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 18:1n-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, monounsaturate-rich diet (LFMR) would result in favorable alterations in the fatty acid composition and oxidative profile of LDL in hypercholesterolemic individuals. Free-living postmenopausal 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.

Effects of age, physical training, and physical fitness on coronary heart disease risk factors in older track athletes at twenty-year follow-up.

OBJECTIVE: To compare current coronary heart disease (CHD) risk factor values in older athletes with mid-life measures and to examine the associations between changes in CHD risk factors with aging, physical training, and physical fitness. DESIGN: Prospective study with three longitudinal evaluation points: initial (T1), 10-year (T2), and 20-year (T3). Subjects were selected because of their elite status in Masters track competition. SETTING: University and medical center laboratories. PARTICIPANTS: Participants were 60 to 92 years of age and included 21 of the initial 27 subjects. At T3, subjects were divided into three groups, based on physical activity levels: high intensity (H), remained elite in national and international competition (n = 9); moderate intensity (M) continued frequent rigorous endurance training but rarely competed (n = 10); and low intensity (L) greatly reduced their training volume and intensity (n = 2). MEASUREMENTS: Smoking history; family history of coronary or cerebrovascular disease; resting blood pressure; resting electrocardiogram (ECG); serum total cholesterol, plasma glucose; body weight, % body fat, body mass index, waist:hip ratio; training pace and mileage; maximal oxygen consumption VO2 max). MAIN RESULTS: Several risk factors (smoking, diabetes, obesity) were never present, and the prevalence of other risk factors (family history of cardiovascular disease, abnormal resting ECG) remained low through T3 (< or = 14% of subjects). Mean systolic and diastolic blood pressure remained low without medication, but diastolic blood pressure measurements had the greatest redistribution between evaluation periods of any risk factor (r = .16, P = .479, T1 to T2). Mean total cholesterol was lower at T2 (-13%, P = .005) and T3 (-14%, P = .019) compared with T1. Change in VO2 max was correlated with changes in body weight (r = -.44, P = .048) and % fat (r = -.52, P = .015) from T1 to T2, whereas age was correlated to changes in systolic blood pressure (r = -.61, P = .003) and total cholesterol (r = -.49, P = .023) from T2 to T3. CONCLUSIONS: The prevalence of CHD risk factors remained low, and mean risk factor values remained low and generally stable in older athletes who had maintained habitual exercise training.

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) (< or = 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 men. 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 < or = 0.01) and low density lipoprotein cholesterol (LDL-C) decreased 12% (182 to 161 mg/dL, P < or = 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-Vapo B ratios. Overall, the LFMR diet was well tolerated and resulted in an improved serum lipid and apolipoprotein profile.

Endurance exercise training improves body composition and plasma insulin responses in 70- to 79-year-old men and women.

Forty-two men and women aged 70 to 79 years were studied to assess the effects of 6 months of endurance or resistance training and subsequent cessation of training on glucose tolerance, plasma insulin responses, serum triglyceride and cholesterol levels, and plasma dehydroepiandrosterone (DHEA) levels. The endurance training group (n = 16) exercised at 75% to 85% heart rate reserve for 35 to 45 minutes three times per week; the resistance training group (n = 17) completed one set of eight to 12 repetitions on 10 Nautilus machines three times per week. No significant changes in any variables occurred in a control group (n = 9). Maximal oxygen consumption (VO2max) increased by 20% with endurance training, but did not change with resistance training. Upper- and lower-body strength increased in the resistance training group, but did not change with endurance training. Neither group changed their body weight with training, but the endurance training group elicited a significant reduction in their sum of seven skinfolds and percent body fat. Neither group altered their glucose tolerance with training; however, the endurance training group had lower plasma insulin responses after training compared with the other two groups. Serum lipid and plasma DHEA levels did not change in either the endurance or resistance training groups. Ten days of no exercise following training did not significantly alter body weight or composition, glucose tolerance, plasma insulin responses, or plasma DHEA levels in either the endurance training (n = 10) or resistance training (n = 14) group.(ABSTRACT TRUNCATED AT 250 WORDS)

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 microL of serum suffices for TC, TG and apoprotein assays; HDL-C requires an additional 100 microL of serum. Advantages of the micromethods include reductions in assay time and in the amount of reagents required.

