Effect of hyperapo B LDL on cholesterol esterification in THP-1 macrophages.

Hyperapobetalipoproteinemia (hyperapo B), a common disorder associated with coronary artery disease, is characterized by an increased number of small, dense, low density lipoprotein (LDL) particles. The cellular mechanisms responsible for early atherosclerosis in hyperapo B are unknown. We tested the hypothesis that hyperapo B LDL may be preferentially metabolized through an LDL receptor independent pathway promoting the accumulation of cellular cholesteryl ester (CE). THP-1 macrophages have little inducible LDL receptor activity after differentiation with phorbol esters and are, therefore, suitable for assessing non-LDL receptor mediated uptake of lipoproteins. LDL isolated from hyperapo B donors was found to have significantly lower total cholesterol to protein ratio (P = 0.03), higher average density (P = 0.0001) and smaller particle diameter (P = 0.016) compared with normal (control) LDL. LDL (250 micrograms lipoprotein-protein/ml) from normal (n = 11) and hyperapo B (n = 18) subjects were incubated for 24 h with THP-1 macrophages. The mean (S.D.) CE accumulation was 6.2 (3.6) for the normal and 6.4 (2.6) for the hyperapo B LDL (P = 0.84). CE accumulation in cells incubated with malondialdehyde modified (MDA) LDL, or without added lipoprotein, was 18.2 (2.0) and 0.6 (0.7), respectively. CE mass accumulation was significantly correlated with time (6-48 h) of incubation and concentration (100-500 micrograms/ml) of LDL protein (P < 0.05); no differences were observed between normal and hyperapo B LDL. Similarly, when the major LDL species was isolated by density gradient ultracentrifugation, mean (S.D.) CE was similar for the normal and hyperapo B LDL (8.7 (1.2) vs. 6.9 (1.5)). There were no differences in the mean (S.D.) incorporation of [14C]oleate into CE (nmol/mg cell protein per 6 h) in THP-1 macrophages incubated with normal or hyperapo B LDL (0.238 (0.045) vs. 0.211 (0.046)); results were comparable in human monocyte-derived (HMD) macrophages (0.298 (0.037) vs. 0.258 (0.022)). Also, mean (S.D.) cellular uptake and degradation (ng 125I/mg cell protein per h) in THP macrophages of normal and hyperapo B LDL were similar (uptake: 18 (14) vs. 12 (6.0); degradation: 58 (32) vs. 44 (8)). In summary: (1) hyperapo B LDL did not stimulate the accumulation of cellular CE via LDL receptor independent pathways in THP-1 macrophages, (2) normal and hyperapo B LDL stimulation of CE synthesis is similar in THP-1 and HMD macrophages and (3) no differences in cellular uptake and degradation of normal and hyperapo B LDL were observed in THP macrophages.

Postmenopausal hormone replacement therapy and cardiovascular risk reduction. A review.

Administration of unopposed postmenopausal estrogen therapy protects against coronary heart disease (CHD) in women. This is mediated, in part, through beneficial effects on lipid and lipoprotein metabolism. Fewer data are available with regard to CHD risk reduction when a progesterone is required in addition to estrogen. Administration of continuous, rather than cyclic, estrogen-progesterone therapy may maintain the beneficial effects of estrogen and yet protect against the increase in endometrial cancer observed with estrogen therapy alone. Clinical guidelines for hormone replacement therapy await the completion of adequate controlled clinical trials.

Glucose tolerance in women: the effects of age, body composition, and sex hormones.

OBJECTIVE: To determine the separate and interactive effects of age, phase of the menstrual cycle, menopausal hormone status, body fat mass, and regional fat distribution on glucose tolerance in healthy women. DESIGN: Retrospective study. SETTING: The Baltimore Longitudinal Study of Aging. PATIENTS: Two hundred sixty healthy women aged 22-89 years. MEASUREMENTS: Plasma levels of estradiol and progesterone, body mass index (BMI), waist-to-hip ratio (WHR), and plasma glucose values in the fasting state (FPG) as well as 120 minutes after 40 gm/m2 of oral glucose (G120) were measured for each participant. RESULTS: We found a progressive decline in oral glucose tolerance of 0.4 mM (6.7 mg/dL)/decade at G120) in women from early to late adult years, with no relationship to phase of the menstrual cycle and no abrupt change associated with the menopause. Multiple regression analysis revealed significant, independent effects of BMI and WHR on FPG and G120. The influence of age (P less than 0.01) on G120 was stronger than that of the BMI or WHR (P less than 0.05). There was no significant relationship between the levels of endogenous sex hormones and glucose tolerance after adjustments for age, BMI, and WHR. However, women taking oral contraceptives, but not those receiving postmenopausal replacement therapy, did exhibit mildly elevated G120 values. CONCLUSIONS: Age per se, and to a lesser extent BMI and WHR, but not levels of endogenous sex steroids, contribute to the physiological decline in glucose tolerance in older women.

