In vitro cellular effects of perfluorochemicals correlate with their lipid solubility

Viktor V. Obraztsov, Gerald G. Neslund, Elisabeth S. Kornbrust, Stephen F. Flaim, and Catherine M. Woods

Department of Biological Research, Alliance Pharmaceutical Corporation, San Diego, California 92121


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preclinical studies comparing perflubron partial liquid ventilation with conventional mechanical ventilation have indicated that perflubron partial liquid ventilation may exert some anti-inflammatory effects. To assess whether these effects were related to the lipid solubility properties of perflubron rather than to nonspecific biophysical properties of the perfluorocarbon (PFC) liquid phase, we studied the effects of PFCs with varying lipid solubilities on the platelet aggregation response to various procoagulants and the erythrocyte hemolytic response to osmotic stress. In both cases, the degree of the response was directly related to the lipid solubility of the PFC. All the perflubron content of erythrocytes was found to be associated with the membrane compartment. The time to reach a maximum effect on hemolysis with perflubron was relatively slow (2-4 h), which paralleled the time for perflubron to accumulate in erythrocyte membranes. The rate and extent of perflubron partitioning into lecithin liposomes were similar to those of erythrocyte membranes, supporting the hypothesis that perflubron was partitioning into the lipid component of the membranes. Thus some of the potential modulatory effects of perflubron on excessive inflammatory responses that occur during acute lung injury and acute respiratory distress syndrome may be influenced in part by the extent of PFC partitioning into the lipid bilayers of cellular membranes.

perfluorocarbon; perflubron; membrane partitioning


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PARTIAL LIQUID VENTILATION (PLV) with biochemically inert perfluorocarbons (PFCs) such as perflubron [perfluorooctyl bromide (PFOB)] is being evaluated for efficacy in supporting gas exchange and pulmonary function compared with standard conventional mechanical ventilation (4, 7, 8, 16, 23, 30). A variety of in vivo studies (3, 7, 8, 16, 21, 26) have indicated that PLV with PFOB may attenuate the excessive lung tissue damage that develops during the localized inflammation that ultimately leads to the development of acute lung injury. These observations provide the basis for PLV with PFOB being developed as a potential lung protective ventilation strategy for patients with acute respiratory distress syndrome (4, 6, 12, 34).

In vitro studies with isolated leukocytes and alveolar macrophages have indicated that PFOB exposure may dampen the extent of their responses to proinflammatory stimuli. Isolated alveolar macrophages when activated with lipopolysaccharide in the presence of PFOB exhibited reductions in cytokine secretion (28), reactive oxygen species production (24), and rate of phagocytosis (27) compared with those in activated cells incubated in control medium. The mechanism(s) underlying these PFC effects on macrophage function is unclear. In the case of human neutrophil interactions with airway epithelial cells, one in vitro study demonstrated that an inhibition of adhesion to lung epithelial cells and subsequent cytolysis was observed only when a liquid PFOB phase was maintained during activation with the proinflammatory stimulus (32). This suggested that in this case, PFOB exerted its anti-inflammatory effect by providing a physical barrier between the effector cells and/or stimulant-containing medium and their corresponding target epithelial cells.

However, we have provided evidence for another potential mechanism of action, showing that PFOB-induced changes in inflammatory responses can occur independently of direct physical contact with the liquid PFC phase. Rather, these inhibitory effects occurred in parallel with diffusion of low levels of PFOB into cellular membranes (24, 35). Based on the biophysical properties of PFOB and its moderate lipid solubility (37 mM in olive oil) (11, 19), it would be predicted to intercalate deep within the lipid bilayer where it would be predicted to cause a nonspecific membrane stabilization effect (1, 10, 17).