The effect of bile salts on carbonic anhydrase.

Bile salts are potent inhibitors of bovine carbonic anhydrase and human carbonic anhydrase I and human carbonic anhydrase II. To further characterize the binding of bile salts to carbonic anhydrase, rate constants for the CO2 hydration reaction in the presence of deoxycholate, cholate, glycocholate and taurocholate were determined using stop-flow experiments. Values for the Michaelis-Menton dissociation constant for bovine carbonic anhydrase, human carbonic anhydrase I and human carbonic anhydrase II were found to be 5.2, 9.2 and 13.2 mmol/L, respectively. The inhibition constant values for the various bile salts tested ranged from 0.1 to 1 mmol/L for bovine carbonic anhydrase, 1.6 to 2.4 mmol/L for human carbonic anhydrase I and 0.09 to 0.7 mmol/L for human carbonic anhydrase II. Our results suggest a mechanism of noncompetitive carbonic anhydrase inhibition for bile salts. Bile-salt binding to carbonic anhydrases as measured by scanning molecular sieve chromatography resulted in an increase in partition radius, molecular volume and surface area. The partition radius increased from 24 A to 28 A in the presence of 2.5 mmol/L sodium deoxycholate at critical micelle concentration. As determined by sedimentation equilibrium measurements, approximately 1 gm of carbonic anhydrase will bind 0.03 gm of deoxycholate, suggesting three to six binding sites for bile salt on the carbonic anhydrase molecule. The conformational changes and inhibition of carbonic anhydrases resulting from bile-salt binding may be important to the regulation of enzymatic activity in tissues along the enterohepatic circulation; by limiting bicarbonate availability this interaction may also contribute to the metabolic derangements seen in patients with cholestatic liver disease.

Carnitine status and lipid utilization in neonatal piglets fed diets low in carnitine.

Forty-eight newborn piglets were assigned to dietary regimens to examine carnitine status and lipid utilization by piglets nursing sows (Diet 1), fed a high-carnitine (1507 mumol carnitine/kg dry matter) casein-whey formula (Diet 2) or fed a low-carnitine (35 mumol carnitine/kg dry matter) egg white protein formula (Diet 3). Four piglets were killed at birth, and four per dietary group were killed at 3, 7, 14 and 21 d of age. Piglets fed Diet 3 had lower (P less than 0.01) carnitine in plasma and liver by d 7 and carnitine remained lower to d 21. Heart carnitine tended to be lower in piglets fed Diet 3, but the differences were not significant. Longissimus muscle carnitine was not affected. In vitro palmitate oxidation was lower (P less than 0.05) on d 7 in the liver from piglets fed Diet 3 but not on d 3, 14 and 21. Low dietary carnitine did not affect in vitro liver beta-hydroxybutyrate production or oxidation of palmitate by longissimus muscle. Plasma glucose and nonesterified fatty acids were higher (P less than 0.05) in piglets nursing sows than those fed Diets 2 or 3. Piglets receiving Diet 3 had reduced carnitine in plasma and certain tissues, and liver lipid oxidation was depressed on d 7 of age, compared with those receiving the other two diets. Feeding the low carnitine diet did not alter glucose status or plasma lipids and ketones.

Differences in [14C]glycerol utilization in normal and familial hypercholesterolemic fibroblasts.

It is known that cultured fibroblasts from familial hypercholesterolemia (FH) patients lack the normal cell receptor for low density lipoprotein (LDL) and that the absence of receptor-mediated transport of LDL cholesterol into these cells results in increased cellular synthesis of cholesterol. After 20 h perincubation in lipid-free medium, cultured FH fibroblasts incorporated significantly greater amounts of [14C]glycerol into cellular lipids than did normal fibroblasts. Relative to the control medium which contained only bovine serum albumin (BSA), preincubation with 5% fetal bovine serum or 50 micrograms LDL/ml decreased [14C]glycerol incorporation by both cell types. FH cells utilized more [14C]glycerol for phospholipid synthesis and less for triglyceride synthesis than normal cells. This study indicates that LDL may be important in the transport of glycerides, as well as cholesterol, to cells.

