Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo.

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.

Sex difference in the saturable binding of low-density lipoprotein by liver membranes in ageing rats.

It is well documented that women of child-bearing age tend to have lower serum low-density lipoprotein (LDL) concentrations than men. In order to explore the metabolic basis of this sex difference, we have compared the saturable binding of 125I-labeled LDL (d 1.02-1.05 g/ml) at 37 degrees C by liver membranes from healthy male and female Wistar rats of different ages (15-213 days). Woolf plots of saturable binding curves over the concentration range 15-65 micrograms LDL protein/ml were linear and compatible with a single class of binding sites. Maximum binding capacity (Bmax) was not significantly different in male and female animals of 15-19 days of age (respectively, 0.331 +/- 0.018 vs. 0.427 +/- 0.044 micrograms LDL protein/mg membrane protein, mean +/- S.E.). Thereafter, Bmax increased in females, reaching a peak of 0.635 +/- 0.042 micrograms LDL protein/mg membrane protein at 60 days. As no increase in Bmax occurred in males, values were significantly higher (P less than 0.02) in females than in males (by a mean of 61-117%) at all ages after 30 days. During ageing, serum cholesterol concentration changed reciprocally with Bmax in females (Pearson's correlation coefficient, r = -0.761, P less than 0.01) and remained essentially constant in males. The equilibrium dissociation constant for 125I-labelled LDL binding to the hepatic membranes was unaffected by both age and sex. These results provide evidence that the sex difference in the plasma total and LDL cholesterol concentrations is related, at least in part, to a greater mean LDL receptor density in the livers of females.

The very-high-density lipoprotein fraction of rabbit plasma is rich in tissue-derived cholesterol.

When plasma from rabbits, which several weeks earlier had been infused with [3H]cholesterol, was subjected to equilibrium density gradient ultracentrifugation, the specific radioactivity of cholesterol in the very-high-density lipoprotein (VHDL) fraction (d 1.22-1.32 g/ml) was three to 8-fold greater (mean, 5.5-fold; P less than 0.001) than that in high-density lipoproteins (HDL; d 1.06-1.21 g/ml). On size exclusion chromatography of plasma, no increase in specific radioactivity was seen in particles smaller than HDL. These findings suggest that those apolipoprotein-lipid complexes that dissociate from HDL during ultracentrifugation to form the VHDL fraction contain proportionately more tissue-derived cholesterol than do those that are more tightly bound to HDL.

Hormonal determinants of apolipoprotein B,E receptor expression in human liver. Positive association of receptor expression with plasma estrone concentration in middle-aged/elderly women.

The relationships of the expression of hepatic low-density lipoprotein (LDL) receptors (apo B,E receptors) to several plasma hormone concentrations were examined in 15 fasted women aged 37-75 years (mean, 57 years), who were undergoing laparotomy for non-neoplastic disease. No subject had clinical or biochemical evidence of familial hypercholesterolemia, renal disease, hepatic disease, or endocrine disease. Hepatic apo B,E receptor expression was quantified in vitro as the EDTA-suppressible binding of 125I-labeled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C; values were 23-75 ng LDL protein/mg cell protein (mean, 47 ng/mg). Receptor expression was strongly correlated with plasma estrone concentration (rs = +0.70, P = 0.035), but was unrelated to the concentrations of testosterone, thyroxine, free triiodothyronine, cortisol, sex hormone-binding globulin (SHBG) or cortisol-binding globulin. Insulin and estradiol concentrations were mostly very low. The correlation of receptor expression with plasma total estrone concentration reflected associations with both the albumin-bound (rs = +0.78, P = 0.014) and unbound (rs = +0.80, P = 0.009) fractions, but not with the SHBG-bound fraction (rs = -0.22, P = 0.574), of this hormone. As the non-SHBG-bound fractions of gonadal steroids are considered to be the biologically active components, these results are consistent with experimental evidence that the synthesis of apo B,E receptors in hepatocytes is stimulated by estrogens, and suggest that circulating estrone may be the major hormonal determinant of receptor expression in fasted middle-aged/elderly women.