To assess whether PFOB could mediate changes in membrane-mediated reactions simply on the basis of its solubility in the membrane lipid compartment, we measured the effect of PFCs with similar gross physical properties but varying lipid solubilities on the subsequent responses of erythrocytes and platelets to osmotic stress and activation, respectively. These have served as classic model systems for assessing the potential for anesthetic effects (20, 22). These cells are enucleated cell types that lack Golgi bodies and endoplasmic reticulum and are devoid of de novo synthesis and membrane trafficking. Therefore, any change in a membrane-mediated stress or activation response is more likely to reflect a primary membrane effect of the perfluorochemical. The effects of PFCs with solubilities in olive oil ranging from 1.2 to 800 mM were assessed in these two model assay systems. We found that the protective or attenuating effects of the PFCs directly correlated with their relative lipid solubility in olive oil. Additional kinetic studies with intact erythrocytes, erythrocyte membranes, and lecithin liposomes were performed, and the results are discussed in reference to modulatory effects of PFOB resulting from accumulation into cellular membranes.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neat perfluorochemicals. Sterile PFOB was supplied by Alliance Pharmaceutical (San Diego, CA); perfluorotributylamine (PFTBA) was obtained from 3M (St. Paul, MN); perfluoromethylcyclohexylpiperidine (PFMCP) was obtained from Perftoran (Pushchino, Russia); bis-perfluorobutyl ethene (F-44E) and perfluorooctyl ethane (PFOE) were purchased from F-Tech (Tokyo, Japan); dibromoperfluorohexane (diBrPFH) was supplied by Exfluor Research (Austin, TX); perfluorohexyl bromide (PFHB) was purchased from Fluorochem; and perfluorooctyl iodide (PFOI) was purchased from Asahi Glass (Tokyo, Japan). Unless otherwise stated, all other chemicals and reagents were obtained from Sigma (St. Louis, MO).

Platelet function assay. Swine blood was collected via a vena cava puncture and mixed 9 parts blood to 1 part 3.8% sodium citrate, pH 7.0. Three-milliliter aliquots of whole blood were incubated with 5% (vol/vol) either PFMCP, PFOE, PFOB, PFHB, PFOI, diBrPFH, or saline alone as a control for 45 min at 37°C on a rotator. The samples were then diluted to ~100,000-200,000 platelets/µl with 10 mM sodium phosphate, 0.12 M sodium chloride, and 2.7 mM potassium chloride, pH 7.4 (PBS). Collagen (1 µg/ml) or ADP (5 µM) was added to samples of the pretreated platelet suspensions to stimulate aggregation. Platelet activation was assessed with a whole blood lumi-aggregometer (impedance mode; Chrono-Log, Havertown, PA) and is expressed as a percentage relative to the saline control for each agonist. Aggregation responses were recorded as the maximal extent of aggregation induced relative to control saline-treated samples. In addition, a regression analysis of aggregation versus log lipid solubility of the PFC was performed.

Red blood cell hemolysis assay. Human red blood cells (RBCs) were isolated from whole blood by sequential washes in Dulbecco's PBS followed by low-speed centrifugation. The erythrocyte pellet was then resuspended in PBS with 5 mM glucose and 1.5 mM adenine to a hematocrit of ~5% as previously suggested for this assay (20). For the experiments, the RBCs were continuously rocked in a test tube with 10% (vol/vol) either PFTBA, PFMCP, F-44E, PFOB, PFOI, diBrPFH, or saline control (0.9% NaCl) for up to 5 h at 37°C. After 0.5, 1, 2, 3, 4, or 5 h of incubation with the PFCs, the RBCs were added to a hypotonic saline solution (0.45% NaCl). The degree of RBC hypotonic hemolysis was determined spectrophotometrically by measuring hemoglobin release at 540 nm.

PFOB uptake into erythrocyte membranes and egg yolk phospholipid liposomes. The liposomes, final concentration 10 mg/ml in PBS, were prepared from lecithin purified from egg yolk (Pharmacia) with a Vibra Cell sonicator (Sonics and Materials, Danbury, CT). Erythrocyte membranes (RBC ghosts) were isolated from the fresh blood of normal volunteers as follows. PBS-washed erythrocytes (2-ml packed cell volume) were lysed in ice-cold 5 mM phosphate buffer (pH 7.4), and the erythrocyte membrane ghosts were concentrated by centrifugation at 50,000 rpm for 20 min in a TLN100 rotor (Beckman Instruments) and then resuspended in 1 ml of PBS. The yield from 2 ml of packed cells (equivalent to 4.2 × 109 cells) was a 1-ml membrane suspension with a protein concentration that was typically on the order of 1.1 mg/ml. When intact erythrocytes were used, washed erythrocytes were resuspended in minimal essential medium (GIBCO BRL).