Effect of threonine and glycine concentrations on threonine aldolase activity of yogurt microorganisms during growth in a modified milk prepared by ultrafiltration.

To evaluate the combined effects of threonine and glycine concentrations during growth on threonine aldolase activity (EC 2.1.2.1) of yogurt microorganisms, Streptococcus thermophilus and Lactobacillus bulgaricus, a modified milk growth medium was prepared using UF to deplete the free amino acid level. Threonine and glycine were added according to a 2x2x2 factorial design at 5 or 195 microg.ml(-1) along with a standard amino acid mixture. Acetaldehyde production and threonine aldolase activity were evaluated utilizing headspace gas chromatography. Results showed that threonine and glycine concentrations did not affect growth or titratable acidity. The high concentration of threonine in combination with low glycine in the growth medium resulted in increased acetaldehyde synthesis by both microorganisms. Conversely, high glycine with low threonine decreased acetaldehyde synthesis. High threonine and low glycine increased threonine aldolase activity of cell-free extracts from S. thermophilus and L. bulgaricus, whereas high glycine and low threonine reduced threonine aldolase activity of both microorganisms.

Lipoprotein-mediated transfer of 2,4,5,2',4',5'-hexachlorobiphenyl into cultured human cells.

1. The very low density, low density and high density lipoproteins (VLDL, LDL, HDL), centrifugally separated from human plasma treated with 2,4,5,2',4',5'-hexachloro[14C]biphenyl (14C-HCB) contained approximately 50% of the 14C-HCB. 2. Normal skin fibroblasts were incubated at 4 degrees C for 37 degrees C for varying times with medium containing 10% serum, LDL or HDL labelled with 14C-HCB. Cellular incorporation of 14C-HCB from serum was temperature-dependent and occurred mainly during the first 30 minutes. Cellular accumulation of 14C-HCB from isolated lipoproteins was also rapid and was more efficient from HDL than from LDL or serum. Accumulation from HDL was concentration-dependent and temperature-dependent. 3. The efflux of 14C-HCB from cells into serum- or lipoprotein-containing medium occurred very rapidly and was most effective in the presence of 20% serum. The order of efficiency in removal of HCB from cells was 20% serum, 50 micrograms LDL protein/ml, and 120 micrograms HDL protein/ml. Little or no efflux from cells occurred into serum-free, lipoprotein-free medium. 4. HDL may be involved in the delivery of HCB to cells, a role in contrast to the generally accepted theory that HDL transports lipids from cells.

[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 Down's syndrome, a disease in which atherosclerosis is rare. Total [14C]acetate incorporation did not differ significantly between normal and Down's 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 Down's 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.

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.

Scanning molecular sieve chromatography of interacting protein systems: nonlinear least-squares analysis of small zone experiments.

A simple routine for nonlinear least-squares analysis is applied to small zone scanning data, where calibration of the gel column requires the use of fully characterized markers of known molecular size. Application of nonlinear least-squares analysis eliminates the difficulty encountered because of scarcity of calibrating markers for gels whose porosities span certain size ranges by providing a sensitive measure of changes in molecular size of the interacting protein system, in order to determine the interaction parameters, size heterogeneity, and the centroid position for time difference chromatographic experiments.

Uptake of [3H]vitamin D3 from low and high density lipoproteins by cultured human fibroblasts.

The plasma distribution and cellular uptake of [3H]vitamin D3 was studied in vitro using cultured human fibroblasts. Incubation of [3H]vitamin D3 (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2-4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotein (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from [3H]1,25-dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of [3H]vitamin D3 from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D3 was time, concentration, and temperature dependent. At a concentration of 50 micrograms LDL/ml of medium, the uptake of [3H]vitamin D3 from LDL at 37 degrees C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D3 absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver.

In vitro uptake of [3H]alpha-tocopherol from low density lipoprotein by cultured human fibroblasts.

