Molar Ratio Of Cholesterol Research Articles

Activation of lecithin-cholesterol acyltransferase by apolipoprotein D: comparison of proteoliposomes containing apolipoprotein D, A-I or C-I

To study the activation of lecithin-cholesterol acyl transferase (LCAT) (phosphatidylcholine:sterol Oacyltransferase, EC 2.3.1.43) by apolipoprotein D in comparison to apolipoproteins A-I and C-I, proteoliposomes with a phosphatidylcholine/free cholesterol molar ratio of 24:1, containing 10–300 μg/ml of apolipoproteins were used. The proteoliposomes were prepared by the cholate dialysis technique. In all proteoliposome preparations we found rouleaux structures and stacked discs. The particles formed with apolipoprotein A-I were the most homogeneous, followed by apolipoprotein D- and apolipoprotein C-I-containing particles. Apolipoprotein A-I was the most potent LCAT activator in our system followed by apolipoproteins C-I and D. The fractional esterification rate observed with apolipoprotein D-containing substrates amounted to 15–48% that of apolipoprotein A-I-containing ones. Neither apolipoprotein A-I- nor C-I-containing proteoliposomes gave linear reaction kinetics with LCAT. Even during the first 15–30 min of incubation, the kinetics deviated strikingly from linearity at all apolipoprotein concentrations. In contrast, proteoliposomes containing apolipoprotein D exhibited linear reaction kinetics up to 60–90 min. At low apolipoprotein A-I concentrations (5 μg/ml), the addition of apolipoprotein D to the incubates resulted in significantly higher esterification rates as compared to substrates containing apolipoprotein A-I only. This was not the case using substrates with high apolipoprotein A-I concentrations (50 μg/ml). From our results we speculate that apolipoprotein D may have some stabilizing effect on the enzyme LCAT.

Plasma membrane of cultured oligodendrocytes: II. Possible structural and functional domains.

An oligodendrocyte plasma membrane-rich fraction, F2.2, was resolved by equilibrium density centrifugation on a linear sucrose gradient from 0.5 M to 1.3 M into three fractions, F2.2a, F.2.2b, F2.2c, and a pellet F2.2p. F2.2a and F.2.2b were enriched 1.5-fold relative to F2.2 in plasma membrane markers at the expense of F2.2c and F2.2p, which became correspondingly impoverished. This gave F2.2a and F2.2b a 42-fold and 37-fold enrichment, respectively, in plasma membrane markers relative to the initial cell homogenate. F2.2c had a sevenfold enrichment in a Golgi marker; together with F2.2p, they contained all the Golgi marker initially present in F2.2. Preliminary data indicated that the F2.2-subfractions differed from one another in their molar ratios of cholesterol to phospholipids and protein to lipids but had similar protein profiles when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Their content of fucosylated glycoproteins appeared also to be different. Morphologically, F2.2a and F2.2b were very similar: they contained large membrane vesicles, membrane sheets, and vesicles entrapped within other vesicles. Membrane-membrane interaction was apparent in these fractions. F2.2c had many of the same elements, but most of the membrane structures contained amorphous material. F2.2p differed morphologically from the other fractions in that it had principally electron-dense structures. It is postulated that F2.2a, F2.2b, and perhaps F2.2c represent different domains of oligodendrocyte plasma membrane. Alternatively, these fractions might correspond to the plasma membrane of oligodendrocyte subtypes.

Cholesterol effects on the interaction of cardiolipin with anti-cardiolipin antibody

(1) Human antibodies to cardiolipin, phosphatidic acid and phosphatidylserine were assessed by binding to nitrocellulose paper and subsequent reaction with an enzyme-linked or radioactively labelled second antibody to human IgG. (2) The addition of cholesterol to constant amounts of cardiolipin impregnated in the nitrocellulose paper resulted in a profound fall in antibody binding beginning at a 0.5 to 1 molar ratio of cholesterol to cardiolipin and stabilizing at about 15% of the original level. (3) Antibody binding to phosphatidic acid and phosphatidylserine also showed extensive cholesterol-induced inhibition beginning at a slightly lower molar ratio of cholesterol to phospholipid. (4) The structural array of neither the cardiolipin alone impregnated in nitrocellulose nor the phospholipid together with cholesterol is known. It is possible that the specific cardiolipin phase structure required for human antibody recognition was disrupted by cholesterol.