Diabetic postprandial triglyceride-rich lipoproteins increase esterified cholesterol accumulation in THP-1 macrophages.

The risk of cardiovascular disease is increased in subjects with insulin-dependent diabetes mellitus (IDDM), although the mechanism remains unclear. To assess whether diabetic postprandial triglyceride (TG)-rich lipoprotein (TGRLP) subfractions (Sf > 400, 100-400, and 20-100) isolated from non-obese, normolipidemic IDDM subjects (n = 14) are potentially more atherogenic than lipoproteins from normal controls (n = 13), we measured cholesteryl ester (CE) synthesis and esterified cholesterol (EC) mass accumulation in THP-1 macrophages incubated with postprandial TGRLP. Diabetic Sf > 400 and Sf 100-400 but not Sf 20-100 significantly increased the mean (+/- SE) rate (pmol/mg cell protein/24 h) of CE synthesis in THP-1 macrophages compared with normal controls (Sf > 400, 673 +/- 26 v 301 +/- 64, P < .025; Sf 100-400, 560 +/- 27 v 298 +/- 39, P < .0005; Sf 20-100, 743 +/- 51 v 831 +/- 45). As well, all three diabetic TGRLP increased the mass of EC (microgram EC/mg cell protein/48 h) as compared with normal controls (Sf > 400, 4.9 +/- 0.61 v 2.9 +/- 0.50, P < .025; Sf 100-400, 5.7 +/- 0.91 v 3.4 +/- 0.34, P < .05; Sf 20-100, 5.4 +/- 0.7 v 3.2 +/- 0.52, P < .05). This effect is sustained for at least 7 hours postprandially and is greater than that of fasting Sf 100-400 (P < .03) and Sf 20-100 (P < .05) and similar to malondealdehyde low-density lipoprotein (MDA-LDL). To assess the mechanisms involved, the chemical composition and cellular degradation of diabetic and control lipoproteins were compared. Postprandial diabetic Sf 100-400 had abnormal composition (phospholipid to protein ratio, 1.86 +/- 0.14 v 1.5 +/- 0.13, P < .05) and in preliminary experiments demonstrated increased cell association (mean +/- SD at 6 hours, 126 +/- 34.3 v 57 +/- 4.2) and degradation (584 +/- 141 v 254 +/- 13) compared with that of normal controls, and may account for the observed increase in EC accumulation. In summary, postprandial diabetic Sf > 400 and Sf 100-400 TGRLP increase CE synthesis and Sf > 400, Sf 100-400, and Sf 20-100 lipoproteins increase EC accumulation in human macrophages compared with normal control lipoproteins. Diabetic Sf 100-400 lipoproteins have abnormal composition and seem to have increased cellular association and degradation compared with normal lipoproteins. Our findings suggest a role for postprandial TGRLP in the increased risk of cardiovascular disease among subjects with IDDM.

Equivalent efficacy of a time-release form of niacin (Niaspan) given once-a-night versus plain niacin in the management of hyperlipidemia.

