Elliott Berlin

Changes in state of water in proteinaceous systems.

Abstract When lyophilized preparations of β-lactoglobulin, bovine serum albumin, and calfskin collagen sorbed at least 0.18 gm H2O per gram of dried protein, it was observed through differential scanning calorimetry (DSC) that the heat of vaporization of the sorbed water was 80–125 cal/gm higher than the ΔHv of liquid water. When less H2O is sorbed, at lower values of P P o for the sorbed water was equivalent to that of free water. These differences in strength of H2O-protein binding may be attributed to the availability of protein surfaces or suitable H2O binding sites. At the higher moisture levels the solid protein matrix has become swollen and possibly conformational changes have occurred in the protein molecules permitting more H2O-surface contacts and the formation of an “icelike” structure. Accordingly extensive water binding was observed in completely wet systems by measuring the heat of fusion of the water associated with wet pellets of ultracentrifugal casein. Water bound in an “ice” structure will not freeze on cooling to low temperatures (−70°C) and may therefore be assessed through DSC. Such bound water was found to correspond to 50%–60% of the dry weight of the protein present.

Calorimetric measurement of the heat of desorption of water vapor from amorphous and crystalline lactose

Abstract The heat required to release and vaporize bound H 2 O from crystalline α-lactose monohydrate and from lactose glass, as determined by differential scanning calorimetry is 12.3±0.7 and 10.8±0.5 kcal·mole −1 of H 2 O, respectively. Water vapor sorption by anhydrous α-lactose leads to the formation of the α-monohydrate. The isotherm, obtained gravimetrically for this process is Langmuir type. β-Lactose is completely non-hygroscopic below 97% relative humidity. Thereafter, it sorbs H 2 O rapidly to form a concentrated solution wherein the lactose is capable of mutarotation. Densites of lactose forms determined pycnometrically by helium displacement are: 1.535 g/cm 3 for α-lactose·H 2 O; 1.547 g/cm 3 for anhydrous α-lactose; and 1.576 g/cm 3 for β-lactose.

Effect of dietary fat on the fluidity of platelet membranes

Dietary fat type was reflected in the phospholipid fatty acid composition of the plasma membrane of rabbit platelets and apparently controlled the fluidity of these membranes. Rabbits were maintained for 6 months on diets that varied in stearic and polyunsaturated fatty acids and thus had different potentials for thrombosis. Microviscosities at 37 C, calculated from the anisotropy of fluorescence from the probe 1,6-diphenyl-1,3,5-hexatriene, were 3.5, 3.4, 2.8 and 2.2. poise for platelet membranes isolated from rabbits whose only source of dietary fat was cocoa butter, milkfat, coconut oil, or corn oil, respectively. The relative fluidities of the membrane isolates were correlated with the polyunsaturated fatty acid contents of the membrane phospholipids.

Platelet membrane fluidity and aggregation of rabbit platelets

Aggregation of rabbit platelets from citrated plasma in response to ADP was directly correlated with platelet plasma membrane fluidity as determined by fluorescence depolarization measurements with the probe diphenylhexatriene. Rabbits were maintained for periods of 200 and 400 days on potentially hyperlipidemic diets (20% fat by weight) with varying levels of saturated and polyunsaturated fatty acids. Dietary variations were effective in modulating the mole percentage distribution patterns of the platelet phospholipid fatty acids. The major chemical control of membrane fluidity was the actual mass of unsaturated lipid in the cells and not simply the relative percentage distributions of such unsaturated fatty acids. Substantially higher phospholipid/protein ratios were observed upon analysis of platelets and platelet membranes from rabbits after 200- than after 400-day diet periods. Accordingly lipid structures were significantly more fluid in either whole platelets or membrane isolates at the end of the shorter diet period. The observations pertaining to the extent of aggregation and membrane fluidity are in consonance with the general role of membrane fluidity in controlling biological activity and support the concept that platelet aggregation is a membrane-associated phenomenon.

