R E T E N T I O N OF A DIALKYLPHOSPHATIDYLCHOLINE IN MYELIN AND OTHER MEMBRANES H. BROCKERHOFF, H. M. WISNIEWSKI, L. C. LIPTON, AND D. S. DESHMUKH Institute for Basic Research in Mental Retardation t050 Forest Hill Road Staten Island, New York 10314
Accepted January 6, 1980
We describe an attempt to incorporate a metabolically inert phospholipid analog into animal membranes, especially myelin, in vivo, with the view of eventual longterm membrane modification or membrane engineering. A sonicated suspension of a mixture of [~"C]phosphatidylcholine and its dialkyl analog, [3H] tetradecyloctadecano(l)phosphocholine, was injected into the brain of weanling rats. Samples were counted of whole brain, myelin, liver, and carcass, at intervals from 1 to 63 days, and the composition of the extracted labeled lipid was determined by thin-layer chromatography. Both lipid labels were found to be cleared from the body at similar rates, but while phosphatidylcholine was metabolized within a day, with the label appearing mainly in the phosphatidylethanolamine fraction and in nonpolar lipids, the dialkylphosphatidylcholine remained intact, with retention in myelin of a small but almost constant amount for a month. Ways will have to be found to enhance uptake of the lipids by the brain.
INTRODUCTION Modifications of the lipid matrix of biological membranes in intact organisms have been achieved by several routes. The hydrophobic membrane core can be altered through the diet; the withholding of linoleic acid, for example, leads to membranes poor in linoleic and arachidonic acid, and, presumably, reduced fluidity (1). The brain is least affected by the diet (2-4). Incorporation of an unnatural analog of an ether-phospholipid in the brain has been achieved by injection of a hexadecyl glycol ether 617 0364-3190/80/0600-0617503.00/0 9 1980 Plenum Publishing Corporation
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(5). The polar regions of a membrane can be made to accept foreign bases, e.g., diethylaminoethyl residues, in the head groups of the phospholipids (6-9). Finally, foreign lipids can be taken up by some microorganisms or cultured cells, e.g., cholesterol and other sterols into Acholeplasma (10-12). The modifications so far achieved are reversible and transient unless maintained through the environment, i.e., diet or culture medium. There is a similarity of these methods, in principle, to drug therapy, which may be considered as membrane modification in the case of those drugs that act in or on the lipid matrix. The reason for the short life span of membrane modifiers is the fast turnover of the membrane components, especially the phospholipids. Our study is intended to be a preliminary investigation into the possibility of a more permanent modification, which might be named membrane engineering in analogy to genetic engineering. Precisely, is it possible to incorporate lipid modifier molecules, with retention of their structure, and maintain their levels, in living membranes for extended periods of time? Such molecules would have to resist rapid catabolic degradation, although they should perhaps not have an indefinite life span. Since the initial breakdown of phosphoglycerides in mammals usually takes place at the carboxyl ester groups, we have chosen a dialkyl analog of phosphatidylcholine for intracerebral injection in rats in order to follow its incorporation, and survival, in myelin and other tissues. For comparison, phosphatidylcholine of analogous structure was injected simultaneously. This lipid was metabolized (mainly converted to other lipids) within a day, while the dialkylphosphatidylcholine stayed intact with a half-time of clearance (whole body) of about a week, and retention in myelin at a steady level for a month. Incorporation into myelin was low, only 0.2-0.3% of the injected dose, and our experimental conditions are not likely to be adequate for modifying the membrane in a perceptible degree, unless the modifier should be a powerful drug. Efforts toward improvement of incorporation will be made in the future.
EXPERIMENTAL METHOD Lipids. Phosphatidylcholine (PC, or diester-PC) as egg phosphatidylcholine (13) was deacylated in position 2 with snake venom phospholipase (14); the lyso compound (0.5 mmol) was reacylated with a mixture (equilibrated overnight) of oleic anhydride (1 mmol) and [1~4C]stearic acid (1 mCi, 0.124 mmol) in 6 ml xylene, with K-oleate (0.15 mmol) added, under vacuum, with rotation, at 95~ for 4 hr (15). Chromatography on silicic acid column (16, 17) yielded diester-PC, 0.39 mmol, pure by thin-layer chromatography, specific activity 0.45 X 10 6 dpm/ixmol. Thus, 2.5% of the lipid contained stearic acid, with the label, in the 2 position.