This study compared the efficacy and safety of a once-a-night, time-release niacin formulation, Niaspan (Kos Pharmaceuticals, Miami Lakes, FL), with plain niacin and placebo for the treatment of primary hypercholesterolemia. The study was conducted in nine academic lipid research clinics in a randomized, double-blind design. Niaspan 1.5 g at bedtime was compared with plain niacin 1.5 g/d after 8 weeks and 3.0 g/d after 16 weeks in divided doses and with placebo. A total of 223 hypercholesterolemic adult men and women participated. Compared with placebo at 8 weeks, Niaspan versus plain niacin at 1.5 g/d showed comparable efficacy, comparably lowering total cholesterol (C) (8%/8%), triglycerides (16%/18%), low-density lipoprotein (LDL)-C (12%/12%), apolipoprotein (apo B) (12%/12%), apo E (9%/7%), and lipoprotein(a) [Lp(a)] (15%/11%), and raising high-density lipoprotein (HDL)-C (20%/17%), HDL2-C (37%/33%), HDL3-C (17%/16%), and apo A-I (8%/6%) (P < or = .05 in all instances). After 16 weeks, the Niaspan effect on LDL-C and triglyceride was unchanged while the plain niacin effect approximately doubled. At equal doses of 1.5 g/d of Niapan versus plain niacin, respectively, AST increased 5.0% versus 4.8% (difference not significant [NS]), fasting plasma glucose increased 4.8% versus 4.5% (NS), and uric acid concentrations increased less, 6% versus 16% (P=.0001). Flushing events were more frequent with plain niacin versus Niaspan (1,905 v 576, P < .001). Flushing severity was slightly greater with Niaspan, but still well tolerated. In conclusion, Niaspan 1.5 g hour of sleep (hs) has comparable efficacy, a lower incidence of flushing, a lesser uric acid rise, and an equivalent hepatic enzyme effect than 500 mg thrice-daily plain niacin in hyperlipidemic subjects. Niaspan may be an equivalent or better alternative to plain niacin at moderate doses in the management of hyperlipidemia.

Detection and management of hyperlipidemia in children and young adults.

The identification and treatment of children and young adults at high risk for adult CHD is recommended as part of the practice of good preventive cardiology. These recommendations are based on the available clinical, biochemical, epidemiologic, and pathologic data.

Biological variability of lipoproteins and apolipoproteins in patients referred to a lipid clinic.

We determined the physiological variability of total cholesterol, high- (HDL) and low-density lipoprotein (LDL) cholesterol, triglycerides, and apolipoproteins A-I and B in fasting blood samples from patients referred to the Johns Hopkins Lipid Referral Clinic. Samples were taken on each of three occasions during baseline evaluation visits before the patients were treated. The median physiological coefficients of variation (CVp) were as follows: total cholesterol, 5.0%; triglycerides, 17.8%; HDL cholesterol, 7.1%; LDL cholesterol, calculated from the previous three measurements, 7.8%; and apolipoproteins A-I and B, 7.1% and 6.4%, respectively. There were no significant differences in CVp between children (less than or equal to 18 years) and adults (greater than 18 years) for any of the measurements. On the basis of our findings, single measurements in serial samples taken on three occasions suffice to establish the patients' usual values with the following precisions (+/- 1 CV): total cholesterol, +/- 4%; triglycerides, +/- 11%; HDL cholesterol, +/- 8%; LDL cholesterol, +/- 6%; and apolipoproteins A-I and B, +/- 7%.

Biological variation of lipids and lipoproteins in fingerstick blood.

The biological variability of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and calculated low-density lipoprotein cholesterol (LDL-C) was determined in three serial (monthly) capillary and venous specimens from 83 subjects. The analytes were quantified with a desktop analyzer. We saw no differences in the coefficient of biological variability (CVb) between capillary and venous specimens for any analyte (TC, 5.2%; TG, 14.7%; HDL-C, 7.2%; LDL-C, 5.4%). The average analytical variability (CVa) for each analyte, determined in quality-control pools, was; TC, 5.0%; TG, 5.2%; HDL-C, 5.8%; and LDL-C, 7.5%. Compared with standardized laboratory measurements, the desktop analyzer exhibited a significant (P < 0.001) positive bias for all analytes (average bias: TC, 5%; TG, 16%; HDL-C, 6%; and LDL-C, 2.4%). Thus, the biological variation of lipids and lipoproteins was the same in fingerstick and venous samples, and the desktop analyzer showed inherently greater analytical variability.

Cell density can affect cholesteryl ester accumulation in the human THP-1 macrophage.

