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PubMed1984

Effects of carbohydrates on membrane stability at low water activities.

The relative effectiveness of a variety of carbohydrates in preserving the structural and functional integrity of membranes at low water activities was studied, using Ca-transporting microsomes from muscle as a model membrane. The order of effectiveness (greatest to lowest) was: trehalose, lactose, maltose, cellobiose, sucrose, glucose, fructose, sorbitol, raffinose, myo-inositol, glycerol. At the highest concentrations of the most effective sugars tested, microsomes were obtained upon rehydration that were similar structurally and functionally to fresh membranes. The least effective carbohydrates, alcohol sugars, all appear to be fusogenic. A structural explanation for relative effectiveness of the sugars was sought, but no clear relationship was found, except that effectiveness does not appear to be related to the number of position of hydroxyl groups available for hydrogen bonding.

Animals↗
PubMed1984

Interactions of phospholipid monolayers with carbohydrates.

Surface pressure studies of phospholipid monomolecular films of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) formed at an air/water interface have been made and the effects on the films studied when various carbohydrates are present in the subphase. The results obtained show that at a given temperature, the area per molecule of DPPC increases with increasing concentration of the carbohydrate in the subphase. The carbohydrate which has the greatest expanding effect on the phospholipid monolayer is glycerol, followed in turn by trehalose, sucrose, glucose, raffinose, and inositol. The mechanism of monolayer expansion by glycerol is different from that observed in other carbohydrates, as the following experiments demonstrate. Below the phase transition temperature of DPPC, the area per molecule of DPPC at a pressure of 12.5 dyn/cm is the same with and without glycerol in the subphase. However, when the monolayer is heated to a temperature above the phase transition temperature for DPPC, the area/molecule on glycerol is considerably greater than the area/molecule on water at the same surface pressure. Cooling the monolayer back to the lower temperature produces an area/molecule of DPPC which is identical on both water and glycerol subphases. Glycerol therefore has no effect on the low-temperature (condensed) monolayers but causes expansion of the high-temperature (expanded) monolayers. By contrast with glycerol, both trehalose and sucrose interact with the DPPC monolayer producing an increased area/molecule over that observed on water, both with low-temperature (condensed) monolayers and with the high-temperature (expanded) monolayers. The efficiency of these carbohydrates at expanding the monolayer films (with the exception of glycerol) shows a strong correlation with their ability to stabilize membrane structure and function at low water contents.

Carbohydrates↗
PubMed1984

Preservation of functional integrity during long term storage of a biological membrane.

Sarcoplasmic reticulum vesicles freeze-dried in the presence of trehalose retain most of their original biological activity for short periods. When the dry vesicles are stored for longer periods in air, Ca2+-transport becomes uncoupled from ATPase activity within a few days. However, when they are stored under vacuum, ATPase activity, Ca2+ transport, and coupling between Ca2+ transport and ATP utilization are maintained essentially intact for at least 110 days.

Adenosine Triphosphatases↗
PubMed1988

Factors affecting the stability of dry liposomes.

Previous studies have shown that liposomes can be preserved in the dry state in the presence of certain sugars, of which trehalose is particularly effective. There have been some discrepancies in results obtained by the various laboratories in which this phenomenon has been studied, both with respect to the efficacy of the sugars tested and the degree to which the dry vesicles can be stabilized. We show here that several factors that affect the stability of the dry liposomes may be responsible for the discrepancies between measurements by different laboratories. These factors include: (1) Size: small, sonicated vesicles are comparatively very unstable, and retain no more than 70% of trapped solute after drying, even in extremely high concentrations of sugars. Very large vesicles are similarly unstable. (2) Charge: a small amount of negatively charged lipid in the bilayer significantly increases stability. (3) Stabilizing sugar: the comparative efficacy of the sugar used varies with the size of the vesicles. (4) Dry-mass ratio. It is the dry-mass ratio between the stabilizing sugar and lipid that is important in the preservation during freeze-drying, not the concentration of either lipid or sugar in bulk solution.

Drug Stability↗
PubMed1988

Trehalose and dry dipalmitoylphosphatidylcholine revisited.

Dry mixtures of sonicated vesicles of DPPC and trehalose which contained a maximum of 0.2 mol water/mol lipid were examined by differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy. Samples of dry DPPC and trehalose prepared from aqueous solution had a minimum Tm of 24 degrees C for the gel to liquid-crystalline transition provided that the vesicles were dried with trehalose while the lipid was in liquid-crystalline phase. This low transition is compared to a transition of 105-112 degrees C for dry pure DPPC and of 42 degrees C for hydrated pure DPPC. The present work is an extension of earlier work from this laboratory using both other lipids and other methods of preparation.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

Effects of free fatty acids and transition temperature on the stability of dry liposomes.