Studies were performed to determine the mechanism of entry of [3H]alpha-tocopherol from low density lipoprotein into cultured human fibroblasts. The cellular uptake of alpha-tocopherol at 37 degrees C was significantly different in time- and concentration-dependent experiments between normal fibroblasts and mutant fibroblasts that lack the membrane receptor for low density lipoprotein. The uptake by normal cells of both 125I-labeled low density lipoprotein and [3H]alpha-tocopherol was significantly lower at 4 degrees C where endocytosis does not occur, compared to 37 degrees C where it does occur. Also, the cellular uptake of both [3H]vitamin E and 125I-labeled low density lipoprotein was much lower when the lipoprotein was modified by methylation, which prevented its binding by the receptor, than when the native lipoprotein was measured. Although passive transfer and exchange cannot be excluded as mechanisms of entry, these data indicate that the majority of the alpha-tocopherol enters the cell with the intact lipoprotein particle. This finding is in contrast to several other hydrophobic compounds which reportedly transfer from the lipoprotein into the cell even in the absence of the lipoprotein receptor.

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.

Relationships between composition and cellular uptake of lipoproteins from normal and hyperlipemic swine.

Young male swine fed a high fat, 1% cholesterol diet for 6 weeks developed a hyperlipemia accompanied by alterations in the composition of the very low density, intermediate density and low density lipoproteins (VLDL, IDL, LDL). High density lipoprotein (HDL) composition remained relatively constant. In addition to compositional analysis, determination of sedimentation coefficients indicated the LDL of hyperlipemic animals was of slightly larger size. The in vitro cellular uptake of normal swine and human 125I-labeled LDL by cultured human fibroblasts was similar in time- and concentration-dependent studies. The receptor-mediated uptake of normal swine 125I-labeled LDL, as well as IDL and VLDL, was significantly greater than that of hyperlipemic swine lipoproteins. Nonspecific uptake of normal and hyperlipemic 125I-labeled LDL was similar, as determined in studies with mutant receptor-negative familial hypercholesterolemic fibroblasts. It is hypothesized that the size of the lipoprotein particle may be a determinant in cellular uptake by physically limiting the number of particles that bind to the receptor site for uptake by the cell.

Effects of dichloroacetate and glyoxylate on low density lipoprotein uptake and on growth of cultured fibroblasts.

The effects of dichloroacetate, a known hypocholesterolemic agent, were studied in cultured growing and confluent human fibroblast cells. Microscopic examination showed no visible adverse effects of dichloroacetate on confluent cells during exposure to concentrations as high as 5 mM for 96 hr. Higher concentrations resulted in cell death after varying periods of incubation. There were no viable cells after 24 hr of exposure to 100 mM dichloroacetate. In contrast, much lower concentrations proved lethal to growing cells; cell growth, as determined by cell numbers at specified times after splitting, was suppressed by 1 mM dichloroacetate and 5 mM concentrations resulted in cell death. Similar effects were noted with glyoxylate. The hypocholesterolemic effect of dichloroacetate is probably not due to any effect on the low density lipoprotein pathway, since concentrations of up to 1 mM dichloroacetate did not affect the cellular binding and uptake of 125I-labeled low density lipoprotein. It is concluded that growing and rapidly metabolizing cells are much more sensitive to the toxic effects of dichloroacetate and glyoxylate than confluent cells.

Up- and down-regulation of insulin receptors. Kinetic models.

A theoretical model for insulin receptor synthesis and degradation in differentiating 3T3-L1 adipocytes is described. This three-step irreversible ordered sequence model explains the up- and down-regulation of receptors in terms of the level of insulin concentration. Kinetic expressions were derived for the model. Numerical solutions for these equations, based on data reported by Reed and Lane (Reed, B.C., and Lane, M. D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 285-289) were used for computer-generated curves illustrating insulin-dependent receptor synthesis and degradation. Results show that this model provides the best fit to the reported data and lend support to the suggestion that the free recycled receptor may differ from the newly synthesized receptor. A possible role for the recycled receptor in signal modulation is suggested.