Concentrations of electrophoretic and size subclasses of apolipoprotein A-I-containing particles in human peripheral lymph.

When cultured cells are exposed to plasma, the initial acceptors of unesterified cholesterol are small lipid-poor apolipoprotein A-I (apoA-I)-containing high density lipoproteins (HDLs) with pre-beta electrophoretic mobility. These are converted by lecithin:cholesterol acyltransferase into larger spheroidal cholesteryl ester-rich HDLs with alpha mobility. To study the determinants of the concentration of small pre-beta HDLs in tissue fluids, we collected prenodal peripheral lymph from 34 fasted normal men. By crossed immunoelectrophoresis, the concentration of pre-beta HDLs in lymph averaged 20% of that in plasma. On multiple regression analysis, pre-beta apoA-I concentration in lymph was directly related to pre-beta apoA-I concentration in plasma and independently to alpha apoA-I concentration in lymph. Similar results were obtained when the same apoA-I-containing particles were quantified by size exclusion chromatography. Lymph pre-beta apoA-I concentration was low in a subject with familial lecithin:cholesterol acyltransferase deficiency, despite a normal plasma pre-beta apoA-I concentration, but was normal in a subject with familial lipoprotein lipase deficiency. These results suggest that the concentration of small pre-beta HDLs in human tissue fluids is determined only in part by the transfer of pre-beta HDLs across capillary endothelium from plasma. Local production, by remodeling of spheroidal alpha HDLs in tissue fluids, may be equally important. Lipolysis of triglyceride-rich lipoproteins by lipoprotein lipase appears to have little effect.

Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice induces hyperlipidemia but reduces atherogenesis.

The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257+/-9, 7.1+/-0.5, and 1.0+/-0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean+/-SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB(48)-containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination.

Evidence that reverse cholesterol transport is stimulated by lipolysis of triglyceride-rich lipoproteins.

The hypothesis that reverse cholesterol transport by high density lipoprotein (HDL) is augmented by lipolysis of triglyceride-rich lipoproteins received support from experiments in rabbits whose tissue cholesterol had been pre-labeled with [3H]cholesterol several weeks earlier. When lipolysis was stimulated by intravenous heparin (which releases lipoprotein lipase from vascular endothelium), reciprocal changes in plasma triglyceride and HDL cholesterol concentrations were accompanied by a rise in the specific radioactivity of HDL cholesterol, indicative of increased transfer of cholesterol into HDL from slowly exchanging cholesterol pools in extra-hepatic tissues.

Do dietary phytochemicals with cytochrome P-450 enzyme-inducing activity increase high-density-lipoprotein concentrations in humans?

Low plasma concentrations of high-density lipoprotein (HDL) are associated with increased risk of coronary heart disease. Several drugs that induce the microsomal cytochrome P-450-dependent enzyme system in liver and intestine, the sites of HDL apolipoprotein (apo) A-I and A-II synthesis, raise plasma HDL concentrations in humans. To test the hypothesis that phytochemicals with cytochrome P-450-inducing activity may also increase plasma HDL concentrations, two controlled dietary trials were undertaken in healthy nonsmoking males aged 20-28 y. One study examined the effect of replacing 300 g glucosinolate-free vegetables with 300 g Brussels sprouts/d for 3 wk. The other study examined the effects of 150 mg eugenol/d in capsule form, using a double-blind, placebo-controlled crossover design. There were no significant increases in plasma apo A-I, apo A-II, HDL cholesterol, or HDL phospholipids. These results suggest that dietary phytochemicals that induce members of the cytochrome P-450 system do not necessarily raise plasma HDL concentrations in humans, but do not exclude the possibility that some phytochemicals may have such an effect.

Plant monoterpenes do not raise plasma high-density-lipoprotein concentrations in humans.

