Rachel B. Shireman

LOW FAT-MONOUNSATURATED RICH DIETS CONTAINING HIGH-OLEIC PEANUTS IMPROVE SERUM LIPOPROTEIN PROFILES

Postmenopausal hypercholesterolemic women are at risk for cardiovascular disease and are encouraged to follow low-fat (LF) (≤30% energy) diets. However, these diets may have undesirable effects on high density lipoprotein cholesterol (HDL-C), apolipoprotein A-I (apo A-I) and triglycerides, whereas diets high in monounsaturated fats do not. Twenty postmenopausal hypercholesterolemic women previously consuming high-fat diets (34% energy) were placed on a low fat-monounsaturated rich diet (LFMR: 26%, 14% energy, respectively) for 6 mon. Sixteen women already eating LF diets (24% energy) were also followed to monitor variations in serum lipids due to seasonal variations. Twenty-five women successfully completed the study (LFMR=12, LF=13). Serum cholesterol decreased 10% (264 to 238 mg/dL, P≤0.01) and low density lipoprotein cholesterol (LDL-C) decreased 12% (182 to 161 mg/dL, P≤0.01) in the LFMR group, but did not change in the LF group. The reduction in serum cholesterol in the LFMR group was greater than estimated by predictive formulas. Serum triglycerides and apo A-I did not change in the LFMR group. A modest decrease in HDL-C, HDL3-C, and apolipoprotein B (apo B) occurred in both groups, but only the LFMR group showed a trend toward beneficial changes in LDL-C/HDL-C and apo A-I/apo B ratios. Overall, the LFMR diet was well tolerated and resulted in an improved serum lipid and apolipoprotein profile.

The absence of a role for the carbohydrate moiety in the binding of apolipoprotein B to the low density lipoprotein receptor

The binding of low density lipoprotein (LDL) to fibroblasts occurs through apolipoprotein B, a glycoprotein. The role of the carbohydrate in binding was assessed in two ways: (1) LDL, freed of sialic acid and most of the glucosamine and hexoses by digestion with a mixture of glycosidases, bound to fibroblasts as does native LDL. (2) The glycopeptides liberated from apoprotein B by trypsin and pronase failed to inhibit LDL binding to fibroblasts. Apparently the carbohydrate moiety of LDL does not interact with the plasma membrane receptor.

Low-fat, monounsaturate-rich diets reduce susceptibility of low density lipoproteins to peroxidation ex vivo

Oxidative modification of low density lipoprotein (LDL) plays an important role in the process of atherosclerosis. The susceptibility of LDL to oxidation and the amount of peroxidation products formed are influenced by the lipoprotein content of 18∶1 n−9, 18∶2n−6, and the 18∶2n−6/18∶1n−9 ratio, which is dependent in part on dietary fatty acids. The purpose of this study was to determine if changing from a typical American diet to a low-fat, monousaturate-rich diet (LFMR) would result in favorable alterations in the fatty acid composition and oxidative profile of LDL in hypercholesterolemic individuals. Free-living postmennopausal hypercholesterolemic women who routinely consumed a diet moderately high in total fat and total saturates (34 and 11%, respectively) followed an LFMR diet (26% fat, 6% saturated fat, and 14% monounsaturated fat) for 6 mon. Sixteen postmenopausal hypercholesterolemic women already following standard low-fat (LF) diets acted as a control for seasonal variations in serum lipids. LDL from randomly selected subjects (LF n=6, LFMR n=5) was evaluated. LFMR diets resulted in LDL with increased concentrations and percentages of 18∶1n−9, reduced 18∶2n−6/18∶1n−9 ratio, and lower percentages of 18∶2n−6. No significant changes in LDL fatty acids occurred in the LF group. Conjugated diene lag time increased in both groups during copper-induced in vitro oxidation. Only the LFMR group experienced an increase in lipid peroxide lag time and a decrease in lipid peroxide formation. The LFMR diet was well tolerated and may be of therapeutic value in the treatment of hypercholesterolemia.

Uptake of [3h]cholesterol from low density lipoprotein by cultured human fibroblasts

The uptake of [3H]cholesterol from low density lipoprotein (LDL) was studied in LDL receptor-positive and receptor-negative human fibroblasts. In both cell lines the uptake depended upon temperature, time of incubation and the concentration of LDL in the medium. Although the incorporation of 125I-labeled LDL was minimal after 2 h of incubation in the receptor-negative (homozygous familial hypercholesterolemia, FH) cells, the uptake of [3H]cholesterol was only slightly less than that of the receptor-positive (WI-38) cells. With longer periods of incubation, a larger difference in labeled cholesterol incorporation was observed; this appeared to be due to a continued accumulation of the steroid in the WI-38 cells. After 8 and 24 h of incubation, some of the [3H]cholesterol was present as the ester in the WI-38 cells, but not the FH cells. Modified (reduced and methylated) LDL did not enter WI-38 cells by the receptor-mediated pathway during 2 h of incubation, as indicated by 125I uptake. [3H]Cholesterol uptake, however, was not significantly different from modified and unmodified LDL. While experiments indicated that significant amounts of cholesterol moved rapidly from LDL to cultured cells with a dependence on time and LDL concentration, no increase in total cell cholesterol was detected in either cell line. FH cells contained less total cholesterol and had a higher 3H specific activity than the WI-38 cells. These data suggest that there may be important mechanisms in addition to the LDL pathway for the movement of lipids into cells.