Intact washed erythrocytes (4.9 × 108/sample), erythrocyte membranes (200 µl of a 1.0 mg membrane protein/ml membrane suspension), or lecithin liposomes (200 µl of a 10 mg/ml liposome suspension) were incubated in the presence of 10% (vol/vol) PFOB for various time points up to 15 (erythrocyte membranes) or 24 (intact erythrocytes or liposomes) h and then were washed free of the liquid PFOB and cell medium mixture. To avoid any possible contamination of membranes by liquid PFOB, all membrane samples were centrifuged over a 0.5 M sucrose density step gradient at 40,000 rpm for 15 min in a TLS55 rotor (Beckman Instruments) after incubation with PFOB. The membrane samples taken from the sucrose gradient were resuspended in 0.3 ml of PBS. Triplicate 0.1-ml fractions of the sample were extracted with isooctane solution containing perfluorodecalin as an internal standard. These were then extracted twice with water, and the PFOB content in the upper isooctane phase was analyzed by gas chromatography with a DB-1 capillary column and an electron capture detector.

Data analysis. Data are presented as means ± SE. The relationships between PFC lipid solubility and the effect on platelet and erythrocyte responses to hypotonic stress were analyzed by regression analysis of the individual data points.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Agonist-induced swine platelet aggregation after PFC exposure. Freshly isolated swine platelets were incubated with 5% (vol/vol) either PFMCP, PFOE, PFOB, PFHB, PFOI, diBrPFH, or saline alone (control) for 45 min at 37°C before being activated with agonist. Initially, responses to both ADP and collagen were monitored per treatment sample. However, at concentrations of 5 µM and 1 µg/ml, respectively, there were no differences noted in the extent of aggregation or time to reach a plateau value within a given treatment sample. This would only be expected if a differential effect on the aggregation compared with the overall aggregation, including that induced by prostaglandin-mediated ATP release, was induced by PFC membrane intercalation. Because this did not appear to be the case in the preliminary studies, all subsequent experiments were carried out with collagen alone and used for the comparison of the different PFC effects. As shown in Fig. 1A, where the treatment groups are plotted in order of increasing lipid solubility of the PFC against the maximal aggregation achieved (relative to control samples), there was an increasing reduction in the level of activation with increasing lipid solubility of the PFC. PFOB pretreatment resulted in a moderate 45% reduction in platelet aggregation relative to the saline control (P < 0.05). In comparison, PFOI (solubility of 98 mM) and diBrPFH (solubility of 800 mM) pretreatment resulted in 74 and 91% suppression, respectively, in platelet aggregation relative to the saline control (P < 0.05).



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Fig. 1.   Agonist-induced swine platelet aggregation after exposure to perfluorochemicals in vitro. Swine platelets were isolated from whole blood and incubated with 5% (vol/vol) either perfluoromethylcyclohexylpiperidine (PFMCP), perfluorooctyl ethane (PFOE), perflubron (PFOB), perfluorohexyl bromide (PFHB), perfluorooctyl iodide (PFOI), dibromoperfluorohexane (diBrPFH), or saline alone (control) for 45 min at 37°C. Subsequent platelet aggregation in response to agonist was assessed with a whole blood lumi-aggregometer, and maximal plateau aggregation value obtained is expressed as a percentage relative to saline control (set at 100% aggregation). A: data presented in order of lipid solubility for each perfluorocarbon (PFC). Values are means ± SE; n, no. of experiments. B: regression analysis of extent of aggregation vs. log PFC solubility.

A regression analysis of the extent of aggregation obtained versus the log lipid solubility of a given PFC indicated that there was a log linear inverse relationship over this range of lipid solubilities, with a correlation coefficient of -0.922 (Fig. 1B). This indicated that for these platelet aggregation responses, the extent of inhibition of platelet aggregation was directly related to the logarithmic lipid solubility of the PFC.

