Effect of n-alkanes on membranes in lipid-water systems

The changes in leaflet thickness and surface area per lipid molecule as a function of added amount of $\text{n-}\text{alkane}$ solvents have been studied in a number of lipid water systems by low angle X-ray diffraction techniques. The most probable site of accumulation of the $\text{n-}\text{alkane}$ is identified as the middle of the leaflet as opposed to intercalation with lipid hydrocarbon chains. Adsorption of $\text{n-}\text{alkane}$ depends on alkyl chain length and the organisation of the lipid hydrocarbon chains in the lipid water phases. Attention is drawn to the possible relationship between these results, the effect of $\text{n-}\text{alkanes}$ on isolated lipid bilayers and their effects as anaesthetics.     

Transepidermal water loss: the signal for recovery of barrier structure and function

Previous studies have demonstrated that perturbations in barrier function stimulate epidermal lipid synthesis and that this increase can be prevented by occlusive membranes. These observations suggest that epidermal lipid synthesis might be related to barrier function and raised the question whether transcutaneous water flux might regulate epidermal lipogenesis. In the present study we first abrogated the barrier with acetone, and then compared the rate of repletion of stainable lipids, barrier recovery, and epidermal lipogenesis in animals covered with occlusive membranes or vapor-permeable membranes versus uncovered animals. Acetone treatment of hairless mice removed stainable neutral lipids from the stratum corneum, with repletion evident both biochemically and histochemically within 48 hr in uncovered animals. In contrast, when the animals were covered with an occlusive membrane, the usual return of stratum corneum lipids was aborted. Since application of vapor-permeable membranes allowed normal lipid repletion, occlusion alone is not responsible for the inhibition of lipid repletion. Acetone treatment also perturbed epidermal barrier function, which returned to normal in uncovered animals in parallel with the reappearance of stratum corneum lipid. However, when animals were covered with an occlusive membrane, barrier function did not recover normally. In contrast, occlusion with vapor-permeable membranes allowed barrier function to recover normally. Finally, whereas occlusive membranes prevented the characteristic increase in epidermal lipid synthesis that follows barrier perturbation, epidermal lipid synthesis was increased in animals covered with a vapor-permeable membrane. These results point to transepidermal water flux itself as the signal that regulates epidermal lipid synthesis, which is associated first with the redeposition of stratum corneum lipids and then the normalization of stratum corneum barrier function.     

Lipokeratinocytes of the epidermis of a cetacean (Phocena phocena)

Summary Biochemical and ultrastructural analysis of epidermis from the porpoise, Phocena phocena, revealed certain similarities and differences between cetaceans and terrestrial mammals. The predominant cell of cetacean epidermis, not found in normal terrestrial mammals, is a lipoker-atinocyte, which elaborates not only keratin filaments, but also two types of lipid organelles: first, lamellar bodies, morphologically identical to those of terrestrial mammals, are elaborated in great abundance in all suprabasal epidermal layers, forming intercellular lipid bilayers in the stratum corneum interstices: and second, non-membrane-bounded droplets appear and persist in all epidermal layers. Although the porpoise lipokeratinocyte morpologically resembles the sebokeratocyte of avians in certain respects, nonmembrane-bounded lipid droplets are not released into the intercorneocyte space as they are in avian stratum corneum. Whereas phospholipid/neutral lipid gradients are similar in porpoise and terrestrial mammals, PAS-positive glycoconjugates, specifically glycosphingolipids, are retained in porpoise stratum corneum, but lost from these layers in terrestrials. The novel, non-polar acylglucosyl-ceramides, which also are lost during cornification in terrestrial mammals, are retained in porpoise stratum corneum. The lipid components of porpoise lipokeratinocytes appear to subserve not only barrier function in a hypertonic milieu, but also underlie the unique buoyancy, streamlining, insulatory, and caloric properties exhibited as adaptations to the cetacean habitat.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Human epidermal lipids: characterization and modulations during differentiation

Using thin-layer chromatography and glass capillary gas-liquid chromatography, we have quantitated the lipids in the germinative, differentiating, and fully cornified layers in human epidermis. As previously noted in nonhuman species, we found progressive depletion of phospholipids coupled with repletion of sterols and sphingolipids during differentiation. The sphingolipids, present only in small quantities in the lower epidermis, accounted for about 20% of the lipid in the stratum corneum, and were the major repository for the long-chain fatty acids that predominate in the outer epidermis. Although the absolute quantities of sphingolipids increased in the outer epidermis, the glycolipid:ceramide ratio diminished in the stratum corneum, and glycolipids virtually disappeared in the outer stratum corneum. Squalene and n-alkanes were distributed evenly in all epidermal layers, suggesting that these hydrocarbons are not simply of environmental or pilosebaceous origin. Cholesterol sulfate, previously considered only a trace metabolite in epidermis, was found in significant quantities, with peak levels immediately beneath the stratum corneum in the stratum granulosum. These studies: 1) provide new quantitative data about human epidermal lipids; 2) implicate certain classes of lipids for specific functions of the stratum corneum; and, 3) shed light on possible product-precursor relationships of these lipids.     

