Biologically active low density lipoprotein in human peripheral lymph.

We have compared the ability of human serum and peripheral lymph to suppress the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), to activate cholesteryl ester synthesis, and to compete with 125I-labeled low density lipoprotein (LDL) for binding to LDL receptors in cultured human fibroblasts. Whole lymph was active in all three tests and the activity per unit volume in lymph was approximately equal to 1/10th that in serum. All three biologic activities in lymph were confined to the d less than 1.063 g/ml fraction. Whole lymph had no significant effect on HMG-CoA reductase activity in fibroblasts from a patient with homozygous familial hypercholesterolemia, whose cells lack LDL receptors. The LDL-like biologic activity per unit mass of immunologically active apoprotein B was approximately the same in lymph as in serum. The current data indicate that functionally active LDL is present in lymph and that the concentration of this lipoprotein is approximately equal to 1/10th that in serum.

Surface binding and catabolism of low-density lipoprotein by circulating lymphocytes from patients with abetalipoproteinaemia, with observations on sterol synthesis in lymphocytes from one patient.

Surface binding of low-density lipoprotein (LDL), degradation of LDL protein and sterol synthesis were investigated in freshly isolated lymphocytes from normal and abetalipoproteinamic human subjects. LDL binding as a function of LDL concentration showed no evidence of the presence of high-affinity binding sites in fresh lymphocytes from either group of subjects. The rate of degradation of LDL protein by lymphocytes from the patients was no greater than that from the normal subjects and, in the fresh lymphocytes of the one patient studied, sterol synthesis was not increased. We conclude that the formation of LDL receptors and the synthesis of sterol in circulating lymphocytes are largely suppressed and that in normal subjects this may be due to the presence of some plasma constituent other than LDL, possibly the apoE protein. This conclusion is discussed in relation to the possible contribution of LDL receptors to the degradation of LDL protein in vivo.

The substrate for cholesterol 7alpha-hydroxylase.

The rates of incorporation of (14C) cholesterol into cholesteryl esters and 5-cholestene-3beta,7alpha-diol (7alpha-hydroxycholesterol) by rat liver microsomes, measured under conditions in which esterification and 7alpha-hydroxylation are varied independently, indicated that cholesterol is the substrate for cholesterol 7alpha-hydroxylase. The specific activities of cholesteryl esters and 7alpha-hydroxycholeste ol in incubations of microsomes labelled with (14C)cholesterol in vitro or in vivo suggest that 7alpha-hydroxycholesterol and esterified cholesterol are not derived from the same pool of free cholesterol.

Concentration of lipoproteins containing apolipoprotein B in human peripheral lymph.

The concentration of apolipoprotein B (apoB) in human serum and peripheral lymph was measured by quantitative immunoelectrophoresis with anti-serum to human low-density lipoprotein. In four normal and six hyperlipidaemic subjects, total lymph apob/ml was 5-10% of total serum apoB/ml in the same subject. These ratios were equivalent to lymph apob concentrations of 60-120 microgram/ml. When the assays were carried out under conditions in which unmasking of immunoreactive sites on lymph and serum apoB was assumed to be maximal (delipidation with Nonidet P40), the lymph/serum apoB concentration ratios in three normal subjects were similar to those obtained with untreated lymph and serum.

Effect of portacaval anastomosis on the activities of hepatic enzymes related to cholesterol and bile acid metabolism in rats.

1. The effect of a portacaval anastomosis on the activities of hepatic enzymes related to cholesterol metabolism was investigated in rats. 2. Portacaval anastomosis led to a fall in body weight and liver weight/body weight ratio, and to a rise in the activities of hydroxymethylglutaryl-CoA reductase and cholesterol 7alpha-hydroxylase per g of liver. The net effect was to maintain a normal activity of both enzymes per 100 g of rat. Diurnal rhythm in the activities of both enzymes was maintained after portacaval anastomosis. 3. The rate of excretion of total bile acids, per 100 g of rat, in bile fistula rats was not significantly decreased by portacaval anastomosis.

The effect of portacaval anastomosis on plasma lipoprotein metabolism in rats.

1. The effect of portacaval anastomosis on the metabolism of plasma lipoproteins was investigated in rats. 2. When compared with sham-operated pair-fed controls, plasma high density lipoprotein cholesterol concentration was decreased, plasma low density lipoprotein cholesterol concentration was increased and plasma total cholesterol concentration was unchanged in the portacaval anastomosis rats. Maximal incorporation of [14C]leucine into the total circulating mass of protein was decreased in the very low density lipoprotein and high density lipoprotein fractions and, possibly, in the low density lipoprotein fraction, but there was no change in maximal incorporation into albumin. It is concluded that portacaval anastomosis diminishes the rate of synthesis of high density lipoprotein and very low density lipoprotein proteins and, possibly, of low density lipoprotein proteins.