Hypotonic hemolysis of human RBCs after PFC exposure. To assess the rate as well as the extent of the effect of PFC lipid solubility on the resistance to hypotonic stress, purified human RBCs were exposed to 10% (vol/vol) either PFTBA, PFMCP, F-44E, PFOB, PFOI, diBrPFH, or saline alone (control) for up to 5 h at 37°C before exposure to hypotonic shock (0.45% NaCl). The subsequent extent of hemolysis relative to control erythrocyte samples (preexposed only to saline, with hemolysis value set to 100%) was a measure of how resistant the different samples were to hypotonic shock. Erythrocytes were chosen as the model system of choice for these kinetic experiments because they are a more robust cell type and readily retained their integrity after prolonged incubation times at 37°C.

As shown in Fig. 2A, there was an increasing effect of PFC preexposure on hemolysis over time observed, which plateaued between 3 and 5 h, with a trend toward increasing protection against lysis with increasing lipid solubility of the PFC. Thus a very minor protective effect was observed with PFTBA (lipid solubility of 1.2 mM; lysis reduced by 2.5%), a moderate effect with PFOB (solubility of 37 mM; lysis reduced by 23%), and a marked effect with diBrPFH (solubility of 800 mM; lysis reduced by 59%). It should be noted that exposure to PFCs did not induce any hemolysis over and above that of the saline control. Rather, the PFC effect was only observed on addition of the hypotonic buffer (data not shown). A regression analysis of the individual data obtained after 5 h of exposure was performed (Fig. 2B), which showed that the effect of PFCs on hemolysis was directly related to the log value of the PFC solubility in olive oil (correlation coefficient = -0.977). If the regression analysis was performed with all data points obtained between 3 and 5 h, the correlation coefficient obtained was -0.974, indicating that the effect was reaching a plateau value after 3 h of incubation.



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Fig. 2.   Relative hypotonic hemolysis of human red blood cells (RBCs) after exposure to perfluorochemicals in vitro. Human RBCs were exposed to 10% (vol/vol) perfluorotributylamine (PFTBA), PFMCP, bis-perfluorobutyl ethene (F-44E), PFOB, PFOI, diBrPFH or saline alone (control) for up to 5 h at 37°C before being exposed to hypotonic saline solution (0.45% NaCl). Extent of RBC hypotonic hemolysis was determined spectrophotometrically by measuring hemoglobin release at 540 nm. A: response over time (relative to control untreated cells). Data are means ± SE; n = 4 experiments. B: regression analysis of data obtained at 5 h preexposure to PFCs.

PFOB uptake into erythrocyte membranes. To assess whether the time course to observe a maximal erythrocyte response reflected the kinetics of PFOB partitioning into the membrane, erythrocyte membranes were exposed to PFOB for varying time intervals between 1 and 15 h, and the PFOB content was measured. As shown in Fig. 3, the rate of uptake of PFOB into the membrane fraction was relatively slow, plateauing between 6 and 15 h at a level of 2 µg PFOB/mg membrane protein. It is important to note that the samples exposed for very short intervals (liquid PFOB added and then immediately washed away) had no detectable PFOB content (data not shown), indicating that these values represented true membrane PFOB content and not residual PFOB being carried along after immersion in PFOB liquid. Parallel studies with intact erythrocytes, which were incubated up to 20 h in the presence of 10% PFOB, showed a steady increase in erythrocyte PFOB content for the first 4 h, reaching a value of 0.98 ± 0.13 µg/108 RBCs by 7 h and remaining constant thereafter, with values of 0.86 ± 0.13 and 0.87 ± 0.14 µg/108 RBCs at 16 and 20 h, respectively.


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Fig. 3.   Uptake of PFOB into erythrocyte membranes. Erythrocyte membranes were prepared by hypotonic lysis and centrifugation and resuspended in PBS at a concentration of 0.5-1.0 mg/ml membrane protein. Membranes were exposed to PFOB for varying lengths of time and then recovered by sucrose gradient centrifugation. PFOB content of membrane samples was determined by gas chromatography. Data are means ± SE; n = 4 experiments.

