulations, suggesting that ceramide intercalation did not alter liposome stability in vivo. In contrast with these results for liposomes, the Vd of ceramide was approximately 20-fold greater (1020 ml/kg), indicating a larger tissue distribution. This high tissue distribution of ceramide relative to liposomes is significant and may indicate that the C6-ceramide liposomes are unstable or, more likely, that rapid release of C6-ceramide occurs and may influence the tissue distribution and ultimately, the therapeutic value of ceramide in vivo.
The nature of the ceramide/bilayer interactions could explain the liposomal ceramide distribution. During the formulation of the ceramide liposomes, ceramide/phospholipid bilayer interactions are favored because of the chemical structure of ceramide, with small hydroxyl head group and saturated hydrophobic chains positioning themselves in the bilayer. This positioning can be envisioned as being similar to the way endogenous ceramide forms in the microdomain of the cellular bilayer membrane (Cremesti et al., 2002; Finnegan et al., 2004). It has been shown that ceramide stabilizes membranes and coassociates, forming lipid rafts (Wang and Silvius, 2003; Megha and London, 2004). Therefore, liposomal instability in the presence of ceramide is unlikely. Correspondingly, long-term storage stability studies (up to 70 days at 4°C) of ceramide liposomes did not result in any particle size change, which is commonly used as an indication of instability (supplemental Fig. 3).
A likely explanation for the loss of ceramide loading in vivo is that rapid ceramide transfer occurs when ceramide comes in contact with other phospholipid bilayer structures (such as red blood cells or other cell membranes) and with plasma proteins, resulting in high ceramide Vd (Fig. 3). Endogenous ceramide bilayer diffusion has been studied extensively owing to its importance in cell signaling processes (Ogretmen and Hannun, 2004). In a study by López-Montero et al. (2005), the half-life of ceramide bilayer diffusion in RBCs was reported to be rapid (within minutes). This diffusion of ceramide in RBC followed Arrhenius kinetics, for which the rate of diffusion was temperature-dependent (López-Montero et al., 2005). The observed preferential distribution of ceramide to well perfused organs, such as liver and lung (Table 3), may be explained by this bilayer exchange mechanism. Liposomes, alternatively, followed a classic RES organ uptake pattern (Table 3).
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The literature supports the ability of lipophilic drugs to pass through the RBC membranes and equilibrate with plasma proteins (Hinderling, 1997). The role of soluble proteins, lipoproteins, and lipid exchange proteins in lipid transfer between cell membranes has been reported for cholesterol, phospholipids, sphingomyelin, 
-to-copherol, and triglycerides (Bell, 1978). For ceramide, a transbilayer diffusion in red blood cells, from the outer monolayer toward the inner monolayer, was reported to be 1 min at 20°C. This rate was too fast to be measured at 37°C (López-Montero et al., 2005). Taken together, the rapid transbilayer diffusion of ceramide and the presence of soluble lipid-associated proteins may explain the observed equilibrium between plasma and whole blood in vitro (Table 4). In vitro release assays that use multilamellar vesicles as a bilayer acceptor compartment have been used to better predict these interactions of liposomal drugs with red blood cells and other cell membranes in vivo (Shabbits et al., 2002).
The rapid release of liposomal C6-ceramide has potential advantages and disadvantages. One of the advantages of this formulation, is that rapid bilayer exchange allows for immediate distribution of ceramide to all well-perfused tissues, which may not result in adverse affects as ceramide appears to exhibit a neoplasm-selective mechanism. Indeed, extensive toxicology studies have shown no biologically significant changes in body weight, organ weight, clinical chemistries, hematology, gross pathology or histology at the 50 mg/kg dose level in the SD rat model, supporting a tumor-selective mechanism (detailed information on the toxicology studies of the "Ceramide Liposomes" can be found at http://ncl.cancer.gov/working_technical_reports.asp). The liposomal ceramide bilayer exchange mechanism also circumvents the lysosomal degradation pathway that might be encountered by following an endocytosis-mediated delivery route. On the other hand, it is desirable in cancer therapy to attain the highest concentration of the chemotherapeutic agent in the target tissue. Maintaining in vivo stability, while ensuring biologically efficacious release kinetics would further increase selective, enhanced permeation and retention-mediated tumor accumulation. Another potential issue is that rapid release of liposomal C6-ceramide could result in a potential disconnect between the perceived potency based on in vitro data and actual in vivo efficacy. In vitro constitutes a closed system in which ceramide release is directed exclusively to the target cell line, but in vivo constitutes an open system with intended and unintended cellular targets.
A possible approach to prevent this rapid bilayer exchange and better utilize the distribution-related advantages inherent in nanotechnology-based delivery systems might be to extend the carbon chain length of the ceramide. It has been reported that extending the carbon chain to 16 increased the association of this long-chain ceramide with liposomes (Shabbits and Mayer, 2003b). However, it should be noted that liposomal C16-ceramide cellular uptake occurs via an endocytosis-mediated mechanism, not bilayer exchange, which may not be as efficient and may result in lysosomal degradation. A major challenge in liposomal delivery to tumor tissue has been the lack of rapid cellular accumulation, allowing liposomes to "wash out" of the interstitial space before incorporation of drug in the target cells and requiring the development of targeted strategies (Hatakeyama et al., 2007). Additionally, variability in tumor vasculature "leakiness" and elevated interstitial pressure can represent a significant barrier to the initial liposome accumulation (McDonald and Baluk, 2002). In this regard, the rapid tissue distribution of the liposomal C6-ceramide may be highly advantageous compared with that of longer chain liposomal ceramide formulations of greater stability. An interesting aspect of the liposomal C6-ceramide is that even after this rapid release of ceramide from the drug delivery vehicle, the PEGylated nanoscale liposomes (which still contains a fraction of loaded ceramide) is intact, displaying favorable kinetics, and does not trigger an immunostimulatory response in animal models. Thus, the C6-ceramide liposome may also be used to deliver an encapsulated therapeutic agent that could work synergistically with ceramide.
This present study highlights the importance of monitoring the in vivo integrity of drug delivery platforms. Although in vitro testing under physiological conditions can give an initial estimate of formulation stability, realistically, it is difficult to simulate the complexity of the biological interplay found in vivo. This necessitates the use of analytical methodologies that can characterize the integrity of drug formulations in biological matrices, to ensure that desired formulation attributes are maintained. The outcome of this approach has identified a unique drug delivery mechanism potentially responsible for the efficacy of short-chain ceramide nanoliposomes despite apparent premature release of C6-ceramide from the drug delivery platform.
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Acknowledgments |
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: PEG, polyethylene glycol; RES, reticuloendothelial system; DSPC, 1,2-distearyl-sn-glycero-3-phosphocholine; C6-ceramide, N-hexanoyl-D-erythrosphingosine; BTS-450, Beckman tissue solubilizer 450; SD, Sprague-Dawley; AUC, area under the time-concentration curve; RBC, red blood cell.
The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Stephan T. Stern, Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD 21702. E-mail: sternstephan{at}mail.nih.gov
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, [3H]DSPC of ceramide liposomes; 
, [3H]DSPC of control liposomes on the y1-axis; 
, [14C]C6-ceramide on the y2-axis. Each point represents the mean ± S.D. from n = 4 to 5 rats.