Cholesterol-phospholipid Ratio Research Articles

Protective effects of Khaya senegalensis stem bark extracts against acetaminophen-induced oxidative damage, dyslipidaemia, and hepatotoxicity in rats

Background Free radical attacks have been implicated in the aetiology of many diseases and several plants are used traditionally for the management of many oxidative-stress related diseases. Khaya senegalensis is used traditionally for the management diseases such as diabetes and for the treatment of infections. However, mechanisms underlying actions of K. senegalensis are poorly understood. Purpose This study aimed at the preliminary determination of the phytochemical constituents and investigation of the antioxidative and hepatoprotective actions of K. senegalensis in acetaminophen-treated rats. Method Aqueous extracts of K. senegalensis were screened for the presence of key phytochemicals. Total flavonoid and phenolic contents were quantified. Wistar albino rats were pre-treated with saline (control) or graded concentrations of K. senegalensis (50 – 200mg/kgbw) for 10 days prior to acetaminophen (2g/kg body weight) administration. Serum levels of vitamin C, thiobarbituric reactive substances, catalase activities, enzyme markers of liver function were assessed. Cholesterol-phospholipid ratio in treated-rats were determined. Results K. senegalensis extract showed the presence of saponins, tannins, cardiac glycosides, alkaloids, flavonoids and phenolic compounds. Total phenolic and total flavonoid contents were determined as 57.14±0.85mgQE/g and 51.72±0.77mgGE/g. Acetaminophen (2g/kg bw) raised serum TBARS (4.7-fold, P<0.001), H2O2 levels (2.3-fold, P<0.001), AST (5.9-fold, P<0.001), ALT (6.6-fold, P<0.001) and ALP (4.2-fold, P<0.001) and reduced serum levels of vitamin C (54%, P<0.001) and catalase activity (74.6%, P<0.001). Treatment of K. senegalensis extracts inhibited effects of acetaminophen on TBARS (18.2% - 46.4%, P<0.05 – 0.001), vitamin C (1.4 – 1.8-fold, P<0.001 – 0.05), H2O2 levels (19.1 – 50.1%, P<0.001-0.05), catalase activities (1.4 – 3.1-fold, P<0.001 – 0.05), AST (27.7 – 62.8%, P<0.001 – 0.05), ALT (35.6 – 57.5%, P<0.001 – 0.05) and ALP (15.9 – 46.2%, P<0.01 – 0.05). The extract reduced cholesterol-phospholipid ratio (21 – 31%, P<0.05). Conclusion These results motivate further development of the therapeutic potential of K. senegalensis

DRUGS CONTAINING GLYCYRRHIZIC ACID IN COMPLEX THERAPY FOR PULMONARY TUBERCULOSIS

The effectiveness of etiotropic chemotherapy is largely determined by the tolerability o the TB drugs (TBD) and the severity of unwanted side effects. It is known about the immunopathological effect of TBD, which can also be attributed to the undesirable effects of anti-tuberculosis chemotherapy. Changes in the structure and composition of mononuclear membranes reflect their functional activity. Secondary disorganization of the lipid bilayer due to the action of TBD is an important element in the development of immunosuppressive conditions in pulmonary tuberculosis and requires specific correction. The paper has the results of a survey of 308 patients with tuberculosis included. The comparison group consisted of 36 healthy volunteers. The membrane destruction coefficient and cholesterol-phospholipid ratio were used in order to assess the state of peripheral blood mononuclear cells and the degree of structural disorganization of their membranes. The prescription of preparations containing glycyrrhizic acid is pathogenetically substantiated and allows improving the state of the membranes of mononuclear cells, thereby reducing the undesirable membrane-toxic effect of first-line anti-tuberculosis drugs, There was an increase in etiotropic therapy intensity on the basis of abacilation at the end of the intensive phase of the main course of chemotherapy from 61% to 72.4% when using Licorice Root, and to 81.3% when using Fosfogliv® in patients with focal and infiltrative pulmonary tuberculosis.