In a subset of samples taken after 4 h of incubation with PFOB, the cells were lysed with 5 mM Tris · HCl (pH 7.4), the membrane fraction was separated from the cytoplasmic fraction by high-speed centrifugation, and the PFOB content in the two fractions was measured. There was no detectable PFOB in the cytoplasmic fraction; rather, as predicted from its biophysical characteristics, all the PFOB was associated with the membrane fraction (data not shown).

To assess how the rate and extent of PFOB uptake into erythrocyte membranes compared with the kinetics of PFOB partitioning into lipid vesicles, lecithin liposomes were prepared and then incubated with PFOB for various time intervals up to 24 h. The rate of uptake into the liposomes was again relatively slow, reaching maximal PFOB levels of 1.2 µg/mg lecithin after 8 h (Fig. 4). This PFOB concentration was in the same range as the levels partitioning into erythrocyte membranes after normalization to the lipid content of erythrocyte membranes.


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Fig. 4.   Partitioning of PFOB into lecithin vesicles. Samples of a 10 mg/ml lecithin vesicle suspension in PBS were exposed to 10% PFOB for varying lengths of time, washed of excess PFOB, and recovered by sucrose gradient centrifugation. PFOB content of vesicle samples was determined by gas chromatography. Data are means ± SE; n = 4 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PFOB is a biochemically inert PFC with high gas solubility properties. It has low lipid solubility (37 mM in olive oil) and is extremely hydrophobic (aqueous solubility ~10-9 M) (11, 19). PFOB (LiquiVent) is under clinical development for use in PLV to improve oxygenation and pulmonary function (4, 6, 12, 34) and has shown some benefit in clinical (4) and preclinical studies at reducing inflammatory responses associated with lung injury in vivo (3, 7, 8, 21, 26) as well as in vitro (24, 25, 27, 28, 32). Two possible mechanisms for the observed anti-inflammatory effects have been proposed. One possibility is that the dense, nonmiscible PFC liquid creates a physical mechanical barrier over the target cell surface. Alternatively, the solubility of PFOB in olive oil (37 mM) (11, 19), although low, is sufficient to expect that some PFOB will partition into the lipid component of cellular membranes. This could affect subsequent membrane protein responses to activation stimuli by nonspecific membrane effects resulting from its localization within the lipid bilayer. Either mechanism, alone or in concert, might be expected to modulate the response of leukocytes to activation stimuli.

Varani et al. (32) have presented evidence in support of a physical barrier mechanism of action for PFOB. Human neutrophil adhesion to and subsequent lysis of cultured lung epithelial cells in vitro were decreased or virtually abolished only when the target cells were maintained in contact with a liquid PFOB phase during activation. When neutrophils or epithelial cells were preexposed to PFOB and then washed before being activated, cell adhesion and subsequent epithelial cell lysis were no different from those in the untreated control cells. However, we have evidence to support that the second mechanism may also play a role, namely that PFOB may also exert some immunomodulatory effects by passive diffusion into cellular membranes (24, 35). PFOB pretreatment did not abolish the activation responses to proinflammatory stimuli but was associated with a significant attenuation of cytokine release by macrophages and superoxide release by neutrophils even when the cells were separated from direct contact with the PFOB phase (24). Likewise, as described in the companion paper (35), indirect exposure of cultured endothelial cells to PFOB resulted in a decrease in the degree to which adhesion molecule expression was upregulated in response to proinflammatory stimuli, which correlated with PFOB partitioning into the cellular fraction. Therefore, we hypothesized that by diffusing into cell membranes, PFOB and other lipid-soluble PFCs may have a generalized protective or attenuating effect on a variety of membrane-mediated cell responses to stress or activation. If this were the case, then one would predict that the extent of the cellular effects observed should correlate with the lipid solubility of the PFC and be dependent on the kinetics of PFC partitioning into the membrane lipid.

