Loss Of Phospholipids Research Articles

Origin of hepatic very-low-density lipoprotein triacylglycerol: the contribution of cellular phospholipid.

When rat hepatocytes were cultured for 24 h in the absence of exogenous fatty acid, the amount of very-low-density lipoprotein (VLDL) triacylglycerol (TAG) secreted (114 +/- 14 micrograms/mg of cell protein) could not be accounted for by the mass of TAG lost from the cells (29 +/- 6.1 micrograms/mg of cell protein) during this period (n = 12). Of the balance (85 +/- 14 micrograms/mg; 94 +/- 15 nmol/mg), a maximum of only 37 nmol/mg of cell protein of TAG could be accounted for by fatty acids synthesized de novo. When labelled exogenous oleate (initial concentration, 0.75 nM) was present in the culture medium, the net gain in cellular plus VLDL TAG (253 +/- 38 micrograms/mg of cell protein per 24 h) was greater than that contributed by the exogenous fatty acid (155 +/- 18.2 micrograms/mg of cell protein, n = 5). Again, the balance (98.8 +/- 18.2 micrograms/mg of cell protein per 24 h) was too great to be accounted for by fatty acid synthesis de novo. In experiments in which cellular glycerolipids were prelabelled with [9, 10(n)-3H]oleic acid, following removal of the labelled fatty acid, there was a net increase in labelled cellular plus VLDL TAG over the next 24 h. That cellular phospholipids are the source of a substantial part of the excess TAG synthesized is supported by the following evidence. (1) The loss of prelabelled cellular phospholipid during culture was greater than could be accounted for by secretion into the medium. (2) During culture of cells prelabelled with 1,2-di-[l-14C]palmitoyl phosphatidylcholine, a substantial amount of label was secreted as VLDL TAG. (3) In pulse-chase experiments, the kinetics of labelled phospholipid turnover were consistent with conversion into a non-phospholipid pool. The enzymology involved in the transfer of phospholipid fatty acids into TAG is probably complex, but the present results suggest that this pathway may represent an important route by which extracellular fatty acids are channelled into VLDL TAG.

Apolipoprotein E Produced by Human Monocyte-derived Macrophages Mediates Cholesterol Efflux That Occurs in the Absence of Added Cholesterol Acceptors

Human monocyte-derived macrophages can efflux accumulated cholesterol without exogenously added cholesterol acceptors (Kruth, H. S., Skarlatos, S. I., Gaynor, P. M., and Gamble, W. (1994) J. Biol. Chem. 269, 24511-24518). Most of the effluxed cholesterol accumulates in the medium as apolipoprotein E-discoidal lipid particles. In the current study, we determined whether and to what degree cholesterol efflux from human monocyte-macrophages depended on apolipoprotein E secretion. Unexpectedly, 2-week-old differentiated monocyte-macrophages secreted similar amounts of apolipoprotein E without or with cholesterol enrichment. Apolipoprotein E mRNA levels in these macrophages were not increased by cholesterol enrichment and were comparable with levels in HepG2 cells. Without cholesterol enrichment, monocyte-macrophages secreted lipid-poor apolipoprotein E with a density >1.21 g/ml. By contrast, cholesterol enrichment of monocyte-macrophages induced the association of apoE with phospholipid and cholesterol to form discoidal particles that floated at densities of 1.08-1.10 g/ml. An anti-apolipoprotein E monoclonal antibody added to the culture medium significantly inhibited cholesterol and phospholipid efflux from the monocyte-macrophages. This showed that apolipoprotein E was required for most of the cholesterol efflux, and that apolipoprotein E did not leave macrophages with lipid but rather associated with lipid after it was secreted. Thus, 1) apolipoprotein E was constitutively secreted by differentiated human monocyte-macrophages, 2) apolipoprotein E only formed discoidal particles following macrophage cholesterol enrichment, 3) apolipoprotein E was necessary for cholesterol efflux to occur in the absence of added cholesterol acceptors and, in addition 4) the level of macrophage unesterified cholesterol was not rate-limiting for this cholesterol efflux, and 5) net phospholipid synthesis occurred in macrophages secondary to apoE-mediated loss of macrophage phospholipid. In conclusion, apolipoprotein E functions in an autocrine pathway that mediates cholesterol efflux from human monocyte-derived macrophages.