Smoking-Induced Alterations in Platelet Membrane Fluidity and Na+/K+-ATPase Activity in Chronic Cigarette Smokers

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis. Platelet adhesiveness and aggregation increases as a result of smoking. Cigarette smoking modifies haemostatic parameters via thrombosis with a consequently higher rate of cardiovascular events, but smoking-induced alterations of platelet membrane fluidity and other changes have not been studied. Thirty experimental and control subjects (mean age 35+/-8) were selected for the study. Experimental subjects had smoked 10+/-2 cigarettes per day for 7-10 years. The plasma lipid profile, platelet carbonyls, sulfhydryl groups, Na(+)/k(+)-ATPase activity, fluidity using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), total cholesterol and phospholipids as well individual phospholipids were determined. Increases in the platelet membrane cholesterol phospholipid (C/P) ratio, phosphotidylethanolamine, phosphotidylserine with decreased phosphotidylcholine, Na(+)/k(+)-ATPase activity, fluidity and no significant change in phosphotidylinositol and sphingomylein, as well as increases in plasma total cholesterol, LDL-cholesterol, protein carbonyls with decreased HDL-cholesterol and sulfhydryl groups were observed in cigarette smokers. Platelet membrane total phospholipids were positively correlated with plasma LDL-cholesterol (r=0.568) and VLDL-cholesterol (r=0.614) in cigarette smokers. Increased plasma LDL-cholesterol, VLDL-cholesterol and total cholesterol might have resulted in the increased C/P ratio and decreased platelet membrane fluidity of cigarette smokers.

Inhibitory effect of morin on DMH-induced biochemical changes and aberrant crypt foci formation in experimental colon carcinogenesis

Morin is a flavonoid present in fruits and Chinese herbs, exhibits various beneficial biological activities. There are numerous evidence suggesting that total dietary fat intake is generally associated with early promotion of colon cancer, the alterations in the lipid profile is important for malignant transformation and tumor development and carbohydrate moieties of glycoproteins reflect the stage of cancer. Aberrant crypt foci (ACF) consisting of morphologically irregular crypts, are thought to be precancerous lesions for colon cancer. Our aim was to study the inhibitory effect morin on aberrant crypt foci and alterations in the levels of lipids, and glycoconjugates in experimental rat colon cancer. Group 1 served as control, groups 2 and 4 received 50 mg/kg b.w. morin orally everyday for 30 weeks. Groups 3 and 4 were given subcutaneous injection of 1,2-dimethylhydrazine (DMH) 20 mg/kg b.w. for the first 15 weeks. Administration of morin at the dose of 50 mg/kg b.w., significantly suppressed the formation of ACF its multiplicity and lowered levels of serum and tissue lipids, cholesterol–phospholipid ratio, glycoconjugate and also increased the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase). These results indicate that morin has a protective effect against DMH-induced colon carcinogenesis.

Cyclooxygenase-2 Inhibition by Etoricoxib Modulates Plasma Membrane Fluidity in Rat Colon

The present study was designed to investigate the effects of a selective cycloogenase-2 (COX-2) inhibitor, etoricoxib, on the membrane-specific enzymes, lipid composition, and changes in fluidity parameters of rat colonic plasma membrane. Two doses of the drug were used, one within its therapeutic anti-inflammatory range as based on the ED50 value in rats (Eto-1), while the other at 10 times higher dose relating to the toxicity studies (Eto-2), which have not been reported so far. The activity of the membrane alkaline phosphatase was found to be increased after treatment with both the doses of etoricoxib as compared to the control. The total lipid and the cholesterol content showed a decrease while an increase in ganglioside sialic acid content was noted. Phospholipid content and the cholesterol-phospholipid molar ratio showed no change in either of the treatment groups. Fluorescence polarization studies with diphenylhexatriene showed that membrane fluidity was least altered in the isolated brush border membrane from colon or in the liposomes prepared from the membrane lipid extracts. Also, the translational diffusion studied with pyrene showed that the fluidity parameter was decreased as measured by the excimer formation. It is concluded that etoricoxib, a new generation COX-2 inhibitor (called the coxibs), appeared to be a safe nonsteroidal anti-inflammatory drug (NSAID) in the colonic membranes.

Carnitine and lipoate ameliorates lipofuscin accumulation and monoamine oxidase activity in aged rat heart

In this study we have focused on the levels of lipofuscin, monoamine oxidase and cholesterol phospholipid ratio in the heart muscle of young, middle aged and aged rats. In parallel, we have also investigated the levels of carnitine and lipoic acid during aging. We observed an increase in lipofuscin accumulation and monoamine oxidase activity in both middle aged and aged rats. Levels of both carnitine and lipoic acid decreased along with a decrease in cholesterol phospholipid ratio. These changes were normalized upon cosupplementation of carnitine and lipoic acid. Our results thus reveal that carnitine along with lipoic acid can be used as an effective supplement against free radical induced damage to the cardiac tissue.