BACKGROUND: Low plasma concentrations of HDLs are associated with an increased risk of coronary artery disease. Two uncontrolled studies suggested that plant monoterpenes may have substantial HDL-cholesterol-elevating activity in humans. Each study used a proprietary mixture of 6 monoterpenes in olive oil. OBJECTIVE: The present study was undertaken to test more rigorously the hypothesis that monoterpenes raise HDL concentrations in men with hypoalphalipoproteinemia. DESIGN: A double-blind, placebo-controlled crossover design was used. Twenty-four men aged 58-68 y (x: 62.3 y) with plasma HDL cholesterol <1.1 mmol/L, plasma triacylglycerols <3.5 mmol/L, and plasma total cholesterol <5.5 mmol/L at recruitment were randomly assigned to 6 capsules daily of a proprietary mixture of 6 monoterpenes in olive oil or 6 capsules daily of olive oil alone for 24 wk, followed by a washout period of 8 wk, and then the alternative capsules for 24 wk. RESULTS: Five men dropped out. In the others, compliance was excellent as judged by capsule counts and urinary menthol glucuronide concentrations. No significant effects were observed on plasma HDL-cholesterol or apolipoprotein A-I concentrations, nor on plasma triacylglycerol, LDL-cholesterol, or apolipoprotein B concentrations. CONCLUSIONS: Plant monoterpenes have no HDL-elevating activity of potential value for coronary artery disease prevention.

Relationship of high density lipoprotein composition to plasma lecithin:cholesterol acyltransferase concentration in men.

The epidemiological associations between the plasma concentrations of several components of high density lipoprotein (HDL) and plasma lecithin:cholesterol acyltransferase (LCAT) concentration have been studied in 101 men aged 52-67 years. Subjects were apparently healthy, and had been selected to provide a wide range of HDL-cholesterol levels. A weak positive correlation was observed between plasma total HDL-cholesterol concentration and LCAT concentration (r = 0.24, P less than 0.02). This reflected an association between HDL3-cholesterol (measured by precipitation) and enzyme concentration (r = 0.21, P less than 0.05). Apoprotein (apo) A-II concentration was also positively correlated with LCAT (r = 0.27, P less than 0.01). HDL2-cholesterol and apo A-I concentration were unrelated to LCAT concentration, as also were the HDL2/HDL3 and HDL-cholesterol/apo A-I ratios. The associations of HDL3 cholesterol and apo A-II with LCAT were strengthened when allowance was made by multiple regression for the effect of log plasma triglyceride; under these circumstances variation in LCAT explained statistically 8% of the variance in HDL3-cholesterol, and 10% of that in apo A-II.

Variations in the apolipoprotein AI-CIII-AIV gene region and in lecithin:cholesterol acyltransferase concentration are determinants of plasma cholesterol concentrations.

We have examined the effects of variation in the region of the apolipoprotein (apo) AI-CII-AIV genes, and in plasma lecithin: cholesterol acyltransferase (LCAT) concentration, on plasma cholesterol concentration in 109 unrelated men aged 52-67 yrs. Restriction fragment length polymorphisms (RFLPs) were detected using the restriction enzymes XmnI, PstI and SstI and individuals were divided into groups using information from all three RFLPs in conjunction. Mean plasma concentrations of both total cholesterol and estimated low density lipoprotein-cholesterol differed significantly (P less than 0.0125) among groups of men with different genotypes. Thus, variation in this gene region may be one of the polygenetic factors involved in determining cholesterol levels in the normal population. In the same subjects, plasma cholesterol was also positively correlated with plasma LCAT concentration (r = 0.55, P less than 0.001), due mainly to an increase in the cholesteryl ester content of apo B-containing lipoproteins with increasing LCAT concentration. Since apolipoproteins AI, CIII and AIV have each been shown to modify the activity of LCAT in vitro, the associations of the RFLPs with plasma cholesterol concentration may reflect changes in LCAT activity secondary to qualitative or quantitative changes in one or more of these apolipoproteins.

Biochemical and pathological features of a modified strain of Watanabe heritable hyperlipidaemic rabbits.