Effects of omega-3 fatty acid and vitamin E supplementation on erythrocyte membrane fluidity, tocopherols, insulin binding, and lipid composition in adult men

Abstract Dietary supplementation with an omega-3 fatty acid preparation (fish oil) together with pharmacologic doses of vitamin E increased both insulin binding and membrane fluidity in erythrocytes from human adult males. Supplementation with fish oil alone induced significant increases in the α- and γ-tocopherol contents of the red blood cell membranes. Forty healthy men were given controlled diets and supplements, which together provided 40% of energy from fat (polyunsaturated:monosaturated:saturated ratio of 0.8:1:1), 360 mg cholesterol/day, and a minimum of 22 mg α-tocopherol (αT)/day for three successive experimental periods of 10, 10, and 8 weeks, during which they were given capsules containing 15 g of a placebo oil/day, 15 g fish oil concentrate (FOC)/day, and 15 g fish oil + 200 IU αT (FOC + αT)/day, respectively. Erythrocyte ghost insulin binding (IB) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence-determined fluidity were significantly increased following the FOC + αT period, however FOC alone had no effect. At the end of each experimental period, IB values, as percentage bound/100 μg ghost protein at 4° C, were 0.96, 0.91, and 1.35, and DPH steady state fluorescence anisotropies were 0.311, 0.303, and 0.296, at 4° C, respectively. Small but statistically significant decreases in fluorescence lifetimes further indicated increased fluidity. FOC supplementation resulted in significantly lower membrane cholesterol:phospholipid ratios and increased membrane tocopherols despite daily vitamin E consumption of only 22 mg as in the placebo period. Membrane incorporation of n-3 fatty acids was, however, limited. Thus, dietary polyunsaturated fatty acids exerted substantial effects on erythrocyte membranes by affecting membrane contents of lipid molecules other than the fatty acids.

Dietary linoleate increases fluidity and influences chemical composition of plasma low density lipoprotein in adult men

Dietary linoleate was effective to increase LDL fluidity in adult men but did not significantly influence VLDL or HDL fluidities. Lipoproteins were isolated ultracentrifugally from plasma of sixteen healthy, free living male volunteers consuming controlled diets formulated from typical U.S.A. foods to have 35 energy % fat with 10 g (diet L) or 30 g (diet H) linoleate per day, 30-50 g saturated fatty acids/day and the balance mainly monounsaturated fatty acids. Calculated cholesterol intakes were 500 mg/day at each calorie level. Changes in LDL fluidity were detected as differences in diphenylhexatriene (DPH) fluorescence polarization upon crossover between the two controlled diets. Thermotropic measurement of DPH fluorescence anisotropy and compositional analyses indicated that LDL and HDL fluidities were dependent upon phospholipid and triacylglycerol concentrations, respectively, and were modulated by the presence of cholesteryl esters. Fatty acid analyses of the major lipid classes of the isolated lipoproteins indicated that changes, upon diet crossover, in DPH fluorescence anisotropy, were a linear function of the incremental change in LDL phospholipid linoleate. The fluorescent probe described an environment corresponding to the fatty acyl moieties of the phospholipids on the LDL periphery, which composition is apparently under dietary control. It is suggested that the diet induced fluidity changes may affect the conformation of the apoprotein moiety on the LDL surface and thus the potential for LDL interaction with cellular LDL receptors.

Heart and liver fatty acid composition and vitamin E content in miniature swie fed diets containing corn and menhaden oils

Female miniature swine, 4-11 yr, were fed 15% fat diets containing n-3 and/or n-6 polyunsaturated fat for 6 months, at 1.95 g fat/kg body weight. Liver lipids from menhaden oil-fed minipigs were elevated in the n-3 fatty acids: 20:5, 22:5 and 22:6, but heart lipids only in 20:5 and 22:6. Liver cell plasma membrane was elevated in 20:5, 22:5 and 22:6 and lowered in the n-6 acids 18:2 and 20:4 in menhaden oil-fed animals, to a greater extent than in the total tissue lipids. Liver alpha-tocopherol tended to decrease upon feeding menhaden oil, but heart alpha-tocopherol concentrations were not affected.