Membrane microdomains in hepatocytes: potential target areas for proteins involved in canalicular bile secretion

The formation of hepatic bile requires that water be transported across liver epithelia. Rat hepatocytes express three aquaporins (AQPs): AQP8, AQP9, and AQP0. Recognizing that cholesterol and sphingolipids are thought to promote the assembly of proteins into specialized membrane microdomains, we hypothesized that canalicular bile secretion involves the trafficking of vesicles to and from localized lipid-enriched microdomains in the canalicular plasma membrane. Hepatocyte plasma membranes were sonicated in Triton and centrifuged overnight on a sucrose gradient to yield a Triton-soluble pellet and a Triton-insoluble, sphingolipid-enriched microdomain fraction at the 5%/30% sucrose interface. The detergent-insoluble portion of the hepatocyte plasma membrane was enriched in alkaline phosphatase (a microdomain-positive marker) and devoid of amino-peptidase N (a microdomain-negative marker), enriched in caveolin, both AQP8 and AQP9, but negative for clathrin. The microdomain fractions contained chloride-bicarbonate anion exchanger isoform 2 and multidrug resistance-associated protein 2. Exposure of isolated hepatocytes to glucagon increased the expression of AQP8 but not AQP9 in the microdomain fractions. Sphingolipid analysis of the insoluble fraction showed the predominant species to be sphingomyelin. These data support the presence of sphingolipid-enriched microdomains of the hepatocyte membrane that represent potential localized target areas for the clustering of AQPs and functionally related proteins involved in canalicular bile secretion.

Acetyl-l-carnitine supplementation restores decreased tissue carnitine levels and impaired lipid metabolism in aged rats

The effects of long-term carnitine supplementation on age-related changes in tissue carnitine levels and in lipid metabolism were investigated. The total carnitine levels in heart, skeletal muscle, cerebral cortex, and hippocampus were approximately 20% less in aged rats (22 months old) than in young rats (6 months old). On the contrary, plasma carnitine levels were not affected by aging. Supplementation of acetyl-l-carnitine (ALCAR; 100 mg/kg body weight/day for 3 months) significantly increased tissue carnitine levels in aged rats but had little effect on tissue carnitine levels in young rats. Plasma lipoprotein analyses revealed that triacylglycerol levels in VLDL and cholesterol levels in LDL and in HDL were all significantly higher in aged rats than in young rats. ALCAR treatment decreased all lipoprotein fractions and consequently the levels of triacylglycerol and cholesterol. The reduction in plasma cholesterol contents in ALCAR-treated aged rats was attributable mainly to a decrease of cholesteryl esters rather than to a decrease of free cholesterol. Another remarkable effect of ALCAR was that it decreased the cholesterol content and cholesterol-phospholipid ratio in the brain tissues of aged rats. These results indicate that chronic ALCAR supplementation reverses the age-associated changes in lipid metabolism.

HYPOTHALAMIC DIGOXIN, HEMISPHERIC CHEMICAL DOMINANCE, AND INTERSTITIAL LUNG DISEASE

The isoprenoid pathway produces three key metabolites--endogenous digoxin, dolichol, and ubiquinone. This was assessed in patients with idiopathic pulmonary fibrosis and in individuals of differing hemispheric dominance to find out the role of hemispheric dominance in the pathogenesis of idiopathic pulmonary fibrosis. All 15 cases of interstitial lung disease were right-handed/left hemispheric dominant by the dichotic listening test. The isoprenoidal metabolites--digoxin, dolichol, and ubiquinone, RBC membrane Na+-K+ ATPase activity, serum magnesium, tyrosine/tryptophan catabolic patterns, free radical metabolism, glycoconjugate metabolism, and RBC membrane composition--were assessed in idiopathic pulmonary fibrosis as well as in individuals with differing hemispheric dominance. In patients with idiopathic pulmonary fibrosis there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels, and low ubiquinone and elevated free radical levels. There was also an increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was an increase in cholesterol phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in patients with idiopathic pulmonary fibrosis. Isoprenoid pathway dysfunction con­tributes to the pathogenesis of idiopathic pulmonary fibrosis. The biochemical patterns obtained in interstitial lung disease are similar to those obtained in left-handed/right hemispheric chemically dominant individuals by the dichotic listening test. However, all the patients with interstitial lung disease were right-handed/left hemispheric dominant by the dichotic listening test. Hemispheric chemical dominance has no correlation with handedness or the dichotic listening test. Interstitial lung disease occurs in right hemispheric chemically dominant individuals and is a reflection of altered brain function.­­­

Isolation of human small intestinal brush border membranes using polyethylene glycol and effect of exposure to various oxidants in vitro.