Previous studies have shown that liposomes composed of phospholipids with low phase-transition temperatures can be stabilized in the absence of water, provided that fusion is inhibited between the vesicles during drying, and that during rehydration the phospholipids do not pass through the gel to liquid crystalline phase transition. These conditions are met by adding certain disaccharides to the vesicles before drying, which inhibit fusion and depress the transition temperature in the dry lipids. The present study shows that preservation can also be achieved with vesicles made from dipalmitoylphosphatidylcholine (DPPC), but that the retention of trapped solute by such vesicles is much less than in vesicles composed of more fluid phospholipids. Addition of free fatty acids to the vesicles before drying destabilizes them; DPPC vesicles containing 15 mol% or more of palmitic acid leaked all their contents during drying, regardless of how much of the stabilizing sugar was added. Unlike the case for more liquid phospholipids, the leakage in DPPC vesicles is due solely to fusion and not to hydration-dependent phase transitions. Addition of free fatty acids results in increased fusion, leading to leakage.

Desiccation↗
PubMed1991

Solution effects on the thermotropic phase transition of unilamellar liposomes.

We have investigated the effect of two monosaccharides, glucose and fructose, and two disaccharides, sucrose and trehalose, on the thermotropic phase transition of unilamellar extruded vesicles of DPPC. All the sugars investigated raise the main transition temperature (Tm) of some fraction of the lipid, but there are differences between the effect of glucose and the other three sugars. At low concentrations of glucose, Tm is lowered. At high concentrations of glucose there are two transitions, one with a low Tm and one with a high Tm. The data suggest that at low concentrations, all of the glucose present may bind to the bilayer and increase headgroup spacing by physical intercalation or increased hydration. The appearance of a Tm above that of pure hydrated DPPC suggests the possibility of the dehydration of some other population of phospholipid molecules. The other three sugars increase Tm, but at high concentrations of trehalose, sucrose, and fructose a second peak occurs at a low Tm. The other sugars appear to dehydrate the bilayer at low concentrations, but may show some binding or increased hydration of some portion of the lipid at very high concentrations. The sugar effects on unilamellar vesicles are strikingly different from the effects of these sugars on multilamellar vesicles.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide.

Dimethylsulfoxide (DMSO) is known to protect isolated enzymes during freezing while destabilizing proteins at high temperatures. This apparent paradox is the subject of a review by Arakawa et al. ((1990) Cryobiology 27, 401-415), who present evidence for a temperature-dependent, hydrophobic interaction between DMSO and non-polar moieties of proteins. The present study investigates the interaction of DMSO with phospholipid bilayers. Phospholipid vesicles containing carboxyfluorescein were exposed to several concentrations of DMSO at various temperatures. Leakage rates increased with DMSO concentration and temperature. This effect was not reduced in the presence of solutes that have been shown to neutralize DMSO toxicity in tissues. The increased leakage rates correlate well with the increased partitioning of DMSO from water to octanol at higher temperatures. Additionally, reductions in the CH2 vibrations of the bilayer are also shown to depend on DMSO concentration and temperature. A similar reduction in CH2 vibrations was observed in solutions of octanol and DMSO, suggesting that this effect is not mediated through an interaction with water. Furthermore, investigation of sulfoxide vibrations indicate that DMSO is not hydrogen bonded to the alcohol moiety of octanol, and therefore the interaction between DMSO and octanol is most likely due to a hydrophobic association. These results are consistent with a destabilization of phospholipid membranes at higher temperatures due to a hydrophobic association between DMSO and the bilayer.

Dimethyl Sulfoxide↗
PubMed1994

Trehalose lowers membrane phase transitions in dry yeast cells.

Recent work has clearly demonstrated a direct correlation between the amount of trehalose present in the yeast Saccharomyces cerevisiae and its ability to tolerate dehydration, but has failed to elucidate the specific role played by trehalose. By using Fourier transform infrared spectroscopy we measured the transition temperature of phospholipids in both intact S. cerevisiae and isolated plasma membranes dried in the presence and absence of trehalose. Our results show that trehalose lowers the temperature of the dry gel to liquid crystal phase transition in yeast from around 60 degrees C to about 40 degrees C, thus allowing yeast rehydrated above 40 degrees C to avoid the damaging effects of passing through a phase transition. These results explain both the need for trehalose and the observation that yeast must be rehydrated with warm water if they are to remain viable. Only when trehalose is present is the dry transition within a physiologically tolerable range and only when the cells are rehydrated above 40 degrees C will they avoid passing through a phase transition.

Cell Membrane↗
PubMed1994

Trehalose inhibits ethanol effects on intact yeast cells and liposomes.