Esterified cholesterol (EC) accumulation was induced in THP-1 macrophages after exposure to acetylated LDL (acLDL), and the extent of accumulation was dependent on cell density. EC mass was 5-fold greater in cells plated at 1.0 x 10(6) cells/35 mm dish compared to cells plated at density 4.0 x 10(6) cells/dish. In addition, [14C]oleate incorporation into EC also increased with decreasing cell number, with 4-fold greater incorporation (6 h: 177 +/- 0.014 vs. 45 +/- 0.001 pmol/mg cell protein, P < 0.001; 24 h: 515 +/- 0.037 vs. 120 +/- 0.012 pmol/mg, P < 0.001) in cells plated less densely compared to cells plated at a higher density. The rate of 125I-labeled acLDL degradation was about 2-fold greater in cells plated at the lower density (105 vs. 60 ng/h per mg cell protein). Northern analysis showed a 2-fold reduction in the expression of human scavenger receptor mRNA in density plated cells, and immunoprecipitation also demonstrated a 2-fold decrease in scavenger receptor protein. Conditioned media did not differentially affect EC formation at either cell density. Fatty acid supplementation increased EC formation and the proportion of esterified sterol content only in cell plated at the higher density. The fatty acid effect was also seen when cells were exposed to beta-VLDL, which induced comparable levels of EC accumulation by non-scavenger receptor-mediated processes in densely plated cells. Foam cell formation in THP-1 macrophages may depend on cell density, which appears to affect both scavenger and non-scavenger receptor activity.

Incorporation of lean red meat into a National Cholesterol Education Program Step I diet: a long-term, randomized clinical trial in free-living persons with hypercholesterolemia.

OBJECTIVE: Clinicians often recommend that intake of all meat, particularly red meat, be reduced in conjunction with a low-fat, low-cholesterol diet to reduce low-density lipoprotein (LDL) cholesterol. This study was designed to determine the long-term effects of lean red meat (beef, veal and pork) compared to lean white meat (poultry and fish) consumption on lipoprotein concentrations in free-living hypercholesterolemic subjects consuming a National Cholesterol Education Program (NCEP) Step I diet. METHODS: A randomized, crossover design was utilized. Hypercholesterolemic men and women (LDL cholesterol between 3.37 and 4.92 mmol/L) (triglycerides <3.96 mmol/L) (n = 145) were counseled to consume > or =80% of their 170 g/d meat intake as either lean red meat or lean white meat for two 36-week phases, separated by a four-week washout period of free meat selection. Subjects were instructed to follow an NCEP Step I diet throughout the study. RESULTS: There were no significant differences in lipid concentrations between the lean red meat and lean white meat phases. LDL cholesterol was 4.02+/-0.04 (SEM) and 4.01+/-0.04 mmol/L in the white and red phases, respectively; this represented a decrease of approximately 2% from baseline concentrations (p < 0.01). Total cholesterol also declined by 1% from baseline (p < 0.05), and high-density lipoprotein (HDL) cholesterol rose over the study period by approximately 2% to approximately 3% from baseline to reach concentrations of 1.37+/-0.03 mmol/L and 1.38+/-0.03 mmol/L in the white and red phases, respectively (p < 0.001). Triglycerides were not altered by treatment. CONCLUSIONS: Consumption of lean red meat or lean white meat, as part of an NCEP Step I diet, is similarly effective for reducing LDL cholesterol and elevating HDL cholesterol concentrations in free-living persons with hypercholesterolemia.

Further insights into the pathophysiology of hyperapobetalipoproteinemia: role of basic proteins I, II, III.

Hyperapobetalipoproteinemia (hyperapoB), a familial lipoprotein disorder characterized by an increase in small, dense, low-density lipoprotein (LDL) particles, is strongly associated with coronary artery disease. There are two metabolic defects in hyperapoB: an increased synthesis of a very-low-density lipoprotein in liver, resulting in an overproduction of LDL, and a delayed clearance of post-prandial triglyceride and free fatty acids. To date, defects in the apolipoprotein B gene do not appear to explain the hyperapoB phenotype. Defect(s) in the uptake or intracellular metabolism of free fatty acids have been found in cells from hyperapoB patients. Three basic proteins (BPs)--BP I (Mr 14,000, pI 9.10), BP II (Mr 27,500, pI 8.48), and BP III (Mr 55,000, pI 8.73)--were isolated from normal human serum. Compared with normal fibroblasts, cultured hyperapoB fibroblasts incubated with BP I, which appears to be the same protein as acylation-stimulating protein (ASP), showed 50% less stimulation of triglyceride acylation and cholesterol esterification, whereas BP II markedly stimulated cholesteryl ester formation, and BP III caused no difference in response vs normal fibroblasts. However, in cultured normal human monocyte macrophages, BP III, but not BP I or BP II, stimulated cholesteryl esterification two- to threefold. BP I, BP II, and BP III may provide new insights into normal metabolism of lipids, lipoproteins, and free fatty acids and the pathophysiology of hyperapoB.