This study presents a method of brush border membrane (BBM) preparation from the human small intestine using polyethylene glycol (PEG) precipitation and also looks at the effect of in vitro oxidant exposure on structural and functional alterations in the membrane. Isolated BBM were relatively pure as judged by 10- to 14-fold enrichment of marker enzymes with less than 1% contamination by other subcellular organelles. These membranes showed uphill transport of glucose and lipid analysis showed a cholesterol-phospholipid (C/P) ratio of 1.19. Isolated BBM were found to be susceptible to superoxide generated by xanthine oxidase (XO), resulting in lipid and protein oxidation along with altered glucose uptake. Superoxide exposure also resulted in phospholipid alterations, especially generation of lyso phospholipids. These changes were prevented by inhibiting XO by allopurinol or scavenging superoxide by superoxide dismutase (SOD). Other oxidants studied did not have significant affect on these membranes. These studies suggest that PEG can be used for preparation of BBM from the human small intestine and these membranes undergo structural and functional alterations on exposure to superoxide.

Developmental regulation and effect of early undernutrition on phosphorylation of rat cortical synaptic membrane proteins

Undernutrition during early postnatal life was employed in rats by restricting the feeding time. The synaptic membrane fraction from cerebral cortex of normal and undernourished rats of various ages was prepared and endogenous protein phosphorylation studied. Many of the synaptic membrane proteins were found to be phosphorylated in an age-dependent manner. Early undernutrition affects the phosphorylation of various proteins in a complex way; most affected were 48-, 52-, 61- and 74-kDa proteins. These proteins were found to have phosphorylations mainly at tyrosine residues. This finding indicates that tyrosine phosphorylations may be affected most by early undernutrition. Adequate nutrition after early undernutrition removes most of the effects of undernutrition on synaptic protein phosphorylation. To address the question of how undernutrition may affect protein phosphorylation, we studied the lipid content of synaptic membrane fraction as it can affect membrane properties, including the fluidity. We found that undernutrition affects phosphorylation of most of the synaptic membrane proteins in the same manner in which it affects the cholesterol–phospholipid ratio of synaptic membrane and, hence, the fluidity of the membrane. This indicates that lipid biosynthesis is one of the ways by which undernutrition can affect synaptic membrane protein phosphorylation.

Effect of colchiceine and ursodeoxycholic acid on hepatocyte and erythrocyte membranes and liver histology in experimentally induced carbon tetrachloride cirrhosis in rats.

Colchiceine and ursodeoxycholic acid (UDCA) are drugs currently in use as therapy for different types of liver damage. We evaluated their ability to reverse the damage induced by carbon tetrachloride (CCl4) in rats. Six groups were analysed: (1) CCl4 (0.4 g kg(-1), i.p., three times a week) for 13 weeks; (2) CCl4 for 8 weeks followed by colchiceine (60 microg kg(-1)) + CCl4 for 5 weeks; (3) CCl4 for 8 weeks and thereafter UDCA (25 mg kg(-1)) + CCl4 for 5 weeks. Groups 4, 5 and 6 were appropriate controls of colchiceine, UDCA and vehicles respectively. Na+,K+- and Ca2+-ATPase activities and the cholesterol-phospholipid (CH/PL) ratio from erythrocyte and hepatocyte membranes were quantified. Membrane enzymatic activities and CH/PL ratios were affected more in group 1 than groups 2 and 3. We concluded that colchiceine and UDCA were effective drugs in this model of liver damage.