Heinz body formation in red cell membrane disorders: its acceleration in membrane lipid abnormalities.

Abnormal Heinz body formation in the presence of acetylphenyl hydrazine was observed in some patients with red cell membrane abnormalities, such as hereditary red cell membrane high phosphatidyl choline haemolytic anaemia, congenital haemolytic anaemias of unknown origin, acquired hyperlipidaemia and paroxysmal nocturnal haemoglobinuria. No abnormality of haemoglobin composition or of oxidation-reduction activities was noted in 66 patients studied. No abnormal Heinz body formation was seen in hereditary spherocytosis, hereditary elliptocytosis or hereditary stomatocytosis with normal membrane lipids, but increased Heinz body formation was observed in some patients with red cell membrane lipid abnormalities. The extent of abnormal Heinz body formation inversely correlated with a decreased molar ratio of free cholesterol to phosphatidyl choline in these red cells. Heinz body formation, therefore, may be abnormal in some red cell membrane disorders, especially when membrane lipid abnormalities exist.

Isolation and characterization of plasma membrane fromAcanthamoeba culbertsoni

A rapid method for preparation of plasma membrane fromAcanthamoeba culbertsoni involving toluene treatment followed by lithium bromide extraction is described. In the plasma membrane preparation, 5′-nucleotidase, Na+ + K+ -ATPase, Mg2+ -ATPase and glucose-6-phosphatase activities were enriched. The membrane preparation was free from nucleic acid, cytochrome P-450 and cytochrome b5. Amino acid (14C-Ieucine) was not incorporated in the plasma membrane in 2 min. Succinic dehydrogenase was not detectable in the plasma membrane preparation. The molar ratio of cholesterol and phospholipids was 0.95 which is characteristics for plasma membranes. Under electronmicroscopy the preparation was homogenous without any other component of the cell. Plasma membrane proteins and glycoproteins were separated on acrylamide gel electrophoresis

Liposome-facilitated enhancement of in vitro immunity to human colon cancer.

Liposome-antigens, in the presence of dialyzable leukocyte extracts (DLE)(1), were tested for their ability to enhance in vitro blastogenic responses to colon tumor antigens. Liposomes made with 6:4:1 molar ratios of phosphatidylcholine, cholesterol, and phosphatidic acid and bearing human colon cancer cell antigens were shown to be immunogenic in vitro, producing specific blastogenic responses. Addition of nonimmune DLE (500 micrograms/5 X 10(5) lymphocytes) in the in vitro culture system enhanced the blastogenic response 3x over liposome-antigen alone (p less than 0.001). Immune DLE (immune to keyhole limpet hemocyanin (KLH) and tuberculin (PPD) antigens) were suppressive (p less than 0.05) in the same study. Blastogenic reactivity to KLH was also generated in nonimmune lymphocytes and enhanced by liposomes with DLE specific to KLH (p less than 0.01). Nonspecific DLE (e.g., to PPD) caused suppression of KLH-induced blastogenesis (p less than 0.05). These findings confirm and extend prior reports noting that DLEs contain both specific and nonspecific lymphocyte blastogenic factors and suggest the use of this liposome-adjuvant system for boosting tumor immune responses.