MEMBRANE INCORPORATION OF INERT LIPID
2-Tetradecyloctadecano(1)phosphocholine (dialkyl-PC, or alkyl2-PC) as the monoenoic analog, 2-tetradecyloctadec- 11-eno(1)phosphocholine (18, 30), was tritiated across the double bond (New England Nuclear); specific activity 15.7 mCi/ixmol. An appropriate quantity of the radioactive lipid was diluted with the monounsaturated lipid and purified on silicic acid thin-layer (16, 17). The resulting mixture had a specific activity of 8.6 • 106 dpm/l~mol. It should be noted that for both lipids the labeled species was fully saturated and the unlabeled diluent (97.5 for diester-PC, 99.7% for dialkyl-PC) was the monoenoic analog. The unsaturation of the carriers results in liquid-crystalline bilayers and ready formation of monoshell liposomes. CH3(CH2) I2CH2
HzC--O---P--O--C H 2---CHz--N(CH3)3
P O - 'H20 Dialkyl-PC
Preparation ofLiposomes. A mixture of 80 i~mol diester-PC and 80 Ixmol dialkyl-PC was freed of solvent and taken up (with help of vortex mixing) in 2 ml of 0.9% sterile aqueous Natl. The emulsion was then sonicated for 4 hr, under argon at 4~ with a Branson Sonifier Cell Disruptor W140D with microtip, at 30 W. Centrifugation for 1 hr at 100,000 g removed any remaining multilamellar liposomes (19). The clear supernatant contained approx. 1 ~mol [14CJPC, 0.45 • 10 6 dpm, and I ~mol [3H]dialkyl-PC, 8.6 x 106 dpm, per 25 t*1. Incorporation and Analysis of Lipids. The liposome solution, 25 t*1, was injected intracerebrally into 17- to 18-day-old rats (COBS outbred, strain CRI CD, Charles River Breeding Lab, Wilmington, Massachusetts) with a 30-gauge needle penetrating 2.5 ram. The animals survived the injection to the end of the experiment without ill effects. They were killed by decapitation in groups of four after 1-63 days and individually analyzed. The blood was not collected. Skin was discarded. Brain was homogenized in 0.32 M sucrose, and myelin prepared according to Norton and Poduslo (20); aliquots were counted by liquid scintillation counting. Figures 1-5 are plotted from the means of data of each group of animals. Lipids were extracted with hexane-isopropanol and washed according to Hara and Radin (21). Liver and carcass were rinsed of blood and individually homogenized in water. Aliquots were taken for counting, and the lipids were extracted from the homogenates (pooled for each date) with hexane-isopropanol. Lipid classes were separated on silicic acid columns with the solvents chloroform (nonpolar lipids), acetone (galactolipids), and methanol (phospholipids) (22). Phnspholipids were further fractionated by thin-layer chromatography (16, 17) and scanned (Berthold TLC-Scanner LB-2760). Spots were collected, eluted, and scintillation counted.
Uptake and Disappearance of Labels. Figures 1-5 show the following results. First, more label appeared in brain, carcass, and body as a whole after two days than had been found after one day. This is most likely the
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100t a ~ ~
Days after im|ectlon
FIG. 1. Retention of labels in whole body (without blood and skin). A - - A : 3H, >95% associated with dialkylphosphatidyl choline; O---Q, ~4C; ~ 4C]phosphatidylcholine; all as percent retained of injected label. Values calculated by adding mean values, for each day, of brain, liver, and carcass.
consequence of our having discarded most of the blood of the animals; we know that monoshell liposomes, especially those of dialkyl-PC, can remain in circulation for several days (23). Secondly, only a few percent of the lipid stayed in the brain (Figure 2), most of it appeared in liver (Figure 4) and carcass (Figure 5) (which included the spleen, probable 2.S.
Days after isjectios
FIG. 2. Retention of labels in brain. A - - A : 3H, >90% associated with dialkylphosphatidylcholine; 0-----0: ]4C; II---B: [J4C]phosphatidylcholine; all as percent of injected label.