Effect of lipid transfer proteins on lipoprotein lipase induced transformation of VLDL and HDL

Lipoprotein lipase-induced lipolysis of human plasma VLDL usually does not yield a complete conversion of VLDL to LDL due to insufficient loss of surface and core lipids and apolipoprotein E. In order to assess the role of lipid transfer proteins in this process human VLDL and apo E free HDL, in approximately physiologic proportions, and with sufficient albumin to bind all released fatty acids, were subjected to 90% lipolysis of triglycerides in 2 h by lipoprotein lipase in the presence or absence of partially purified human cholesteryl ester and phospholipid transfer proteins. Lipoprotein lipase caused a partial transfer of VLDL unesterified cholesterol (16%) and phospholipid (11%), apo E (19%) and almost complete transfer of apo CII and CIII to HDL. VLDL remnants possessed excess apo E and surface and core lipids when compared to plasma LDL, and densities ranging from that of VLDL/IDL to LDL. With addition of the lipid transfer proteins to the lipolysis incubation there was an increased transfer of phospholipid and unesterified cholesterol (2-fold) and apo E (1.6-fold) to HDL over that for lipoprotein lipase incubations. The source of transferred material was primarily from remnants which isolated in the LDL density range in lipoprotein lipase incubations. This transfer resulted in LDL-like particles which had a smaller particle size but lighter density compared to those in lipoprotein lipase incubation. Transfer of cholesteryl esters to VLDL from HDL in exchange for triglyceride was absent or substantially reduced in incubations containing lipoprotein lipase and lipid transfer proteins compared to incubations with only lipid transfer proteins. It is concluded that during rapid lipolysis lipid transfer proteins promote the loss of phospholipid, unesterified cholesterol and apo E from VLDL remnants but do not promote the transfer of cholesteryl ester from HDL to VLDL.

PHOSPHOLIPIDS AND NUCLEAR RNA

It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of phospholipids in DNA synthesis and RNA transcription.

Alterations of erythrocyte morphology and lipid composition by hyperbilirubinemia

Morphology and membrane lipid composition of erythrocytes from neonates (jaundiced and healthy) and adults (before and after incubation with bilirubin) were studied. The morphological index, expressing the relative proportions of the different stages of cell distortion, and the membrane cholesterol, phospholipids and cholesterol/phospholipids molar ratio, were determined. In jaundiced neonates a significant increase in the morphological index ( P < 0.01) was found. After incubation with bilirubin, adult erythrocytes also showed an increase in the morphological index ( P < 0.01). Hemolysis occurred under these conditions, and the red cell ghosts obtained (vesicles) showed a rounded morphology. Higher cholesterol/phospholipid ratio and lower phospholipid content were found in jaundiced neonates compared with healthy babies ( P < 0.05) and adults ( P < 0.01), as well as in the cells ( P < 0.05) and vesicles ( P < 0.01) obtained after bilirubin incubation. Bilirubin cytotoxicity may occur in a stepwise manne: deposition of bilirubin in membrane produces echinocytosis, which is followed by disintegration of the lipid bilayer with loss of phospholipids from the membrane.

Changes of Endogenous Antioxidants and Fatty Acid Composition in Irradiated Rice Bran during Storage

The effects of γ-irradiation (5, 10, and 15 kGy) on antioxidants and lipid composition of rice bran were studied. The free fatty acid (FFA) level in rice bran irradiated at 5 kGy was not significantly different from that of raw rice bran. Increases in irradiation doses to 10 and 15 kGy resulted in an increase in FFA levels. γ-Irradiation at 15 kGy resulted in greater (p ≤ 0.05) loss of phospholipids in rice bran during storage. Also, γ-irradiation had deleterious effects on lipid stability, E vitamers, and oryzanol in rice bran during irradiation and subsequent storage. The decomposition of individual E vitamers increased with an increase in irradiation level. The loss of total E vitamers and oryzanol occurred in two stages: 50−82% and 12−33% immediately following irradiation and a further 10−35% and 39−42% during storage. Keywords: Rice bran; γ-irradiation; vitamin E vitamers; fatty acids

Effects of Ca++ and glycine on lipid breakdown and death of ATP-depleted MDCK cells