Radiopaque Liposomes: Effect of Formulation Conditions on Encapsulation Efficiency

Liposomes containing sodium ioxitalamate were prepared by sonication. Suitable amounts of purified soybean phosphatidylcholine and cholesterol were used at various molar ratios. Stearylamine or dicetylphosphate were added to this lipid composition when charged liposomes were required. After sonication and removal of unencapsulated solute, this manufacturing process yielded small multilamellar vesicles as confirmed by electron microscopy. These liposomes did not exhibit a narrow range of size distribution; the mean particle size varied from 135 to 145 nm. With respect to the efficiency of encapsulation, two parameters were distinguishable: the volume of encapsulated aqueous space per unit of lipid weight and the percentage of the contrast agent added that became encapsulated in the liposomes. Investigation of the preparative parameters revealed that increased molar ratios of cholesterol yielded higher aqueous volume and iodine contents in the liposomes, which were attributed to a reduction of the liposome permeability to the contrast agent. However, the inclusion of cholesterol into the bilayer liposomal membrane was limited, probably by solubility restrictions. Negatively and positively charged liposomes had higher rates of encapsulation than did neutral liposomes. This result was expected since efficient encapsulation of polar compounds requires formation of large aqueous spaces within the vesicles per mole of lipids. Increase of the lipid fractions at a constant ratio of phosphatidylcholine-cholesterol, with all the other factors kept constant, reduced the aqueous volume entrapped per millimole of lipid and, consequently, the iodine content in the liposomes. However, an increase in the initial sodium ioxitalamate concentration diminished the aqueous volume entrapped in the liposomes but increased the iodine content.

Myelinogenic cycle and myelination status of the brain of the catfish, Heteropneustes fossilis

The lipid composition of the brain of the catfish, Heteropneustes fossilis (Bloch) was studied over the age period of 2–12 months, during which time the body weight increased from 2.5 to 33 g. The brain weight increased from 28 to 84 mg between 2 and 9 months of age, with a further increase of only 7 mg during the following three months. The concentration of cholesterol increased slowly up to 5 months of age, from which time the concentration began to increase rapidly and attained adult values at 8 months. The concentration of total lipid-P increased steadily up to the age of 7 months and remained almost constant thereafter. In contrast, there was very little increase in the concentration of the galactolipids (cerebroside + sulphatide) until month 5, after which the concentration increased rapidly to reach the adult level at 8 months of age. The concentration of cholesterol esters decreased during development; however, there occurred a transient rise at 5 months of age, which continued up to 8 months. These data indicate that the period between months 5 and 8 is one of active myelination in the brain of this species. The mole ratio of cholesterol, phospholipids, and galactolipids in the adult fish brain was 31:53:1, much higher than that in adult mammalian brain. Moreover, within the phospholipids, ethanolamine phosphoglyceride comprised < 10%, as against the corresponding value in the mammals of about 40%. These data therefore suggest a low level of myelination in the brain of catfish, as compared to that in higher species.

Biochemical correlates of the mechanism of salt uptake and excretion in the gills of freshwater field crab on adaptation to higher salinity

Male adult paddy field crabs (Oziotelphusa senex senex) were adapted to salinity of 34%. After 3 months the gill lamella of the crab was sectioned and examined under an electron microscope and compared with that of freshwater crabs. The apical region nearest to cuticular boundary showed numerous foldings projecting into the cytoplasm of the epithelium. This brush border appearance disappeared in salinity-adapted crabs. Mitochondria also reduced in number in the sections of salinity adapted crabs. In seawater medium the crabs showed a hypo-osmotic regulation. This regulation is said to be involved in active sodium excretion through gills, in contrast to the active Na+ uptake found in freshwater crabs. The ultrastructural changes have been correlated with significant reduction in cholesterol and phospholipid molar ratios of the gill as well as Na+ + K+ ATPase and divalent ion ATPase activities of particulate system of the crab. The univalent ion ATPase activity served as a marker to show the reduced uptake of Na+ on adaptation to higher salinity. The reduced Ca2+ ATPase activity in the gills of seawater adapted crabs has been correlated with the reduced phospholipid content of the gill tissue.

Cholesterol-induced effects on the viscoelasticity of monoglyceride bilayers

Changes in the viscoelastic properties of glycerol monooleate bilayers resulting from the incorporation of cholesterol into the membranes have been measured. The interface tension increases with the cholesterol concentration, reaching saturation for a 4.2:1 mole ratio of cholesterol:lipid in the film-forming solution. Incorporation of cholesterol in the membrane causes the appearance of a large intrinsic viscosity; this also increases with the sterol content of the membrane. Molecular models of lipid-sterol interactions and packing are considered to explain both the observed changes in membrane properties and similarities with comparable lipid systems.