Murraya koenigii and Brassica juncea — Alterations on lipid profile in 1–2 dimethyl hydrazine induced colon carcinogenesis

The influence of the two commonly used spices Murraya koenigii (curry leaf) leaf and Brassica juncea (mustard) seeds on the levels of lipids, fecal bile acids and neutral sterols was studied in rats administered 1,2-dimethyl hydrazine (1,2 DMH). The levels of cholesterol and phospholipids decreased in the experimental groups when compared with the control. The cholesterol phospholipid ratio showed an elevated level in the DMH treated control compared with the species group. Bile acids and neutral sterols showed a sharp increase in the spices treated groups in liver and feces when compared with the control. Morphological and histological studies revealed that the mean number of neoplasms in the colon and intestine were significantly low in the spices fed groups.

Effects of bile salt hydrophobicity on crystallization of cholesterol in model bile.

Precipitation of cholesterol crystals is an essential step in gallstone formation. In the present study we found much faster and more extensive precipitation of various cholesterol crystal shapes in whole model biles containing the hydrophobic bile salt taurodeoxycholate than in biles containing the relatively hydrophilic taurocholate. Addition of taurodeoxycholate to isolated cholesterol-phospholipid vesicles also induced more crystallization than taurocholate. Crystallization behaviour in whole model biles and in vesicles after addition of corresponding bile salts was very similar. The very hydrophilic bile salts tauroursodeoxycholate and taurohyodeoxycholate never induced crystallization from vesicles, and crystallization in corresponding whole model biles did not occur. These bile salts also reduced crystallization dose dependently after addition of taurodeoxycholate to vesicles. Ultracentrifugation experiments suggested a higher vesicular cholesterol-phospholipid bile salts. These findings indicate that bile salt hydrophobicity influences shape of cholesterol crystals and extent of crystallization, possibly by modulating the vesicular cholesterol-phospholipid ratio.

Non-insulin dependent diabetes and reverse cholesterol transport

<h2>Abstract</h2> Quantitative and qualitative changes are observed in high-density lipoproteins (HDL) in patients with non-insulin dependent diabetes mellitus (NIDDM), and, more generally, in states of insulin resistance combined with central obesity. Reduced levels of HDL cholesterol are observed in patients with NIDDM, this decrease being correlated with the degree of insulin resistance. Qualitative changes in HDL are characterised by an increased triglyceride content, changes in the free cholesterol-phospholipid ratio, and an increase in the number of glycosylated apolipoprotein A-I molecules, giving rise to major variations in the viscosity of HDL particles. The transport of cholesterol is reduced when HDL is glycosylated and the transfer activity of cholesterol esters is increased. There is also a reduction in the level of HDL lipid peroxidation. These abnormalities in the lipid profile cause changes in reverse cholesterol transport which may be involved in the genesis of the accelerated atherosclerosis observed in patients with NIDDM.

Non-insulin dependent diabetes and reverse cholesterol transport

Quantitative and qualitative changes are observed in high-density lipoproteins (HDL) in patients with non-insulin dependent diabetes mellitus (NIDDM), and, more generally, in states of insulin resistance combined with central obesity. Reduced levels of HDL cholesterol are observed in patients with NIDDM, this decrease being correlated with the degree of insulin resistance. Qualitative changes in HDL are characterised by an increased triglyceride content, changes in the free cholesterol-phospholipid ratio, and an increase in the number of glycosylated apolipoprotein A-I molecules, giving rise to major variations in the viscosity of HDL particles. The transport of cholesterol is reduced when HDL is glycosylated and the transfer activity of cholesterol esters is increased. There is also a reduction in the level of HDL lipid peroxidation. These abnormalities in the lipid profile cause changes in reverse cholesterol transport which may be involved in the genesis of the accelerated atherosclerosis observed in patients with NIDDM.

Characterization of a Small Vesicular Cholesterol Carrier in Human Gallbladder Bile