A modified strain of heritable hyperlipidaemic rabbit has been produced by crossing male albino rabbits homozygous for low density lipoprotein receptor deficiency into a coloured commercial colony with strong breeding characteristics. The genetic deficiency has been preserved in the resulting offspring through many generations. Litter numbers, live weight gains and energy intake are similar to normal rabbits. Free and esterified cholesterol in serum, and total cholesterol in very low density plus low density lipoproteins, are markedly increased in homozygote, but only slightly raised in heterozygote, animals. High density lipoprotein-cholesterols show an opposite trend but with less marked differences between the genetic strains. Liver total and esterified cholesterol levels were substantially increased in homozygotes, and the ability of liver membranes to bind human 125I-LDL was markedly reduced, owing to a reduction of the number of high-affinity binding sites. All animals with serum cholesterol values greater than 14 mmol/l at weaning developed extensive aortic atherosclerosis within 16 weeks. The early lesions had the histological appearances of fatty streaks and progressed to complicated disease at 6-12 months. A distinctive pattern of calcific arteriosclerosis, quite different from atherosclerosis, was observed in most aging heterozygote animals and was associated with extensive renal calcium deposition. Corneal arcus developed in some homozygotes but there was no evidence of cerebral atherosclerosis. We conclude that homozygotes of this modified strain can be used for macroscopic studies of the progression of aortic atherosclerosis in the first 4 months after weaning but after this period a combination of macroscopic and microscopic techniques are required. Heterozygotes are unsuitable for studies of this nature.

Plasma lipoprotein lipids in relation to the MspI polymorphism of the apolipoprotein AII gene in Caucasian men. Lack of association with plasma triglyceride concentration.

Digestion of the human apolipoprotein (apo) A-II gene with the endonuclease MspI produces fragments of 3.0 or 3.7 kb, reflecting the presence or absence of a polymorphic site within an Alu sequence 3' to the gene. Patients with hypertriglyceridemia have been shown to have an increased prevalence of the 3.0 kb allele. To explore this observation further, plasma lipoprotein lipids were studied in a random sample of fasted middle-aged Caucasian men, of which 59 were 3.0 kb homozygotes, 24 were heterozygotes, and 2 were 3.7 kb homozygotes. After adjusting for the effects of age, height, weight, alcohol intake and cigarette consumption by covariance analysis, no statistically significant associations were present between genotype and the concentrations of triglyceride in whole plasma or the d less than 1.019 g/ml fraction of plasma (i.e., VLDL + IDL). Nor were the cholesterol concentrations in VLDL + IDL, low density lipoprotein (LDL, d = 1.019-1.063 g/ml), high density lipoprotein (HDL), HDL2 or HDL3 related to genotype. In an independent comparison of eight 3.0 kb homozygotes and eight 3.7 kb homozygotes (all Caucasians) drawn from a different community, genotype was unrelated to the triglyceride or cholesterol concentrations in VLDL (d less than 1.006 g/ml), IDL + LDL (d = 1.006-1.063 g/ml) or HDL, after adjustment for the effects of covariates. These results suggest that the MspI polymorphism of the apo A-II gene is not associated with genetic variation that significantly affects triglyceride transport in the majority of men.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemostatic factors in human peripheral afferent lymph.

Peripheral afferent lymph was obtained by cannulation of a collecting vessel in 17 healthy men (mean age 26 years). Lymph/plasma ratios of all vitamin K-dependent factors were lower than expected from molecular weight. Factor VII, factor IX and tissue factor pathway inhibitor (TFPI) lymph/plasma activity ratios were higher than antigen ratios. Activated factor VII (FVIIa) and TFPI-Xa complex concentrations were higher in lymph than plasma, and the raised FVIIa did not appear to be due to cannulation. The fibrinogen lymph/plasma activity (Clauss) ratio averaged about 20% of the antigen ratio. The result of an ELISA for D-dimer was higher in lymph than plasma, often more than five-fold. This high level in lymph was not explored but may indicate proteolysis of fibrinogen and fibrin with release of D-like and D-dimer-like fragments in interstitial fluid.

