Phyllis G. Kliman

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.

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.

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.

Effect of dietary corn, coconut, and menhaden oils on lipoprotein, liver, and heart membrane composition in the hypercholesterolemic rabbit

Abstract The relative capacities of n-3 and n-6 polyunsaturated fatty acid-containing diets (PUFA) to modify chemical composition in plasma lipoproteins, liver membrane, and heart phospholipids and to thereby modulate lipoprotein and membrane fluidity were studied in the rabbit. Stock diet-fed New Zealand rabbits were made hypercholesterolemic by feeding a casein-based, semi-purified diet containing coconut oil. Subsequent replacement of the coconut oil with corn or menhaden oil caused reduction or elevation, respectively, of blood cholesterol levels. Very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) became progressively less fluid following transfer from the PUFA containing low fat stock diet to the coconut oil diet. Subsequent corn oil feeding induced significant increases in VLDL and LDL, but not HDL, fluidities. Menhaden oil feeding did not affect lipoprotein fluidity. Liver membranes were less fluid in corn oil-fed and menhaden oil-fed than in coconut oil-fed rabbits. Despite these differences in lipoprotein and membrane fluidity the amount of rabbit 125I-LDL capable of binding to liver membranes from the animals was not affected by the dietary fat modifications. Fatty acyl compositions were affected by the diets. Heart and liver phospholipids showed extensive incorporation of n-3 fatty acids and deletion of linoleate in the menhaden oil-fed animals. Dietary effects on lipoprotein phospholipid fatty acids were statistically significant but not as pronounced as in these tissues.

Influence of age and sex on composition and lipid fluidity in miniature swine plasma lipoproteins

Age- and sex-related differences were observed in the plasma cholesterol level, the plasma concentrations of certain lipoprotein components, and the HDL lipid phase fluidity in miniature swine from post-weaning (6 weeks) through puberty (6 months), maturity (2-6 years), and old age (10-12 years). Age effects were more dominant in the males, with VLDL protein; LDL protein, triacylglycerol, and phospholipid; and HDL triacylglycerol, phospholipid, cholesterol, and polyunsaturated fatty acids showing statistically significant negative correlations with age. These effects were not observed in females. HDL cholesterol was positively correlated with age in females. Total plasma cholesterol decreased with age in males only, but plasma triacylglycerol was not influenced by age in either sex. Higher concentrations of all lipoprotein lipids were observed in the female minipigs regardless of age. HDL lipids became less fluid with age in the males alone suggesting a physical chemical basis for the lower incidence of heart disease among females. The more fluid HDL circulating in the female may be more capable of mobilizing peripheral tissue cholesterol for catabolism thus protecting her from developing atherosclerotic lesions.

Effect of sorbed water on the heat capacity of crystalline proteins

Abstract Heat capacity measurements by differential scanning calorimetry (DSC), performed with anhydrous samples of ovalbumin, yielded a value for Cp of 0.267 ±0.033 cal/g/°C at 12°C; and the extrapolation of data from hydrated samples to zero water content yielded a similar value for Cp of 0.244±0.011 cal/g/°C. The heat capacity of anhydrous β-lactoglobulin is 0.273 ±0.027cal/g/°C, and the value obtained by extrapolation of data for hydrated samples is 0.284±0.019 cal/g/°C. A linear relation between specific heat and moisture content was observed with the hydrated samples which contained 0.03–0.21 g sorbed water per gram of protein. No temperature dependence of specific heat was observed in the interval scanned (0–25°C). The computed, apparent, partial specific heats of the proteins are 0.245 ±0.010 cal/g/°C for ovalbumin and 0.283±0.02 cal/g/°C for β-lactoglobulin; and the partial specific heat of the sorbate is 1209±0.103 cal/g/°C for water sorbed by ovalbumin, and 0.947±0.137 cal/g/°C for water sorbed by β-lactoglobulin.

Effect of dietary cholesterol on the lipoprotein profile and binding of radioiodinated lipoproteins to hepatic membranes in the cockerel (gallus domesticus)

1. Cockerels fed a cholesterol-supplemented diet experienced a marked elevation of lipoprotein particles of density less than or equal to 1.006 g/ml (VLDL) and a diminution of lipoprotein particles of density 1.02-1.05 g/ml (LDL). 2. Unlike VLDL of some cholesterol-fed animals, cholesterol-fed cockerel VLDL did not display beta-mobility on agarose gel electrophoresis. 3. [125I]LDL and [125I]HDL binding to cockerel liver membranes was not affected by cholesterol feeding. 4. Different lipoprotein types appear to bind to a common site on cockerel liver membranes. 5. The results suggest that liver cells of cockerels may not possess LDL binding sites that are analogous to those of mammalian species.

Blood Risk Factor Metabolites Associated with Heart Disease and Myocardial Fatty Acids in Copper-Deficient Male and Female Rats

Abstract Intact and castrated males and intact and ovariectomized female rats were fed a copper-deficient diet in order to establish whether the protection provided in females against cardiovascular pathology and mortality is due to endogenous sex hormones, and different levels of blood lipids and/or myocardial fatty acids. Seventy-three male and female rats were assigned to a copper-deficient diet (0.6 μg of copper/g diet) containing 62% fructose for 8 weeks. Twelve of the male rats underwent castration and 12 of the females were ovariectomized. All animals exhibited high levels of plasma cholesterol, triglycerides, and uric acid, which were neither affected by the sex of the rat nor by the surgical treatment. The composition of fatty acids of the myocardium was similar in males and females. Except for those animals that were sacrificed by us, all other male rats died of heart pathology. In contrast, none of the female rats exhibited heart pathology and none died of the deficiency. It is suggested that heart pathology and mortality in copper deficiency are sex related and not due to high levels of plasma cholesterol, triglycerides, and uric acid or to differences in myocardial fatty acid composition.

Effect of Dehydration on the Specific Heat of Cheese Whey

Differential scanning calorimetry was used to determine the specific heat of cheddar cheese whey as a function of water content and thereby provide fundamental data useful for the further development of dried whey products. The specific heat of fluid cheddar cheese whey, which contains 7% solids, was 0.951 ± .036 cal/g/°C at 12°C. A linear relationship was maintained between specific heat and moisture content when dried whey solids were re-hydrated to moisture levels between 3 and 93% H2O. The apparent partial specific heat of the whey solids was 0.328 cal/g/°C and that of the water was 0.995 cal/g/°C, a value close to that of bulk water. An inflection, however, was noted in the relation between specific heat and water content at 50% H2O when the specific heat data were obtained with concentrated whey samples prepared by evaporation of water from fluid whey. These data yielded apparent partial specific heat values for water of 0.966 cal/g/°C above 50% H2O and 1.203 cal/g/°C below 50% H2O. Apparently the water is in a more structured form in concentrated systems provided that the solids are initially fully hydrated. This conforms to the concept that a critical amount of water must be present in a proteinaceous system for the water to be held in a quasi-solid or “icelike” structure.