To test this hypothesis, we treated a suspension of platelets and erythrocytes with PFCs of varying lipid solubilities (1.2-800 mM in olive oil) (19) for various incubation times (0.5-5 h) and then measured their subsequent responses to activation and hypotonic stress, respectively. Erythrocytes and platelets were chosen as model cells because they represent simple enucleated cell types that lack extensive intracellular organelle systems. Consequently, any observed responses represent a direct membrane-mediated response(s) without the potential for complications due to downstream transcriptional and translational events or potential interference with membrane trafficking.

The data presented in this report clearly demonstrated a relationship between PFC lipid solubility and the extent of reduction in agonist-induced platelet aggregation or hemolysis. Whereas pretreatment with PFMCP (lipid solubility of 2 mM) resulted in a minimal protective effect against hemolysis under hypotonic conditions or on platelet aggregation, the moderately soluble PFOB (37 mM in olive oil) induced a 23% reduction in hemolysis and a 45% reduction in platelet aggregation compared with 59 and 91% reductions, respectively, observed with the highly lipid-soluble diBrPFH (800 mM in olive oil). As seen in Fig. 1, the platelet response to agonists appeared to show a log linear correlation with PFC lipid solubility. PFOI, which is slightly more than twice as lipid soluble as PFOB (98 mM), resulted in a 75% reduction compared with a 45% reduction in aggregation, but the highly lipid-soluble diBrPFH (lipid solubility of 800 mM) resulted in a further decrease of only 16% (overall, a 91% reduction) in aggregation. This also suggests that even with the highly lipid-soluble diBrPFH, the membrane response could not be completely abolished.

To confirm that PFOB did indeed partition into cellular membranes when cells are incubated in the presence of liquid PFOB, erythrocyte membranes were incubated in the presence of PFOB for up to 15 h and PFOB content was measured. The kinetics of uptake were relatively slow and plateaued at 2 µg/mg membrane protein (~0.85 × 108 RBC equivalents) between 6 and 15 h (Fig. 3). By comparison, the antihemolytic effect increased over the first 3 h of incubation, reaching a maximum between 3 and 5 h of incubation (Fig. 2A). Intact erythrocytes reached a plateau PFOB concentration of 0.98 µg/108 RBCs by 7 h of incubation, with PFOB levels remaining constant thereafter. This close correlation between PFOB content of membranes and intact RBCs on a per cell basis indicates that the cellular PFOB was all associated with the membrane compartment as originally predicted from its high log P (partition coefficient between octanol and water phases). Furthermore, the kinetics of PFOB uptake into lecithin vesicles (Fig. 4) were very similar to the kinetics of uptake by erythrocytes. This was confirmed by taking a subset of intact erythrocytes that had been incubated for 4 h in the presence of PFOB, fractionating them into cytoplasmic and membrane fractions, and then measuring the PFOB content. All the PFOB was observed in the membrane fraction (data not shown).

The effects we report here on platelet and erythrocyte responses have been well described for general anesthetics (5, 20, 22, 31). The EC50 for the antihemolysis effect has been shown to correlate with anesthetic potency. Diffusion of anesthetics into membranes has also been shown to modulate activation responses of endothelial cells (9) and leukocytes (15, 29, 33). However, not all lipid-soluble agents have anesthetic activity, and no anesthetic-like effects have been observed with PFOB. Compounds that produce anesthesia in animals or humans, e.g., perfluoromethane (CF4), halothane (C2F3HClBr), and isoflurane (C3F5H2OCl), generally follow the Meyer-Overton hypothesis that predicts that anesthetic potency correlates with lipid solubility. However, the deviation from this hypothesis becomes more pronounced as the chain length of perfluoroalkanes increases even though these compounds are soluble in oil (2, 10, 14). In addition, the replacement of larger halogen atoms for fluorine has been shown to eliminate anesthetic activity. Therefore, a "cutoff" in potency has been established that generally predicts polyhalogenated or PFC molecules to behave as anesthetics when they possess up to two-carbon chain lengths, although there are notable exceptions (2, 14).