Cholesterol phospholipid vesicles play an important role in the nucleation of cholesterol in bile. Recent studies have identified an additional vesicle population in human bile. In this study, the role of these small vesicles as cholesterol carriers was examined. Gallbladder bile was obtained from 60 patients at cholecystectomy. Large vesicles, small vesicles, lamellae, and mixed micelles were separated using gel filtration chromatography. Small vesicles were present in bile from the majority of patients both with and without cholesterol gallstones, whereas the void volume vesicle fraction was found almost exclusively in bile from patients with cholesterol gallstones. Both large vesicular and small vesicular cholesterol increased as total bile cholesterol concentration increased; however, the cholesterol-phospholipid ratio in the large vesicle fraction from patients with cholesterol stones was significantly greater than the ratio in small vesicles (1.6 +/- 0.3 vs. 1.0 < or = 0.1, p < 0.05). Whole bile cholesterol crystal appearance time was correlated significantly with the percentage of cholesterol transported by large vesicles (r = 0.63, p < 0.001) but not with the percentage of cholesterol present in small vesicles. Finally, large vesicles isolated by gel filtration chromatography formed cholesterol crystals faster than small vesicles (5.3 +/- 2 vs. 17.4 +/- 4 days, p < 0.01). These data suggest that a heterogenous population of vesicles is present in human gallbladder bile. As bile becomes saturated with cholesterol, it increasingly is solubilized by both small and large vesicles. The small vesicles have relatively less cholesterol and are more stable than the larger variety, from which cholesterol is most likely to precipitate.

Effect of ethanol on rat gastric surfactant: A fluorescence polarization study

Background: Gastric surfactant is believed to protect gastric mucosa by the hydrophobic properties of its phospholipidic component, which are reflected in the fluorescence polarization of a lipophylic fluorescent probe. The present study aimed to observe the consequences of intragastric administration of 40% ethanol on the physical properties of rat gastric surfactant. Methods: Fluorescence polarization studies and lipid composition of gastric mucosal surface scrapings were performed. Results: Time course experiments indicated that the ulcerogenic action of ethanol occurred along with a fluidization of the surface scrapings followed by secondary rigidification. The fluidizing effect of ethanol was related to modifications of the molecular dynamics of lipid structures. The rigidifying effect of ethanol was a result of an increase in the cholesteroltriglyceride and cholesterol-phospholipid ratios and an increase in the percent composition of phosphatidylethanolamine of surface scrapings. Conclusions: The results suggest that alcohol could alter the gastric mucosal barrier by its disorganizing effect on the molecular dynamics of the gastric surfactant. The second rigidifying effect of ethanol could be a part of the damage repair phenomenon.

Mechanisms of ascorbic acid-induced inhibition of chemical transformation in C3H/10T1/2 cells

Ascorbate irreversibly inhibited morphological transformation induced by methylcholanthrene in C3H/10T1/2 cells. To determine the mechanisms of this inhibition, we studied ascorbate uptake, redox potential, matrix proteins, and lipid composition of 10T1/2 cells. Ascorbate (16.8 nmol/dish) saturated cells and reduced the NADH-to-NAD+ ratio. Daily treatments with ascorbate, 28 nmol/dish, maintained intracellular ascorbate and reduced NADH by half. Matrix collagen and glycoproteins were stimulated by ascorbate, Iso-ascorbate, and dehydroascorbate in a dose-dependent manner. Both ascorbate and dehydroascorbate reduced total lipids with time; neutral lipids increased but were released into the media, phospholipids were modified, cholesterol-phospholipid ratios declined, and an inverse relationship between unsaturation index and cholesterol-phospholipids was apparent. Lipophilic bodies gradually accumulated. Our data suggest that inhibition of transformation by ascorbate, Iso-ascorbate, or dehydroascorbate may be associated with regulation of the redox potential, glycoproteins, and lipids in 10T1/2 cells.

Modulation of Cellular Cholesterol and Its Effect on Cornified Envelope Formation in Cultured Human Epidermal Keratinocytes

When cultured human epidermal keratinocytes (NHK) reach confluence they start to differentiate and an increase in the total cellular cholesterol content is observed. This increase parallels the appearance of a characteristic feature of terminal keratinocyte differentiation, the spontaneous formation of cornified envelopes (CE). Synthesis of CE is catalyzed by the plasma membrane-associated transglutaminase (TGm). Supplementation of the medium with inhibitors of cholesterologenesis suppressed increase in cholesterol levels and CE formation but did not interfere with TGm expression or TGm activity. Modulation of the plasma membrane cholesterol-phospholipid ratio of confluent NHK cultures using either pure phospholipid liposomes or liposomes enriched in cholesterol strongly affected spontaneous CE formation. Pure phospholipid liposomes completely inhibited CE formation, whereas cholesterol-enriched liposomes ensured envelope formation, even in the presence of inhibitors of cholesterol synthesis. From these results we conclude that in differentiating NHK an increase in the cellular cholesterol level is part of the differentiation program and is essential for the spontaneous CE formation.