MEMBRANE INCORPORATION OF INERT LIPID 0.4-
~0~ o . 1
Days after injecties
FIG. 3. Retention of labels in myelin, A--,A: 3H, >90% associated with dialkylphosphatidylcholine; 0 - - - 0 : ~4C total; II---m: [J4C]phosphatidylcholine.
site of rapid capture of liposomes). This result is a manifestation of rapid drainage of the liposome preparation from the site of injection. Third, the rates of uptake and disappearance of 3H and '4C were very similar; this is true for brain (Figure 2) as well as carcass (Figure 5), and in myelin especially the rates were virtually identical for a month (Figure 3). This unexpected result is clearly seen in Figure 6, which gives the ratio of 3H/ 14C labels in the tissues and shows that only after 2 months there began
14r I |
Days after i~jection
FIG. 4. Retention of labels in ]~ver. A - - A : 3H, >95% associated with d~alkylphosphatidylcholine; 0 - - - 0 : ~4C;D - - ~ , [~'Clphosphatidylcholine; all as percent retained of injected label.
BROCKERHOFF ET AL.
Days a l t a r injection
FIG. 5. Retention of labels in carcass (body without brain, liver, skin, blood). A - - A : 3H, >95% associated with dialkylphosphatidylcholine; ~ - - Q : ~4C; gl---I: [~4C]phosphatidylcholine; all values as percent of injected label.
o .0 ac
Days a f t e r injactioa
FIG. 6. Ratio 3H/~4C in myelin, A - - A ; liver, 0 - 0 ; brain follows myelin ratio closely.
Nonpolar lipid Galactolipid PS + PI + PA SM AIk2-PC PC PE
Day after injection
TABLE 1 DISTRIBUTION OF 3H AND ~4C OVER LIPID FRACTIONS FROM MYELIN, IN PERCENT"
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a preferential retention of 3H (i.e., dialkyl-PC) in the myelin, and of 14C in the carcass; the liver, however, cleared 14C much faster than 3H (Figure 6). The half-time of disappearance of label was around five days for the whole animal (Figure 1). In the myelin, the label (especially 3H) persisted much longer, with a half-life time of 1-2 months (Figure 3). Such slow rates are typical for the lipids of myelin (24-29). Metabolism of Lipids. Although the disappearance rates of ~4C and 3H in the whole animals were very similar (Figure 1), the metabolic turnover rates of the lipids were by no means identical. After one day, in all tissues, only half or less of the ~4C activity was found in diester-PC (Figures 2-5), while 90% or more of the 3H was still associated with the dialkyl-PC after 16 days. This is shown in Table I, which gives the results of lipid analyses of myelin from 1 to 16 days. In carcass and liver, there was even less conversion of dialkyl-PC label into other lipids (data not shown here). Figures 2-5 and Table I show that the concentrations of remaining [~4C]diester-PC had already reached the level of dynamic equilibrium after 1 day; this means, first, that the half-time of turnover cannot have been larger than several hours; second, that the "remaining" diester-PC is not, in fact, the originally injected lipid but also a product of lipid turnover, i.e., reutilization of the fatty acid.
Incorporation of a Modifier Lipid into Membranes. The lipid chosen, dialkyl-PC, had been synthesized to resemble natural palmitoyl-oleoyl (diester) phosphatidylcholine as closely as possible, except for the absence of the carboxyl ester groups (18). The effective hydrophobic chain length (30, 31) is the same, 28, for both lipids, and dialkyl-PC forms monolayers and multishell liposomes with properties close to those of PC (18, 30). Sonication yields monoshell liposomes for both lipids. For injection we prepared mixed-lipid monoshell liposomes. This allows us to decide if the modifier lipid has actually been incorporated into the tissue membranes or if it perhaps remains in the form of liposome, trapped outside of cells or inside (after endocytosis). The results show that 50% of the lipid of the liposomes is completely metabolized within a few hours; this is possible only if the liposomes have fused to the endoreticular membrane system or if the diester-PC has been completely degraded to a lyso compound, which itself is fusogenic and destabilizes the liposome (32, 33). Much of the ~4C label of the injected diester-PC is certainly, after a while, no longer associated with the original stearic acid but with products
MEMBRANE INCORPORATION OF INERT LIPID