Epidermal differentiation: the role of proteases and their inhibitors

Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency infurless mice, targeted ablation of the serine protease Matriptase/MTSP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent findings on the role of cystatin M/E and legumain as a functional dyad in skin and hair follicle cornification, a paradigm example of the regulatory functions exerted by epidermal proteases, will be discussed.     

A dynamic x-ray diffraction study of anaesthesia action. Changes in myelin structure and electrical activity recorded simultaneously from frog sciatic nerves treated with n-alkanes

Changes induced in the structure and electrical activity of myelin were recorded simultaneously from frog sciatic nerves treated with $\text{n-}\text{alkanes}$. The results suggest that the effect of $\text{n-}\text{alkanes}$ seems to be two-fold: (a) there is an initial reversible phase, in which a significant modification of the X-ray diffraction patterns, concomitant with the continuous fall of the action potential, is observed; (b) there is a final phase which is irreversible. This occurs some time after the complete abolition of the electrical activity. At this stage, further changes of the X-ray diffraction patterns are detected, the most significant of them being in the $\text{n-}\text{pentane-treated}$ myelin, and consist of an increase in the membrane bilayer thickness.     

Sphingolipid activator proteins are required for epidermal permeability barrier formation

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton $\text{T}{}_1$ NMR and obtained a value of 4.0 · 10^?3 cm · s^?1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.     

Comparison of the promoting activity of pristane and n-alkanes in skin carcinogenesis with their physical effects on micellar models of biological membranes

In 1976 (Horton, A.W., Butts, C.K. and Schuff, A.R. (1976) Colloid Interface Sci. 5, 159–168) we assayed pristance (2,6,10,14-tetramethylpentadecane) in a model interfacial system that has given excellent correlation with cocarcinogenic activity among $\text{n-}\text{alkanes}$, as tested in cycloalkane diluents. It was predicted that this branched-chain derivative of the diterpenes would have higher activity than $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$, one of the most cocarcinogenic of the $\text{n-}\text{alkanes}$ in such diluents. Pristane was compared with $\text{n-}\text{C}{}_{18}\text{H}{}_{38}$ and two other $\text{n-}\text{alkanes}$ for their promoting activities in cyclohexane for C3H male mice after a single application of 7,12-dimethylbenz$\text{[a]}\text{anthracene}$. The branched-chain alkane proved to be more active. 20% $\text{n-}\text{tetracosane}$ in cyclohexane was inactive, which correlated with its effects in this diluent in the physical assay system. The promoting activity of 75% $\text{n-}\text{octane}$ in cyclohexane, predicted by the physical assay, was confirmed by tests on mice. The combined by-products of the synthesis of tetracosane, including C₁₂ alkanes and alkenes, C₁₉ and C₂₀ alkylbenzenes, and C₂₄ alkenes, proved to be a very active promoter. However, a mixture in cyclohexane of purified tetracosane with this composite of potential impurities was inactive. From the alkanes behavior in physical systems involving vatious membrane phospholipids and steroids, it is hypothesized that the primary step in their biological activity is a chain-chain interaction with membrane lipids that alters the properties of liquid-crystalline phases at aqueous interfaces. Resulting changes in the microfluidity of the lipid phase and the lateral mobility of critical hormone receptors and enzyme systems, such as the nucleotidyl cyclases, would perturb control systems that maintain the normal behavior of the initiated cell. Thus, its progression to a proliferating neoplasm may be favored.     

The interaction of spectrin · actin and synthetic phospholipids

Using differential scanning calorimetry and freeze fracture electron microscopy interactions were studied between lipids and a spectrin · action complex isolated from human erythrocyte membranes. With dispersions of $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$, $\text{1,2-}\text{dimyristoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphoglycerol}$ and mixtures of these two compounds, which for experimental reasons were chosen as the lipid counterpart, such an interaction could clearly be deduced from changes in the temperature and the enthalpy of the phase transition. Furthermore it was demonstrated that the interaction with this membrane protein protects the bilayer against the action of Ca²⁺ and Mg²⁺ and prevents fusion of lipid vesicles which easily occurs in some of the systems when divalent ions were added to the pure lipid vesicles.     