The relationships between cytosolic free calcium (Caf), cell associated glycine, phospholipid hydrolysis and cell death were investigated in Madin-Darby canine kidney (MDCK) cells injured by depletion of adenosine triphosphate (ATP). Glucose free incubation for three hours with a mitochondrial uncoupler resulted in progressive loss of glycine from cells. However, they were not lethally injured unless a perturbation of Ca++ homeostasis was also induced. Exposure to a Ca++ ionophore and uncoupler in 1.25 mM Ca++ medium (+Ca) resulted in accelerated cell death. ATP depleted cells with ionophore in 100 nM Ca++ medium (-Ca) were also lethally injured, but after a significant delay. Depletion of glycine preceded death in both groups of cells. Exogenous glycine (5 mM) protected +Ca cells against lethal membrane damage, but the beneficial effects were lost over a period of time. In contrast, -Ca cells were completely protected throughout. Phospholipid mass and radioactive label in lipid fractions of cells prelabeled with 3H-oleic acid were measured. Accelerated death of +Ca cells was accompanied by large decreases of phospholipid mass, loss of phospholipid label, and accumulation of unesterified labeled fatty acid. These changes were greatly decreased by incubation in -Ca medium. On the other hand, protection by glycine could not be attributed to modifications of either the massive breakdown of phospholipids that occurred in +Ca cells, or the modest changes seen in -Ca cells. In +Ca cells, the deleterious effects of increased Caf and phospholipid breakdown ultimately prevailed over protection by the amino acid. Thus, separate pathways of cell death associated with increased Caf and decreased glycine were defined in ATP depleted, Ca(+)- permeabilized MDCK cells. Calcium excess and massive phospholipid loss are features of a damage process that occurs independently of whether cells are protected by glycine or not. Conversely, the glycine sensitive component of injury is expressed regardless of whether intracellular Ca++ is increased, or large phospholipid losses occur. ATP depletion in -Ca medium provides a system to study mechanisms of glycine cytoprotection uncomplicated by Ca++ toxicity.

Effect of endothelin-1 induced ischemia on peroxidative damage and membrane properties in rat striatum synaptosomes

Synaptosomes obtained from rat striata lesioned by central injection of endothelin-1 (ET-1) were analyzed for the levels of lipid peroxidation products, the susceptibility to lipid peroxidation, the phospholipid and free fatty acid composition and the activity of Na+,K(+)-ATPase one hour after ET-1 treatment. The intrastriatal injection of ET-1 promoted an increase of endogenous thiobarbituric reactive substances (TBARS), as index of free radical mediated lipid damage, and a greater susceptibility to iron/ascorbate-induced lipid peroxidation. The pattern of free fatty acids showed a significant decrease of arachidonic and docosahexaenoic acid consequent to ET-1 treatment. The analysis of lipid composition showed a significant loss of phospholipids: among phospholipid species, sphingomyelin and phosphatidylethanolamine plasmalogen were particularly reduced by ET-1 treatment. The activity of membrane-bound Na+,K(+)-ATPase was also significantly reduced in synaptosomes obtained from ET-1 lesioned striata. Taken together these results indicate a significant modification of synaptosomal membrane of ET-1 treated rat striata, possibly due to a free radical mediated damage.

Effect of glibenclamide on the metabolism of fatty acids in cultures of newborn rat heart cells under normoxic and hypoxic conditions

Several deleterious biochemical alterations have been observed in myocardial cells during ischemia, including perturbations of transmembrane ion equilibria, production of noxious oxygen-derived radicals and loss of membrane phospholipids. Although the precise relationship between these alterations and the reduction of oxygen and glucose supplies is not fully understood, the decrease of intracellular ATP content appears to be a key event in the cascade. Recent evidence suggests that opening of ATP-sensitive K + channels may constitute an endogenous protective mechanism during ischemia. We have thus tested the effects of glibenclamide, a channel blocker, and aprikalim, a channel opener, on the metabolism of membrane fatty acids in cultures of newborn rat heart cells under normoxic and hypoxic conditions. We showed that glibenclamide partially blocks the loss of membrane phospholipids induced by oxygen deprivation in contractile myocytes, whereas aprikalim fails to alter this metabolism under either normoxic or ischemic conditions. In cultures of fibroblast-like heart cells neither drug was able to modify fatty acid metabolism.