Influence of saturated and unsaturated fats on platelet fatty acids in cholesterol-fed rabbits

Feeding natural fats varying in contents of palmitate (16:0), stearate (18:0), oleate (18:1), and linoleate (18:2) to rabbits resulted in modulation of platelet phospholipid fatty acyl composition. Rabbits were fed high fat semipurified diets containing 2% corn oil (CO) + 18% CO, cocoa butter (CB) or milkfat (M) for periods of up to 300 d. Platelet phospholipid linoleate contents corresponded to diet levels with 18:2 highest in CO-fed rabbits and following the sequence CO greater than CB greater than M. Stearate was highest in CB-fed rabbits, corresponding to high 18:0 levels in CB, but palmitate levels were not affected by diet. Both CB and M-fed rabbits were higher than CO-fed rabbits in oleate. Though CO is highest in 18:2, the accepted 20:4 precursor, arachidonate was highest in M-fed rabbits. Adding cholesterol (0.2%) to the diets did not affect platelet phospholipid fatty acyl composition except to elevate 20:4 in M-fed rabbits. CO-fed rabbits showed uniquely high levels of tetracosadienoate (24:2). Fatty acyl composition data were essentially constant between 200 and 300 d on diet. Phospholipid fatty acyl unsaturation was apparently homeostatically controlled as mole percent unsaturate to saturate ratios were independent of diet. The observed homeostasis resulted in minimal diet influences on platelet membrane fluidity and ADP or collagen stimulated platelet aggregation. Platelet fluidity, determined by fluorescence polarization, was a function of oleate and linoleate contents of the cells. Cholesterol feeding generally lowered platelet fluidity and altered the dependence of fluidity on fatty acyl composition.

Fatty acid modification of membrane fluidity in Chinese hamster ovary (TR715-19) cells.

Dietary saturated fatty acids, especially lauric (12:0), myristic (14:0) and palmitic (16:0) acids, which are hypercholesterolemic, influence cell membrane fatty acid composition and affect LDL receptor function. When membrane phospholipid fatty acids in Chinese hamster ovary cells, containing the human LDL receptor, were modified (Hannah J. S. et al., 1995 Metabolism 44, 1428-1434), LDL receptor function was affected, but correlations with DPH-determined membrane fluidity were weak. The role of fluidity in various membrane domains with respect to the LDL receptor is examined here. Membrane fluidity was assessed by measuring steady-state fluorescence polarization of diphenylhexatriene (DPH) and its polar propionic acid (DPH-PA) and trimethylammonium (TMA-DPH) derivatives from 38 to 4 degrees C in fatty acid modified Chinese hamster ovary cells. Fatty acid changes modulated mid-bilayer fluidity as determined with DPH, but fluidity in phospholipid headgroup domains, assessed with DPH-PA and TMA-DPH, was independent of fatty acyl composition. The DPH fluidity was related to membrane unsaturation (P < 0.02), oleate contents (P < 0.009) in particular, but inversely related (P < 0.0002) to the longer chain (> or = 20 C atoms) unsaturated fatty acids with from four to six double bonds. The LDL binding was independent of fluidity, but there were weak relations between LDL internalization and DPH-PA anisotropy and between LDL degradation and TMA-DPH anisotropy. It was concluded that LDL binding was not related to mid-bilayer fluidity, but the results with the polar probes suggest a role of fluidity in modulating vertical displacement of the LDL/LDL receptor complex across the plasma membrane.

Influence of dietary fats on the fluidity of the lipid domains of rabbit plasma lipoproteins

The effects of dietary stearic and other saturated fatty acids on the fluidity of the plasma lipoproteins were assessed with fluorescence polarization techniques. Rabbits were maintained on diets containing either cocoa butter, milkfat, coconut oil, or corn oil as the only source of fat. Microviscosities eta, of the lipid regions of plasma very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) were determined by measuring the anisotropy of fluorescence from the probe 1,6-diphenyl-1,3,5-hexatriene. The microviscosity values followed the sequence eta HDL greater than eta LDL greater than eta VLDL when the lipoproteins were isolated from the plasma of rabbits fed cocoa butter, milkfat, or corn oil, HDL and LDL consist of an invariant phase in the temperature range 0--50 degrees C regardless of diet. VLDL from rabbits fed milkfat, corn oil, or cocoa butter displayed monophasic behavior in the same range, while VLDL, from rabbits fed coconut oil showed a phase transition at 31.9 +/- 3.7 degrees C. Lipoproteins were less fluid in fasted than in non-fasted rabbits and VLDL and LDL from fasted milkfat-fed rabbits showed phase transitions. Despite the fatty acid compositions of the dietary fats, VLDL and LDL were more fluid from rabbits fed cocoa butter than from rabbits fed corn oil; apparently metabolism influences microviscosity.