Human stratum corneum lipid organization as observed by atomic force microscopy on Langmuir-Blodgett films

The barrier function of skin ultimately depends on the physical state and structural organisation of the stratum corneum extracellular lipid matrix. Ceramides, cholesterol and a broad distribution of saturated long-chain free fatty acids dominate the stratum corneum lipid composition. Additionally, smaller amounts of cholesterol sulfate and cholesteryl oleate may be present. A key feature determining skin barrier capacity is thought to be whether or not different lipid domains coexist laterally in the stratum corneum extracellular lipid matrix. In this study, the overall tendency for lipid domain formation in different mixtures of extracted human stratum corneum ceramides, cholesterol, free fatty acids, cholesterol sulfate and cholesteryl oleate were studied using atomic force microscopy (AFM) on Langmuir-Blodgett (LB) films on mica. It is shown that the saturated long-chain free fatty acid distribution of human stratum corneum prevents hydrocarbon chain segregation. Further, LB-films of human stratum corneum ceramides express a pattern of connected elongated domains with a granular domain interface. The dominating effect of both cholesterol and cholesterol sulfate is that of increased ceramide domain dispersion. This effect is counteracted by the presence of free fatty acids, which preferentially mix with ceramides and not with cholesterol. Cholesteryl oleate does not mix with other skin lipid components, supporting the hypothesis of an extra-endogenous origin. In the system composed of endogenous human ceramides and cholesterol plus 15 wt% stratum corneum distributed free fatty acids, i.e., the system mimicking most closely the lipid composition of the stratum corneum extracellular space, LB-films on mica express lateral domain formation.     

Evaluation on the hydrolysis of methylumbelliferyl-tetra-N-acetylchitotetraoside by various glucosidases. A comparative study

• 1.1. In human plasma, an enzyme is present which hydrolyzes 4-methylumbelliferyl-tetra-N-acetylchitotetraoside. The function of this enzyme is unknown.
• 2.2. We have examined whether hyaluronidase, neutral endoglucosaminidase, $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-}\text{hexosaminidase}$, aspartylglucosaminidase, β-d-glucosidase, and chitobiase could hydrolyze MU-TACT. The results obtained are detailed below.
• 3.3. A purified commercial preparation of hyaluronidase does not hydrolyze MU-TACT.
• 4.4. Substrate specificity requirements, pH optimum and subcellular localization indicate that neutral endoglucosaminidase is distinguishable from MU-TACT hydrolase. Also commercial neutral endoglucosaminidase D and H have no affinity towards MU-TACT.
• 5.5. $\text{N-}\text{Acetyl}\text{-β-}\text{d}\text{-}\text{hexosaminidase}$ is different from MU-TACT hydrolase for the following reasons: (a) a purified enzyme preparation does not hydrolyze MU-TACT; (b) there is no correlation in the activity of the enzymes; (c) MU-TACT hydrolase is not deficient in cells of a patient with a deficiency of total $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-}\text{glucosaminidase}$; and (d) the 2 enzymes have very different Chromatographic characteristics and Con A binding properties.
• 6.6. Enzyme characteristics, substrate structural requirements and a lack of correlation with MU-TACT hydrolase activity suggest that aspartylglucosaminidase, β-d-glucosidase, and chitobiase are not involved in the hydrolysis of MU-TACT.
• 7.7. None of the enzymes which we have considered corresponds to MU-TACT hydrolase. The exact nature and the function of the enzyme remains an enigma.

     

Individual variation of human plantar stratum corneum lipids, determined by automated multiple development of high-performance thin-layer chromatography plates

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skin's lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl₂H₂SO₄ at 130°C or a solution of CuSO₄H₃PO₄ at 140°C allowed visualization of the lipids and quantification. The MnCl₂H₂SO₄ solution stained saturated fatty acids less intensity. Therefore, the CuSO₄H₃PO₄ solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.     

Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide

Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO₄-fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate.     

An alpha-L-fucopyranosyl-myl-inositol in normal human urine.

An α-$\text{L}$-fucopyranosyl-$\text{myo}$-inositol has been isolated from normal urine of ABH-secretors. The compound was also present in small amounts in the urine of ABH-non-secretors. After ingestion of 20 g of $\text{myo}$-inositol, two secretors increased their excretion of α-$\text{L}$-fucopyranosyl-$\text{myo}$-inositol three and thirteen times respectively. The same $\text{myo}$-inositol diet did not give rise to any increased excretion of α-$\text{L}$-fucopyranosyl-$\text{myo}$-inositol in the urine of two non-secretors.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems.Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, an observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition.The results are discussed in terms of the observed low concentrations of cholesteorl in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.