Sterile filtration of liposomes: Retention of encapsulated carboxyfluorescein

Multilamellar vesicles incorporating carboxyfluorescein (CF) were calibrated through polycarbonate membranes of 0.1, 0.2 or 0.4 μm pore size. After removal of unencapsulated material, the vesicles were passed through 0.2 μm sterilizing filters constituted of polycarbonate, cellulose acetate or polyvinylidene fluorure (PVDF). The liposomes were readily filtered without significant change in the vesicle size and loss of phospholipids. The leakage of encapsulated material was only slight, lower than 5% in all cases, except for vesicles of 300 nm sterilized through polycarbonate membranes of 0.2 μm pore size. However, the same preparation was easily passed through 0.2 μm acetate cellulose membranes without significant leakage of the incorporated dye. In spite of the small size of the liposomes, trapping efficiencies up to 33% were achieved, depending on the vesicle mean diameter, lipid concentration, bilayer composition and inclusion of freeze-thawing cycles.

Lipid changes in mature‐green bell pepper fruit during chilling at 2°C and after transfer to 20°C subsequent to chilling

Lipid composition and pigment content in bell pepper (Capsicum annuum L. cv. Bell Tower) fruit that were freshly harvested, chilled 14 days at 2° C. or chilled and then transferred to 20 °C for 3 days (“rewarmed”) were determined. There was slight to moderate loss of membrane glycerolipids during chilling, with much greater losses after chilled fruit was rewarmed. Galactolipid (GL) loss exceeded that of phospholipid (PL). The ratio of monogalactosyl ‐to digalactosyl‐diacylglycerol did not change in chilled or in rewarmed fruit, and there was no chlorophyll loss, but the amount of neutral carotenes declined during chilling and dropped further alter rewarming. Only minor changes in total membrane sterols (TMS = free sterols + steryl glycosides + acylated steryl glycosides) were noted in chilled and in rewarmed fruit (a small increase followed by a small decrease), but major changes occurred in sterol glycosylation and esterification. The ratio of stigmasterol to sitosterol increased during chilling and rose further after rewarming. Due to PL loss, the ratios of TMS and free sterols to PL increased in rewarmed fruit. The ratio of linolenate (18:3) to linoleate (18:2) rose during chilling and after rewarming in all fatty‐acyl lipids (GL. PL. and acylated steryl glycosides), but the unsaturation index increased only in GL. These results indicate that most membrane damage occurs after rewarming of chilled fruit and that the chloroplasts are especially chilling sensitive.

Protection from Cold Injury by Deferoxamine, an Iron Chelator

The presence of hydroxyl radical (OH°) has been implicated in the pathogenesis of cold injury. Since iron is known to catalyze the OH° formation responsible for cellular injury, this study was designed to examine whether an iron chelator such as deferoxamine can salvage a tissue from cold injury. Cold injury was induced in the hind limbs of rabbits. The experimental group received 0.6 mM of deferoxamine through the femoral vein prior to cooling of the limbs. Deferoxamine reduced the tissue injury, as evidenced by the decreased release of lactate dehydrogenase, a nonspecific marker for cellular injury. In addition, this drug inhibited OH° formation and lipid peroxidation when examined by monitoring the formation of conjugated dienes and malonaldehyde, presumptive markers for lipid peroxidation. Rewarming of the cooled limbs was also associated with the loss of membrane phospholipids, with the corresponding accumulation of lysopho-sphoglyc erides and free fatty acids, especially linoleic and arachidonic acids. Deferoxamine prevented the loss of phospholipids and inhibited the accumulation of amphipathic lipid products. These results indicates that deferoxamine salvaged the tissue from cold injury, possibly by preventing the formation of OH° presumably by chelating iron, thus protecting the phospholipids from free radical attack.

Phospholipid loss in dying platelets.

The death of a cell results in a large amount of membrane lipid, predominantly phospholipids and cholesterol, that must be eliminated. In this study, we have examined what happens to phospholipids in dying rat platelets. Rat platelets were incubated for up to three days following their activation with thrombin. Platelet death occurred during the first day of incubation. This was indicated by a complete loss of platelet lactate dehydrogenase into the incubation medium. The platelets progressively lost over one-half of their phospholipid content during the three days of incubation. Cholesterol and sphingomyelin (the phospholipid with the highest affinity for cholesterol) were not lost during the same period. Our findings suggest that significant degradation of cellular non-sphingomyelin phospholipid can be triggered by cell death. The preservation of sphingomyelin in dying platelets, may be an adaptive response to maintain cholesterol in a solubilized state within dying cells.