The effect of ethanol on stability of intact yeast cells has been investigated. Several strains with differences in trehalose metabolism were examined for their ability to survive in the presence of 10% (v/v) ethanol. A positive correlation was observed between cell viability and trehalose concentration. When leakage of electrolytes from the cells was recorded by observing changes in conductivity of the medium, we found that ethanol increases leakage, but the presence of trehalose reverses that effect. Similar studies were done with liposomes of similar composition to those seen in intact cells in log and stationary phases. In the presence of ethanol, carboxyfluorescein trapped in the liposomes leaked to the medium. When trehalose was added inside, outside or on both sides of the membrane, the ethanol-induced leakage was strongly inhibited. More leakage was observed in liposomes in gel phase state than in liquid-crystalline phase, suggesting that the thermotropic behavior of the lipids in the plasma membrane, together with trehalose, plays a role in enhancing ethanol tolerance.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

Interaction of cord factor (alpha, alpha'-trehalose-6,6'-dimycolate) with phospholipids.

We previously reported that cord factor (alpha,alpha'-trehalose-6,6'-dimycolate) isolated from Nocardia asteroides strain GUH-2 strongly inhibits fusion between unilamellar vesicles containing acidic phospholipid. We chose to study the effects of this molecule on liposome fusion since the presence of N. asteroides GUH-2 in the phagosomes of mouse macrophages had been shown to prevent phagosomal acidification and inhibit phagosome-lysosome fusion. A virtually non-virulent strain, N. asteroides 10905, does not prevent acidification or phagosome-lysosome fusion and, further, contains only trace amounts of cord factor. In the present paper, we have investigated the effects of cord factor on phospholipid bilayers that could be responsible for the inhibition of fusion. We show that cord factor increases molecular area, measured by isothermal compression of a monolayer film, in a mixed monolayer more than would be expected based in its individual contribution to molecular area. Cord factor, as well as other glycolipids investigated, increased the overall hydration of bilayers of dipalmitoylphosphatidylcholine by 50%, as estimated from the unfrozen water fraction measured by differential scanning calorimetry. The effect of calcium on this increased molecular area and headgroup hydration was measured by fluorescence anisotropy and FTIR spectroscopy of phosphatidylserine liposomes. Both techniques showed that cord factor, incorporated at 10 mol%, increased acyl chain disorder over controls in the presence of Ca2+. However, FTIR showed that cord factor did not prevent headgroup dehydration by the Ca2+. The other glycolipids tested did not prevent either the Ca(2+)-induced chain crystallization or headgroup dehydration of phosphatidylserine bilayers. These data point to a possible role of the bulky mycolic acids of cord factor in preventing Ca(2+)-induced fusion of liposomes containing acidic phospholipids.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Is vitrification involved in depression of the phase transition temperature in dry phospholipids?

Recent literature has suggested that the depression of the phase transition temperature (Tm) in dry phospholipids by sugars may be ascribed to vitrification of the stabilizing solute, rather than by the direct interaction between sugar and phospholipid we have proposed. Koster et al. ((1994) Biochim. Biophys. Acta 1193, 143-150) claim that the only necessity is that the glass transition (Tg) for the sugar exceed Tm for the lipid. Evidence is presented in the present paper that this is not sufficient. Based on the vitrification hypothesis of Koster et al., the predicted order of effectiveness in depressing Tm in dry dipalmitoylphosphatidylcholine (DPPC) is dextran > or = hydroxyethyl starch > stachyose > raffinose > trehalose > sucrose > glucose. In fact, the opposite order was seen. The effect of raffinose, sucrose, or trehalose on Tm in dry DPPC depends on the thermal history of the sample, as we have reported previously. When DPPC dried with trehalose is heated for the first time, Tm is about 55 degrees C, but on the second and subsequent heating scans Tm falls to about 25 degrees C. Koster et al. suggest that this effect is due to heating the sample above Tg rather than to melting the hydrocarbon chains. We present evidence here that all that is required is for the chains to be melted. Further, we show that retention of residual water by DPPC dried with trehalose depends on the drying temperature, but is independent of drying temperature with glucose, a finding that is consistent with direct interaction. We conclude that vitrification is not in itself sufficient to depress Tm in dry phospholipids.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Arbutin inhibits PLA2 in partially hydrated model systems.