Microplate methods for determination of serum cholesterol, high density lipoprotein cholesterol, triglyceride and apolipoproteins

Microtiter plate methods were developed for the enzymatic determination of serum total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and triglyceride (TG), and for the turbidometric determination of apolipoproteins. The micromethods resulted in accurate, precise values that were in good agreement with the conventional spectrophotometric assays. The coefficient of variation for TC determinations was 4.5% or less and bias was 5% or less. The lipid micromethod assays are sensitive to 10 mg/dL or less, and the apolipoprotein assay to 1 mg/dL. Less than 100 μL of serum suffices for TC, TG and apoprotein assays; HDL-C requires an additional 100 μL of serum. Advantages of the micromethods include reductions in assay time and in the amount of reagents required.

Uptake of 2,3,7,8-tetrachlorodibenzo-p-dioxin from plasma lipoproteins by cultured human fibroblasts

Tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to human plasma in vitro associated with the plasma lipoproteins. The effects of plasma and lipoproteins on cellular uptake of dioxin were studied using normal human skin fibroblasts and mutant fibroblasts from a patient with homozygous familial hypercholesterolemia. The latter cells lack the normal cell membrane receptor for low density lipoprotein (LDL). The time- and temperature-dependent cellular uptake of [3H]dioxin was greatest from LDL, intermediate from high density lipoprotein (HDL) and least from serum. A significantly greater uptake from LDL by the normal cells compared to the mutant cells indicated the involvement of the LDL receptor-mediated pathway. Concentration-dependent studies indicated that the cellular uptake at 37 degrees C of [3H]dioxin varied linearly with dioxin concentration at constant LDL concentration. Thin-layer chromatography (TLC) showed that conversion to more polar compounds may have occurred after 24-h incubation with cells. [3H]Dioxin could be removed from cells efficiently by incubation with 20% serum greater than HDL greater than LDL. Since the vehicle of delivery may influence subsequent location and metabolism of this compound in cells, it is concluded that the physiologic vehicles (either serum- or LDL-associated dioxin), rather than organic solvents, should be used in experiments with cultured cells or perfused organs.

Association of aflatoxin B1 with plasma components in vitro

The in vitro association of [3H]aflatoxin B1 with plasma from man, pig and rat was compared. The uptake of aflatoxin B1 into plasma was rapid and did not differ between species. Its association with lipoprotein fractions was minimal when incubated with whole plasma; column chromatography of plasma from all 3 species resulted in the elution of the aflatoxin B1 with the low Mr plasma proteins. It is concluded that although aflatoxin B1 is a hydrophobic compound, it does not partition into the plasma lipoproteins and that its in vitro uptake into plasma does not differ among the 3 species studied. Plasma transport is probably not one of the factors determining species specificity for carcinogenesis by aflatoxin B1.

Low density lipoprotein receptor regulation: Kinetic models

The macromolecular species distribution in a receptor-mediated endocytotic pathway was computer simulated based on kinetic data reported in the literature. In the proposed model, the rapidity with which the recycled receptor is shuttled to the cell surface is indicated by the magnitude of k-3, the shuttling constant. The magnitude of k-3 will vary with the experimental conditions, but when this value is large, the internalized receptor is shuttled back to the cell surface with a traverse time of 14 min. Under steady-state conditions, after the cells have been incubated in the presence of LDL for 5 h (M.S. Brown and J.L. Goldstein, Cell 9 (1976) 663), the time required for a receptor to traverse the entire endocytotic pathway is 52 min. Our simulation suggests that normal LDL binding in such a short-term experiment may be independent of receptor synthesis. Thus, the degradation of LDL and resultant build-up of cholesterol would have no apparent inhibitory effect on the down-regulation of receptor synthesis.

Removal of benzo[a]pyrene from cells by various components of medium

Benzo[a]pyrene is removed from cells in culture by various additions to the medium. During post-treatment incubation, WI-38 fibroblasts were incubated with a low density, very low density and high density lipoproteins, delipidated or complete serum or plasma, or serum albumin. The time course of removal was followed. Increasing concentrations of lipoproteins resulted in increasing percentages of removal of benzo[a]pyrene from cell membranes. The most efficient addition was 10% complete human plasma. These results indicate that benzo[a]pyrene remains at or close to the plasma membrane for at least several hours and readily redistributes to medium components.

[14C]Acetate incorporation by cultured normal, familial hypercholesterolemia and Down's syndrome fibroblasts☆

Fibroblasts from patients with homozygous familial hypercholesterolemia (FH), a disease characterized by accelerated atherogenesis, are known to lack functional low-density-lipoprotein receptors, which ultimately results in increased cholesterol biosynthesis in the cultured cells. [14C]Acetate incorporation in these cells was compared to that of normal fibroblasts and to fibroblasts from patients with Downs syndrome, a disease in which atherosclerosis is rare. Total [14C]acetate incorporation did not differ significantly between normal and Downs fibroblasts, nor did its partitioning into the hexane-extractable and aqueous fractions of the cell hydrolysates. [14C]Acetate incorporation was much greater in FH cells in both the aqueous and hexane-extractable fractions. Preincubation in fetal bovine serum increased acetate incorporation only by FH cells, while 50 micrograms low-density lipoprotein/ml medium depressed acetate incorporation in all three groups. A C27 sterol, identified by gas chromatography-mass spectrometry as a probable isomer of cholesterol, was present in small amounts in FH fibroblasts, but was not detectable in the normal or Downs cells. The absolute amounts of [14C]acetate incorporated into the non-sterol lipids were greater in the FH fibroblasts, indicating that these cells may have to synthesize, in addition to cholesterol, other required cellular lipids which are delivered to the normal cells by low-density lipoproteins.