PFCs such as the ones used in this study are predicted to diffuse into cell membranes because of their lipid solubility but because of their relatively large carbon chain lengths would not be expected to induce anesthesia. Another key determinant for why these PFCs are not capable of producing general anesthesia may be their specific localization within the membrane lipid bilayer. PFCs with anesthetic properties appear to localize closer to the polar headgroup rather than deeper into the bilayer. This has been shown to be the case with two closely related chlorofluorocarbons cyclic C4H4ClF3, and cyclic C4Cl2F6. The anesthetic C4H4ClF3 with its higher dipole moment localized at the membrane interface near the polar headgroups, whereas the nonanesthetic C4Cl2F6 with the lower dipole moment localized preferentially deep within the bilayer interior (1, 17). The most likely mechanism to explain the moderate effects of the moderately lipid-soluble PFCs that we observed in this study is that they have lower dipole moments compared with the nonperfluorinated hydrocarbons. Therefore, PFCs such as PFOB, with its low solubility in aqueous phases (log P ~7), are more likely to partition in the center of the bilayer compartment of the membrane and, as such, exert a minimal effect on lipid fluidity and ordering within the membrane bilayer. Such a localization could partially explain the nonspecific attenuating or protective effects of PFOB and PFCs seen here and by other investigators for a variety of cell model systems (24, 25, 27, 35) and why the effects reach a set maximum plateau for any given PFC. Indeed, preliminary NMR studies have confirmed that PFOB does partition into model lipid bilayers but does not induce any changes in membrane dynamics or chain order (J. Ellena and D. Cafiso, personal communication). This is consistent with the hypothesis that PFOB would exert only a general membrane stabilization effect and would not be expected to induce changes in membrane dynamics or headgroup order that underlie the more pronounced in vivo effects of anesthetic agents.

The platelet aggregation response discussed in this report was used as a standard model system because it is used as a standard assay for assessing whether or not an agent with moderate lipid solubility can impact membrane responses. It is important to note that these PFOB effects we describe were observed under conditions where platelets were exposed directly to saturating concentrations of PFC. During PLV in vivo, equilibration with the systemic circulation occurs across the lung-blood barrier and, as such, platelet exposure to PFOB is indirect. Furthermore, phase I clinical studies with Oxygent, an emulsified PFOB formulation containing 58% PFOB, have been conducted to determine the effects on coagulation and immune function. Doses up to 1.8 g/kg were administered directly into the circulation. No untoward effects on bleeding time, coagulation responses, or immune function responses were observed nor were any clinical signs of immune dysfunction observed in the 14-day period after dosing in which the subjects were monitored (Leese PT, Noveck RJ, Shorr JS, Woods CM, Flaim KE, and Keipert PE, unpublished data; Noveck RJ, Shannon EJ, Leese PT, Woods CW, Shorr J, Flaim KE, and Keipert PE, unpublished data). These data combined with the fact that PFC exposure to the circulation is indirect during PLV would suggest that the effects we report here are unlikely to occur in vivo during PLV therapy.

In conclusion, PFOB caused a moderate protection in vitro against osmotic hemolysis in human erythrocytes and a moderate decline in the aggregation of human platelets compared with the marked responses observed with the highly lipid-soluble diBrPFH. In contrast, the marginally lipid soluble PFCs PFMCP and PFOE (lipid solubility 1-2 mM) had negligible effects on these cellular responses. Overall, there appeared to be a log linear relationship between the magnitude of the PFC effects and their relative lipid solubilities. The PFC pretreatment of erythrocytes required 3 h of incubation to achieve maximum protection from hemolysis, which reflected the relatively slow kinetics to reach equilibrium between the PFC phase and cell lipid phase. These observations taken together may explain, in part, the possible mechanisms behind the beneficial anti-inflammatory effects of PFOB seen with the in vivo and in vitro models of cellular stress and acute inflammation.


    ACKNOWLEDGEMENTS

We are grateful to Glen Luena and Ron Clemente for technical assistance with the gas chromatography analysis of perflubron content.


    FOOTNOTES

Present address of S. F. Flaim: Galileo Laboratories, Inc., 5301 Patrick Henry Dr., Santa Clara, CA 95054.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. M. Woods, Dept. of Biological Research, Alliance Pharmaceutical Corp., 3040 Science Park Rd., San Diego, CA 92121 (E-mail: cmw{at}allp.com).

Received 18 May 1999; accepted in final form 1 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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