Arbutin is a glycosylated hydroquinone found at high concentrations in certain plants capable of surviving extreme and sustained dehydration. In this paper, we examine a potential role of this molecule in anhydrobiosis. We have studied its effects on the physical properties of phospholipids and on preservation of liposomes during drying. Arbutin depresses the gel to liquid crystalline phase transition temperature of dry phospholipids, as measured by differential scanning calorimetry, with a pattern similar to that seen in phospholipids dried with the disaccharide trehalose. Unlike trehalose, however, arbutin does not protect dry liposomes from leaking their contents. Also, using Fourier transform infrared spectroscopy, we found an increase in the vibrational frequency of the phosphate asymmetric stretch in partially hydrated phospholipids in the presence of arbutin. Trehalose, by contrast, depresses the frequency of the phosphate in dry phospholipids, indicating that the modes of interaction of trehalose and arbutin with the bilayer are different. Previously, we have shown that phospholipases can be active in liposomes with surprisingly low water contents. Based on the structural similarity of arbutin to a known inhibitor of phospholipase A2 (PLA2), it appeared possible that arbutin might serve as an inhibitor of phospholipases. Liposomes of varying composition were lyophilized in the presence and absence of phospholipases. When the liposomes were partially rehydrated at 76% relative humidity, arbutin inhibited PLA2, but did not inhibit phospholipases B or C. Accumulation of enzyme product in the liposome membranes was measured by analytical thin layer chromatography, and was taken as a measure of enzyme activity. Arbutin did not inhibit any of the enzymes in the presence of excess water. Based on these data, hypotheses are presented concerning the mechanism of PLA2 inhibition by arbutin in the mostly dehydrated state.

Arbutin↗
PubMed1985

Degradation of functional integrity during long-term storage of a freeze-dried biological membrane.

Trehalose, and to some extent a few other carbohydrates, is capable of stabilizing the structure and function of isolated biological membranes during lyophilization. In this paper the results of investigations into the long-term stability of the lyophilized membrane-carbohydrate mixtures were reported. The effects of varying water content, oxygen level, and light on the rates of oxidation, browning, and degradation of biological activity were reported. The efficiency with which three carbohydrates stabilized membrane structure was also reported, with glucose shown to be less efficient than maltose or trehalose. Increased water content accelerated loss of biological activity, possibly because, under the same conditions, nonenzymatic browning and photooxidation were accelerated also. Glucose-containing samples were especially unstable at elevated humidities. Efficiency of preservation could be maximized by storage under conditions of low oxygen, low humidity, and dark, and by the inclusion of high levels of trehalose.

Animals↗
PubMed1987

Stabilization of phosphofructokinase during air-drying with sugars and sugar/transition metal mixtures.

Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after air-drying and rehydration. The addition of trehalose, maltose, or sucrose to the enzyme solution prior to rapid drying results in a recovery of almost 70% of the original activity, whereas about 30% is recovered during slow drying. Similar stabilization is seen with up to 200 mM lactose, but at higher concentrations the sugar comes out of solution during drying, and there is a dramatic drop in the activity recovered. Glucose at concentrations up to 500 mM is ineffective at protecting air-dried PFK. Addition of ionic zinc to enzyme-sugar mixtures prior to drying greatly enhances the stabilization imparted by the above sugars, but zinc alone affords no protection. Several other organic solutes (proline, glycine, trimethylamine N-oxide, glycerol, and myo-inositol) that afford cryoprotection to PFK, an effect enhanced by the addition of zinc, do not stabilize the enzyme during air-drying, even if zinc is present. The addition of ionic copper, cobalt, or nickel to trehalose-PFK solution prior to rapid drying results in a large increase in the activity recovered, and the presence of cadmium or manganese leads to a minor increase. Magnesium and calcium are ineffective in this respect. During slow drying, the presence of cadmium or calcium leads to increased preservation, magnesium and manganese have no influence on stabilization, and copper and nickel inactive the enzyme.

Air↗
PubMed1989

Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy.

Lipid phase transitions in membranes are thought to be a major damaging event during cooling of cells prior to cryopreservation or during warming after freeze-thaw has been completed. Although there is abundant evidence that such transitions occur in isolated phospholipids, the evidence that they are found in membranes in intact cells is less clear, due largely to technical difficulties in detecting such transitions in the complex mixtures of lipids and proteins found in natural membranes. We show here that Fourier transform infrared spectroscopy provides a rapid, convenient method for detecting these transitions in intact cells. We have used intact pollen grains of cattail (Typha latifolia) as a primary experimental subject. Spectra taken of the intact pollen grains show most of the features commonly seen in natural membrane vesicles or pure phospholipids. Shifts in the vibrational frequency and width of the CH2 bands with temperature can be used to detect lipid phase transitions. Biochemical analysis, coupled with the spectroscopy, was used to assign transitions to nonpolar and polar lipids. Finally, although assignment of the melting lipid unambiguously in other cells has not yet been made, we show that the transitions can nevertheless be detected in other intact cells, including those of four plant species and sperm of three animals.

Freezing↗