Release of choline from rat brain under hypoxia: contribution from phospholipase A 2 but not from phospholipase D

Moderate hypoxia induced in rats by inhalation of 10% oxygen led to an increase of the concentration of free choline in the brain and caused a large net-release of choline from the brain into the venous blood as determined by the measurement of the arterio-venous difference. In hippocampal slices from rat brain, hypoxia increased the release of choline into the superfusion medium. The activity of phospholipase D, as measured by the formation of phosphatidylpropanol in the presence of propanol, was not stimulated under these conditions. However, the mobilization of choline was completely depressed by lowering extracellular calcium and by 0.1 mM mepacrine. We conclude that hypoxia leads to a selective activation of phospholipase A 2 in the brain and, consequently, to a net loss of choline-containing phospholipids and membrane structures.

LIPID CHANGES IN BELL PEPPER FRUIT DURING CHILLING AND REWARMING

Lipid composition and pigment content in bell pepper fruit that were freshly harvested, chilled 2 weeks at 2C (CH), or chilled then rewarmed to 20C for 4 days (RW), were determined. There was slight to moderate loss of glycerolipids during chilling, with much greater losses after rewarming. Loss of galactolipid (GL) exceeded that of phospholipid (PL). The ratio of the GL, MGDG:DGDG, did not change in CH and RW fruit, and there was no loss of chlorophyll or change in the Chl a:b ratio, but neutral carotenes declined ca 30 % after rewarming. Only small changes in total membrane sterols (TMS) were noted in CH and RW fruit, but major changes in sterol glycosylation and esterificatian occurred. The stigmasterol:sitosterol ratio increased during chilling and after rewarming. Due to PL loss, the ratios of TMS and cerebrosides to PL increased sharply in RW fruit. The ratio of 18:2 to 18:3 declined with chilling and with rewarming in all acyl lipids, but total unsaturation increased only in GL. These results indicate that most membrane damage occurs after rewarming and that the chloroplasts are especially chilling sensitive.

Identification and characterization of plasmalogen fatty acids in swine heart

Reperfusion of ischemic swine myocardium is associated with the loss of sarcolemmal phospholipids resulting in the accumulation of amphiphilic metabolites, lysophosphoglycerides and free fatty acids, especially arachidonic acid, causing electro-physiological dysfunction and cell death. Recently, phospholipids containing a vinyl ether bond at the Sn-1 position, commonly known as plasmalogens, have been identified as major constituents of heart, which contain a large amount of arachidonic acid in the Sn-2 position. Because of the potential importance of plasmalogens, the fatty acid composition of the choline and ethanolamine phosphoglycerides in swine heart was determined. Lipids were extracted from the left ventricular biopsies from swine heart, phosphoglycerides were separated from the neutral lipids by thin layer chromatography, converted into methyl derivatives and analysed by GC. The peaks for fatty acid methyl ester (FAME) and dimethylacetal (DMA) derivatives of choline and ethanolamine phosphoglycerides were confirmed using GC-MS. The results showed high amounts of 18:1 (17 mol %), 18:2 (24 mol %) FAME in choline phosphoglycerides in contrast to the occurrence of a high amount of 20:4 (28 mol %) FAME in ethanolamine phosphoglycerides, suggesting that plasmenylethanolamine, and not plasmenylcholine, may serve as the depot for arachidonic acid in swine heart.

Ageing of Cucumber and Onion Seeds: Phospholipase D, Lipoxygenase Activity and Changes in Phospholipid Content

Cucumber (Cucumis sativus L. cv. Mesa) and onion (Allium cepa L. cv. Rijnsburger Heldis) seeds were rapidly aged at 40 o C a 74% relative humidity. Onion seeds were also slowly aged at 40 o C with 15% relative humidity for 11 months and one more month at 28% relative humidity. Significant loss of total and individual phospholipids was an early event during both storage treatments. With slow ageing of onion, loss of phosphatidylcholine occurred several months before loss of viability and vigour was detected. Phosphatidic acid, the lipid product of phospholipase D action, increased during rapid ageing of both cucumber and onion. Phosphatidic acid was present in onion seeds before the ageing treatments and its content remained unchanged in the slowly aged seeds

The number of amphipathic alpha-helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles.