Familial defective apolipoprotein B-100: a review, including some comparisons with familial hypercholesterolaemia.

Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder caused by the substitution of glutamine for arginine at position 3500 in apo B-100. The presence of mutant apo B-100 in low-density lipoproteins (LDL) markedly reduces their affinity for the LDL receptor, leading to hypercholesterolaemia and increased proneness to coronary artery disease. In some FDB heterozygotes the clinical picture is indistinguishable from that in heterozygous familial hypercholesterolaemia (FH). In European and N. American populations the frequency of FDB is at least as high as that of FH. In most lipid clinics, 2-5% of patients given a clinical diagnosis of FH have FDB, not FH. Most FDB heterozygotes respond well to drugs that lower plasma LDL levels by inducing receptor activity. This may be due partly to increased receptor-mediated hepatic removal of mutant and normal precursors of LDL, using apo E as recognition element. Several important lessons can be learnt from the study of FDB.

The affinity of low-density lipoproteins and of very-low-density lipoprotein remnants for the low-density lipoprotein receptor in homozygous familial defective apolipoprotein B-100.

We have identified two familial defective apo B-100 (FDB) homozygotes by DNA sequencing and have measured affinity of low-density lipoproteins (LDL) and very-low-density lipoprotein (VLDL) remnants for the LDL receptor in vitro. The patients were a 66-year-old man with coronary heart disease (plasma cholesterol level, 9.5 mmol/l before treatment) and his 69-year-old sister, without signs of cardiovascular disease (plasma cholesterol, 12.0 mmol/l before treatment). In both patients, treatment with statins caused a marked fall in plasma cholesterol level. Binding affinity of LDL from the two patients was 10%-20% of normal at 4 degrees C and 37 degrees C. Binding affinity of VLDL remnants was normal. We conclude that (1) residual affinity of LDL in homozygous FDB is high enough to permit significant catabolism via the LDL-receptor pathway, and (2) normal affinity of VLDL remnants permits normal hepatic clearance of precursors of LDL and increased clearance of LDL precursors when receptor activity is stimulated by statins. Residual affinity of LDL and normal affinity of remnants could explain why expression of the FDB mutation is generally milder than that of LDL receptor mutations causing familial hypercholesterolaemia.

Loss of cholesterol from muscle and skin of monkeys treated with nicotinic acid.

In Rhesus monkeys, nicotinic acid given daily by subcutaneous injection for 5 weeks brought about a reversible decrease in total cholesterol concentration in skeletal muscle and skin to about half the normal value. The decrease in cholesterol concentration was due to a net loss of cholesterol from muscle, since the treatment had no effect on the water content or on the percentage of DNA or protein in fresh tissue. In muscle, free cholesterol was lost in preference to esterified cholesteol, but in skin both cholesterol fraction were affected to about the same extent. Analysis of the cholesterol content of subcellular fractions of homogenates of muscle showed that loss of cholesterol occurred mainly from the soluble fraction and the 800-g sediment, with no significant loss from the mitochondrial or microsomal fractions.

Sterol balance in a patient with abetalipoproteinaemia.

Total-body cholesterol synthesis was measured in a woman with abetalipoproteinaemia and in a normal woman of similar age. The rate of synthesis of cholesterol was 15.4 +/- 4.1 mg/kg/day in the patient and 14.3 +/- 2.6 mg/kg/day in the control subject, indicating that cholesterol synthesis in the whole body is not increased in the complete absence of plasma low density lipoprotein.

The effect of nicotinic acid on the metabolism of the plasma lipoproteins of rhesus monkeys.

The effect of nicotinic acid was investigated in Rhesus monkeys. Subcutaneous injections of nicotinic acid lower the plasma very low density lipoprotein (VVLDL) and low density lipoprotein (HDL) concentration. The fall in LDL concentration is not accompained by any change in the lipid or protein composition of either lipoprotein. Analysis by Sephadex gel chromatography and polyacrylamide-gel electrophoresis showed that the proteins of monkey VLDL and LDL are qualitatively similar to those of human VLDL and LDL, although there are differences in the proportions of the various proteins present in the two species. Subcutaneous injections of nicotinic acid diminish the maximum incorporation of 14C from [14C]threonine into VLDL and LDL apoproteins, but have no effect on incorporation into albumin or HDL apoprotein. Peak incorporations into the apo-B and apo-C of VLDL are diminished to about equal extents by nicotinic acid. Comparison of the amount of 14C lost from apo-B of VLDL after the peak of incorporation, with that gained by apo-B of LDL during the same period, suggests that some of the circulating apo-B of LDL IS DERIVED FROM SOURCES OTHER THAN CIRCULATING VLDL.

The binding of very low density lipoprotein remnants to the low density lipoprotein receptor in familial defective apolipoprotein B-100.