such as the vinyl ether chain of plasmalogens and the very long-chain fatty acids of the cerebrosides (34, 35). (We have followed the fate of this label no further). It is the more surprising that the rate of disappearance of both 3H and ~4C should be so similar in brain and carcass (Figure 6), since the [3H]dialkyl-PC is clearly, for the largest part, completely oxidized in one sweep rather than converted to other lipids. The similarity of these rates might be accidental or might be explained by assuming that, in the growing animals, none of the lipids, neither the resistant [3H]dialkylPC nor any of the different carriers of the ~4C label, is oxidized in the course of individual turnover, but that all disappear together by elimination of whole chunks of the membranes in which they are contained, probably through degradation by the lysosomal apparatus. The constant ratios of the retained labels alone (in brain and carcass) make it seem that the 3H in dialkyl-PC survives no longer than the 14C of diester-PC, but the lipid analyses reveal a fundamental difference: the 14C label has been assimilated by incorporation into natural membranespecific lipids, but the [3H]dialkyl-PC remains what it was when injected. The level of this membrane modifier attained under our experimental conditions is small: about 2 txg (i.e., 0.2% of 1 ~xmol injected, Figure 3) in about 20 mg of myelin (at 20 days) containing about 8% PC, i.e., about 0.1% of the total phosphatidylcholine was the dialkyl analog (assuming that saturated labeled and monounsaturated carrier dialkyl-PC turnover at similar rates). However, this level is maintained for many weeks, and if the modifier were the carrier of pharmacological potential, say, an anticonvulsant, a physiological effect might already be noticeable. Obviously, though, ways must be found to increase the level of incorporation in the brain. Possibly, positively or negatively charged liposomes would be more efficiently retained, or the opening of the blood-brain barrier, e.g., by hypertonic injection, might be beneficial. Retention of up to 50% of a labeled lipid in brain has already been achieved by others (5, 36, 37), although in those cases the lipids were injected in micellar rather than liposomal state, in much smaller doses, and were metabolized by the tissue rather than inserted as such into the membranes. Judging from our results, myelin is the most promising candidate for membrane engineering. The modifier disappeared much faster from the other tissues, although the results for the liver (Figure 6) are interesting insofar as they show that the artificial phospholipid can indeed survive much longer than the natural membrane components. Much longer survival times might be achieved with modifiers even more resistant to degradation. If the breakdown of a modifier phospholipid should start with co-oxidation of the alkyl chains, ~o-substitution, e.g., with halogen, or o~branching, might lengthen survival; initial breakdown at a phosphate ester
BROCKERHOFF ET AL.
linkage might be stopped by employing phosphonium analogs. Such modifier lipids, if resistant to breakdown by lysosomes, might, after digestion even of their whole membrane, reappear in another membrane of the same kind. For all the difficulties lying ahead, the goals of membrane engineering might be easier to reach, and faster to bring therapeutic advances with them, than the goals of genetic engineering. For example, a condition such as epilepsy, which is known to respond to drugs that act on the lipid bilayer, might become better manageable if a drug could be semipermanently anchored in the bilayer of the neuronal membranes.
ACKNOWLEDGMENTS This study was supported by research grants GM 21875 and NS 14480 from the National Institutes of Health, U.S. Public Health Service.
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32. LucY, J. A. 1974. Lipids and membranes. FEBS Lett. 40:S105-Sl11. 33. VAN DEN BESSELAAR, A. M. H. P., VAN DEN BOSCH, H., and VAN DEENEN, L. L. M. 1977. Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes. Biochim. Biophys. Acta 465:454-465. 34. GOZLAN-DEVILLIERRE, N., BAUMANN, N., and BOURRE, J. M. 1978. Distribution of radioactivity in myelin lipids following subcutaneous injection of [t4C]stearate. Biochim. Biophys. Acta 528:490-496. 35. COHEN, S. R., and BERNSOHN, J. 1978. The in vivo incorporation of linolenic acid into neuronal and glial cells and myelin. J. Neurochem. 30:661-669. 36. TJIONG, H. B.~ GUNAWAN, J., and DEBUCH, H. t976. On the biosynthesis of plasmologens during myelination in the rat. VIII  Incorporation of 1-[1-~4C]alkyl-2-acyl3-sn-glycerophosphoethanolamine with different fatty acids. Hoppe-Seyler's Z. Physiol. Chem. 357:707-712. 37. PALTAUF, F. 1971. Biosynthesis of plasmalogens from alkyl- and alkyl-acyl-glycerophosphoryl ethanolamine in the rat brain. FEBS Lett. 17:118-120.