The objective of this work was to determine the role of the amphipathic alpha-helical structural units of human apolipoproteins A-I, E, and A-IV in defining the sizes and reactivities with lecithin:cholesterol acyltransferase (LCAT) of their reconstituted lipoprotein particles. We prepared reconstituted high density lipoprotein (rHDL) particles with each of the three apolipoproteins in two weight ratios with lipid: 2.7/0.07/1 and 1.35/0.04/1, palmitoyloleoylphosphatidylcholine/cholesterol/apolipoprotein, by the sodium cholate dialysis procedure; and examined the rHDL product sizes and distributions by nondenaturing gradient gel electrophoresis. The rHDL particles were also incubated with low density lipoprotein (LDL), and with LDL plus LCAT, to observe any structural modifications due to phospholipid transfers to LDL and to cholesterol esterification by LCAT. In addition, we examined the average structural properties of the original rHDL by several fluorescence methods and circular dichroism spectroscopy, and determined their reaction kinetics with LCAT. The results indicate that the diameters of the largest rHDL particles, containing two apolipoproteins per particle, correlate with the maximum number of putative amphipathic alpha-helical segments in their sequences, and that smaller particles of this class may arise from the removal of one or more alpha-helical segments from contact with lipid. Furthermore, the larger particles may be converted into the smaller ones upon loss of phospholipid to LDL, and may form one or two well defined products when reacted with LCAT. In general, the subclasses of particles have distinct spectroscopic properties, consistent with a different apolipoprotein folding in particles containing different proportions of phospholipid to apolipoprotein. Furthermore, the different apolipoprotein structures lead to significant differences in reactivity with LCAT.

Protection from cold injury by deferoxamine, an iron chelator.

The presence of hydroxyl radical (OH) has been implicated in the pathogenesis of cold injury. Since iron is known to catalyze the OH formation responsible for cellular injury, this study was designed to examine whether an iron chelator such as deferoxamine can salvage a tissue from cold injury. Cold injury was induced in the hind limbs of rabbits. The experimental group received 0.6 mM of deferoxamine through the femoral vein prior to cooling of the limbs. Deferoxamine reduced the tissue injury, as evidenced by the decreased release of lactate dehydrogenase, a nonspecific marker for cellular injury. In addition, this drug inhibited OH formation and lipid peroxidation when examined by monitoring the formation of conjugated dienes and malonaldehyde, presumptive markers for lipid peroxidation. Rewarming of the cooled limbs was also associated with the loss of membrane phospholipids, with the corresponding accumulation of lysophosphoglycerides and free fatty acids, especially linoleic and arachidonic acids. Deferoxamine prevented the loss of phospholipids and inhibited the accumulation of amphipathic lipid products. These results indicates that deferoxamine salvaged the tissue from cold injury, possibly by preventing the formation of OH presumably by chelating iron, thus protecting the phospholipids from free radical attack.

Ethinylestradiol increases volume and decreases sinusoidal membrane surface in the rat liver: a stereological analysis.

Structural alterations of liver parenchyma caused by ethinylestradiol, a synthetic estrogen known to induce cholestasis and to act as a tumor promoter factor, were investigated. Male rats treated with 17 alpha-ethinylestradiol (5 mg/kg body weight for 5 days) were compared with controls (n = 5 each). After perfusion fixation and systematic random sampling, paraffin sections, semithin sections and thin sections were examined observing standard stereological techniques. Ethinylestradiol treatment induced an increase in liver volume by 65% (p less than 0.001), which was caused more by hypertrophy (volume of singular hepatocyte +35%, p less than 0.001) than by hyperplasia (number of hepatocytes +23%, p less than 0.001). A decrease in sinusoidal membrane surface density (-43%, p less than 0.005) associated with a decrease in sinusoidal microvillar volume density (-50%, p less than 0.005) were both compensated for by the increase in liver volume. No canalicular alterations were observed. Thus changes in hepatocytes detectable with stereological techniques affect the sinusoidal pole where decreased sinusoidal membrane surface is associated with or reflects a substantial loss of membrane phospholipids. The increased liver volume may constitute an adaptive response compensating for the relative decrease in sinusoidal membrane surface and displays characteristics comparable to those of preneoplastic hepatocytes.