Structural characteristics of tetanolysin and its binding to lipid vesicles.

Tetanolysin binding to lipid vesicles was found to depend on the molar ratio of cholesterol to phospholipid, being low in vesicles containing up to 20 mol% cholesterol and high in vesicles containing more than 33 mol%. High concentrations of purified tetanolysin preparations formed arc- and ring-shaped structures. The structures were not readily detectable in diluted preparations unless incubated with lipid vesicles containing high molar ratios of cholesterol to phospholipid. It is suggested that the toxin is concentrated on the vesicles to local concentrations high enough to form the arcs and rings.

Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity.

Proteoliposome vesicles containing apoA-I, lecithin, and cholesterol (including labeled cholesterol) were prepared from various molar ratios of the three components by the cholate dialysis technique. Comparative studies on the sensitivity and efficiency of these proteoliposomes to serve as substrate for lecithin:cholesterol acyltransferase (LCATase) indicated that the proteoliposome with apoA-I:lecithin:cholesterol molar ratio of 0.8:250:12.5 was ideal for assaying LCATase activity of both plasma and purified enzyme. This proteoliposome was shown to be comparable in size by gel filtration (radius, 131.9 +/- 4.8 A, n = 6) and by electron microscopy (radius, 123.4 +/- 5.1 A, n = 100). The proteoliposome preparation was stable as LCATase substrate for at least 3 and 5 weeks, respectively, when stored at 4 degrees C and -20 degrees C, and was a better substrate for the enzyme activity assay than were lecithin-cholesterol liposomes incubated with apoA-I. Under the standardized assay system LCATase activity was a linear function of plasma enzyme added and was independent of the amount of plasma cholesterol added to the proteoliposomes in the range of 3 to 20 microliters of plasma. The mean LCATase activity by this method was 95.1 +/- 14.0 (range 76.5-122.5) nmol/hr per ml of plasma from fifteen normal human subjects. This method of substrate formation using the cholate dialysis technique permits the preparation of large amounts of stable, efficient, homogeneous, and well-defined substrate that is suitable for measuring low levels of enzyme activity, comparative studies, and large scale investigations of plasma LCATase, as well as studies of the mechanism and regulation of LCATase reaction.

Lipid composition of transverse tubular membanes from normal and dystrophic skeletal muscle

Vesiculated fractions of transverse tubular (TT) membranes were isolated from normal and genetically dystrophic breast muscle of the chicken. The lipid composition of the TT was compared to the lipids of highly purified sarcoplasmic reticulum (SR) membranes and enriched fractions of sarcolemma (SL) membranes. Normal TT membranes contained a significantly higher molar ratio of cholesterol to phospholipid phosphorous (0.86) when compared to both SR (0.17) and SL (0.38) membranes. The phospholipid phosphorous-to-protein ratio was highest for SL (1.55 μmol phosphorous/mg), intermediate for TT (1.09), and lowest for the SR (0.79) membranes. TT membranes contained lower amounts of phosphatidylcholine and higher amounts of sphingomyelin when compared to SR while the TT phospholipid distribution was not significantly different from that of the SL. These data indicate that our putative TT fraction displays a lipid composition distinctly different from that of SR and SL and confirms other biochemical and morphological data which indicate that they are of TT origin. All three membrane classes isolated from dystrophic muscle displayed significant alterations in the composition of various lipids. Cholesterol levels were elevated most notably in SL (2.4-fold) followed by the SR (1.3-fold) but not TT membranes. Dystrophic TT and SL but not SR displayed elevated levels of phospholipid phosphorous. Few differences in the distribution of the individual phospholipid classes were found among the dystrophic membranes except for a 2.2-fold increase in the sphingomyelin content of the SR. The observed lipid abnormalities may be the underlying cause of reported alterations in excitation-contraction coupling and suggest that avian dystrophy may result from a generalized membrane defect.

Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions.

Micellar complexes of human apolipoprotein A-I and phosphatidylcholine, with or without cholesterol, were prepared by adding apolipoprotein A-I (apo A-I) to sodium cholate-lipid mixtures. Cholate was removed by dialysis and the apo A-I.lipid complexes were isolated by gel filtration chromatography or by density gradient ultracentrifugation. The lipid mixtures consisted of dipalmitoylphosphatidylcholine or egg yolk phosphatidylcholine in the presence of various molar ratios of cholesterol. The formation of complexes was examined at different phosphatidylcholine (PC)-to-apo A-I ratios, PC-to-cholate ratios, and cholate concentrations. Yields of complexes were maximal when incubation and dialysis were performed near the transition temperature of the PC. Upon lipid binding and complex formation, apo A-I experienced a significant increase in alpha-helix content, and a blue shift in the intrinsic tryptophan fluorescence. In all lipid-protein incubation mixtures, from 600:1 to 75:1, PC/apo A-I (molar ratios), relatively small, stable complexes were present which gave maximum yields at incubation ratios similar to their isolated stoichiometries of 75:1 to 140:1, PC/apo A-I (molar ratios). For the isolated complexes, molecular weights were determined by sedimentation equilibrium to be in the range from 220,000 to 260,000; fluorescence polarization using the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene showed a broadened and shifted gel to liquid-crystalline phase transition, characteristic of micellar complexes of apo A-I with PC. Complexes prepared using apo A-I, covalently labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride, had an overall particle rotational relaxation time of 530 ns. On electron micrographs, the complexes, negatively stained with phosphotungstate, appeared as lamellar, discoidal particles.

Erythrocyte osmotic fragility in protein-energy malutrition: Cholesterol, phospholipid, and Ca 2+, Mg 2+ adenosine triphosphatase

Erythrocytes from children suffering from protein-energy malnutrition (kwashiorkor) are shown to have increased resistance to osmotic lysis. Cholesterol as well as phospholipid in the erythrocyte membranes of children suffering from kwashiorkor were significantly higher as compared to those in controls. The molar ratio of cholesterol:phospholipid was lowered by 23% in these subjects ( P < 0.01). The specific activity of Ca 2+, Mg 2+-ATPase was decreased by about 50% in children suffering from kwashiorkor ( P < 0.001). In a relationship between the median corpuscular fragility of erythrocytes, the cholesterol:phospholipid ratio, and Ca 2+, Mg 2+-ATPase, the possibility is raised of an accumulation of intracellular Ca 2+ as a factor responsible for increased resistance of these cells to osmotic lysis in protein-energy malnutrition.

A new approach for the immunogenic presentation of membrane-bound human colon tumor antigens.

Liposomes bearing human tumor membrane vesicles were effective immunogenic complexes for inducing antibodies in rabbits. Multilamellar liposomes (MLV) at 7:4:1 molar ratios of phosphatidylcholine, cholesterol and phosphatidic acid were prepared with sonicated membrane (MN) isolated from LS174T colon tumor cells. MLV liposomes prepared together with MN (i.e., MN-MLV antigens) or MN added to preformed MLV (i.e., MN+MLV antigens), and MN antigens alone were used as immunogens. Rabbits were immunized i.v. with 100 g protein of each antigen group. Boosters (i.v.) were at days 13 and 29. Binding assays were performed by indirect radioimmunoassay on viable tumor cell targets. The MN+MLV groups were distinguished by earlier reactivity and greater specificity to the colon tumor antigens.

A morphological and biochemical study of myelinogenesis in fish brain.

Myelinogenesis in different brain parts of trout was studied comparatively by means of histochemistry and biochemistry. A more detailed approach, including ultrastructural analysis, to characterize this important differentiation process, particularly in the optic tectum, revealed a good correlation between the structural development of myelinated axons and the lipid composition of membranes. The molar ratio of cholesterol to phospholipids and galactolipids proved to be most suitable for monitoring myelination in fish CNS.