Factor VII activation, apolipoprotein A-I and reverse cholesterol transport: possible relevance for postprandial lipaemia.

Postprandial lipaemia is associated with activation of factor VII (FVII) and efflux of cholesterol from tissues to nascent plasma high density lipoproteins (HDL) containing apolipoprotein A-I (apo A-I). To determine whether FVII activation and cholesterol efflux occur together in other situations, the responses to intravenous infusion of HDL-like apo A-I/phosphatidylcholine discs were measured in 10 healthy men. Disc infusion (40 mg apo A-I/kg body weight) over 4 h was followed by increases in HDL cholesteryl ester and plasma apo A-I (p <0.0001). Significant activation of FVII was apparent during infusion in fasting subjects (p = 0.03), activated FVII averaging 123% of baseline value by 12 h (p <0.0001). Plasma thrombin-antithrombin (TAT) complex increased to 156% of baseline level by 12 h (p >0.05) but individual responses differed considerably. Peak TAT post-infusion was associated inversely with peak HDL triglyceride concentration (p = 0.004). The coagulation responses to disc-infusion may be due to transfer of phosphatidylserine to cell surfaces during cholesterol efflux.

Very low activated factor VII and reduced factor VII antigen in familial abetalipoproteinaemia.

Abetalipoproteinaemia is a rare disorder of apolipoprotein B metabolism associated with extremely low plasma concentrations of triglyceride. To discover whether the general positive association between factor VII and triglyceride levels extends to this condition, 5 patients were compared with 18 controls. All patients had a triglyceride below 100 micromol/l. Plasma unesterified fatty acid concentration was normal. Although factor IX activity was only slightly reduced (mean 88% standard) and factor IX antigen was normal, mean activated factor VII in patients was strikingly reduced to 34% of that in controls, a level similar to that found in haemophilia B. The patients' mean factor VII activity and factor VII antigen were also significantly reduced to 54% and 63% of those in controls, respectively. Mean factor XI activity and tissue factor pathway inhibitor activity were reduced in patients to 70% and 75% of control values respectively, while factor XII, factor XI antigen, factor X, prothrombin and protein C were normal.

Plasma lipoproteins and adrenocortical hormones in men--positive association of low density lipoprotein cholesterol with plasma cortisol concentration.

The associations of plasma lipoprotein lipids with the plasma concentrations of two adrenocortical hormones (sampled at 09.00-11.00 h) have been investigated in a random sample of 70 men aged 52-67 yr (mean, 59 yr). Plasma low density lipoprotein (LDL) cholesterol concentration was found to be positively correlated with plasma cortisol, independently of the concentrations of plasma triglyceride, high density lipoprotein cholesterol and androstenedione. After adjusting for covariates, plasma cortisol explained approximately twelve per cent of the variance in LDL cholesterol. These results suggest that plasma cortisol may significantly influence the metabolism of LDL in healthy humans.

A steady-state gel filtration method on micro-columns for the measurement of percentage free testosterone in serum.

A method is described for measuring the percentage of free testosterone in serum using steady-state gel filtration on micro-columns of Sephadex G50 (fine). The method has a coefficient of variation of 4.0% and up to 20 samples can be processed in a day. Reference ranges have been established for normal women at different stages of their menstrual cycles and for normal men; there is good agreement with values published by workers using other methods. This method combined with a specific and precise method for measuring total testosterone opens up the possibility for the routine measurement of free testosterone concentration in serum.

Plasma free testosterone--is an index sufficient?

Only about 50% of women who are clinically hirsute have a raised total plasma testosterone concentration; in those cases where the total testosterone is normal the free testosterone may, in fact, be raised. Since the measurement of free testosterone is tedious, workers have used an androgen index or a calculated free testosterone concentration from the measurements of total testosterone and sex-hormone-binding globulin. We have examined the correlation between measured free testosterone, a derived free testosterone and an androgen index in hirsute women, normally menstruating women and non-hirsute women on oral contraceptive therapy. These three measures of the free testosterone concentration in blood gave similar results in all cases.