Lipid analysis of isolated plasma membranes of lymphoid cells in benign thymomas of Buffalo/Mna rats.

Plasma membranes were isolated from lymphoid cells of benign thymomas obtained from inbred BUF/Mna rats (21 mo old) and from normal thymocytes obtained from young rats (7 wk old) of the same strain. The isolated plasma membranes were electron microscopically pure, and the specific activities of Na+, K+-ATPase, and 5'-nucleotidase were enhanced. The lipid compositions of the plasma membranes from these two sources were analyzed and compared. The cholesterol and plasmalogen contents of membranes from both sources were similar, but the phospholipid content of the benign thymoma lymphoid cell membranes was slightly lower than that of the normal thymocytes, resulting in a somewhat higher molar ratio of cholesterol to phospholipid. The plasma membranes of the thymoma lymphoid cells also exhibited a slightly higher microviscosity as measured with fluorescence polarization. No significant differences were observed in the phospholipid compositions of the two membrane preparations.

Application of highly purified anti-galactocerebroside antibody to the analysis of lipid-lipid or lipid-protein interactions. Significance of lipid matrix.

The significance of the lipid matrix in the reaction of liposomal antigen, antibody and complement (Ag-Ab-C) was analyzed using purified anti-glactocerebroside antibody and synthetic lipids and the following results were obtained. 1. For the optimal Ag-Ab-C reaction it was necesary that galactocerebroside (galxCMH) and lecithin molecules were well dispersed by virtue of cholesterol (Chol) and that the molar ratio of cholesterol to the sum of galactocerebroside and lecithin was more than one. 2. The Ag-Ab-C reactivity changed depending upon the chain length of lecithin, and the maximal reaction was observed in the case of dilauroylphosphatidylcholine. When the fatty acyl chain of lecithin was either shorter or longer than that of dilaurosylphosphatidylcholine, the reactivity was reduced. 3. The Ag-Ab-C reactivity was increased by elongation of the fatty acyl chain of galactocerebroside and an abrupt change was found to be around the carbon number 8 to 10 of the fatty acyl chain. 4. The Ag-Ab-C reactivity was elevated by the increase in unsaturation of fatty acyl moiety. 5. There is a tendency that an increase in the charge of the lipid matrix leads to the reduction of the Ag-Ab-C reactivity. 6. The results suggest that the physicochemical properties of lipids and especially the lipid-lipid interaction in the hydrophobic region of the lipid matrix play an important role in the Ag-Ab-C reaction.

Increased fluidity of serum lipids and development of spontaneous mammary tumors in C3H mice

The degree of lipid fluidity (LFU) was quantitatively monitored by fluorescence polarization analysis of the hydrocarbon fluorescent probe diphenylhexatriene when embedded in artificial and biological lipid complexes. The results have shown a marked increase in LFU in serum lipids associated with the development of spontaneous mammary tumors in C3H mice. An increase in serum LFU was observed during the initiation of primary tumors. The serum LFU further increases as a function of increase in the tumor volume. This increase was not observed when animals were given transplants of syngeneic, spontaneously arising mammary tumors or following implants of antigenically inert glass beads. Since the serum LFU in C57BL/6, NZB and A/JAX non-tumor-bearing mice was found to be significantly lower than in C3H non-tumor-bearing mice, it is suggested that alterations in the dynamics of serum lipids in the C3H system may have a direct relation to the induction and/or growth of spontaneous tumors in these mice. Moreover, experiments carried out with an artificial membrane model system have shown that the dynamics of a lipid complex are directly determined by its lipid composition. The three major parameters that determine the LFU of a lipid domain are: (a) the relative amounts of different phospholipids; (b) the molar ratio of cholesterol to phospholipids; and (c) the degree of saturation of the fatty acids. It is therefore suggested that alterations of this kind may occur in serum lipids during spontaneous mammary tumor development in C3H mice.