We have compared the affinity for low density lipoprotein (LDL) receptors of LDL and very low density lipoprotein (VLDL) remnants from patients with familial defective apo B-100 (FDB) with that of LDL and VLDL remnants from normal subjects. The binding affinity of FDB LDL was markedly reduced in all 14 FDB patients examined, hut the affinity of FDB remnants did not differ significantly from that of remnants prepared from normal subjects. Since the mutant form of apo B-100 present in FDB is recognized by LDL receptors with greatly reduced efficiency, we suggest that apo B plays only a minor role in the receptor-mediated uptake of VLDL remnants by the liver in man. These results are consistent with our previous suggestion that the ability of drugs that stimulate hepatic receptor activity to lower the plasma LDL level in FDB is due in part to increased hepatic uptake of lipoprotein precursors of LDL, including remnant particles with normal apo B-100 and those with mutant apo B-100.

Metabolism of apolipoprotein B-containing lipoproteins in familial hypercholesterolaemia: effects of plasma exchange.

The turnover of apolipoprotein B (apo B) in very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) was investigated in 2 homozygous and 3 heterozygous patients with familial hypercholesterolaemia. The effects of a marked reduction in plasma LDL concentration, brought about by plasma exchange, upon apo B turnover were studied in 4 patients. Specific activity-time curves for the the plasma apo B after intravenous radioactive VLDL before plasma exchange indicated that in the heterozygotes all IDL-apo B was derived from VLDL and all LDL-apo B was derived from IDL, but the curves from the homozygotes showed that a significant fraction of the LDL in the plasma was not derived from IDL. Plasma exchange did not increase the rates of synthesis of LDL-apo B or VLDL-apo B and had no significant effect on the precursor--product relationship between IDL-apo B and LDL-apo B in heterozygous or homozygous patients. These findings provide no support for the hypothesis that apo B synthesis is controlled by the plasma LDL.

The distribution of cholesterol and apoprotein A-I between the lipoproteins in plasma and peripheral lymph from normal human subjects.

The lipoproteins of peripheral lymph and plasma from normal human subjects were separated according to their density by sequential ultracentrifugation and according to their size by gradient gel electrophoresis and gel exclusion chromatography. High density lipoproteins (HDL) carried a higher proportion of the total cholesterol in lymph than in plasma. Within the HDL fraction, the less dense and more lipid-rich component (HDL2) carried a higher proportion of the total HDL cholesterol in lymph than in plasma. Gradient gel electrophoresis showed (1) a higher proportion of large to small HDL particles in lymph than in plasma and (2) the presence of at least three populations of apo A-I-containing lipoproteins with Stokes diameters larger than the Stokes diameter of HDL2. Separation by gel exclusion chromatography showed that the proportion of large HDL particles with a high cholester: apo A-I ratio was greater in lymph than in plasma. In view of the sieving effect of the blood capillaries, which favours the passage across the capillary walls of smaller vs larger particles, we suggest that the higher ratio of large to small HDL particles in lymph than in plasma is due to the conversion of small to large HDL in the interstitial fluid by incorporation of cholesterol and other lipids from extravascular cells into the smaller particles.

The metabolism of very low density and intermediate density lipoproteins in patients with familial hypercholesterolaemia.

The metabolism of apolipoprotein B (apoB) in very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) was studied in normal subjects and in patients with familial hypercholesterolaemia (FH) after an intravenous injection of autologous VLDL labelled with 125I. There were no significant differences in half life, pool size and turnover rate (mg/kg/h) of VLDL-apoB between the normal subjects, the FH heterozygotes and the FH homozygotes. IDL-apoB metabolism in the FH patients differed significantly from that in the normal subjects. In the FH patients, the rise to the maximum of the specific activity curve was slower, the half life of the descending limb of the specific-activity curve was longer, the fractional rate of turnover was lower and the plasma concentration was higher than in the normals. The effect of cholestyramine on IDL-apoB metabolism in the normal subjects did not differ from that in the FH heterozygotes and homozygotes, though cholestyramine is known to stimulate hepatic uptake of low density lipoprotein (LDL) by the LDL receptor. It is suggested that in normal human subjects the LDL receptor makes some contribution to the hepatic uptake of IDL-apoB derived from VLDL, but that IDL uptake is mediated partly by a separate receptor that recognizes apolipoprotein E but not apoB.

Further evidence for the role of high density lipoprotein in the removal of tissue cholesterol in vivo.

The lipoproteins of human peripheral lymph and plasma were separated according to particle size by polyacrylamide gradient gel electrophoresis. All samples of lymph contained lipoproteins that moved to the same positions on the gel as plasma LDL and plasma HDL. Some samples of lymph also contained lipoproteins with the mobility of VLDL and IDL. The lymph lipoproteins corresponding to plasma LDL reacted with anti-LDL serum and those corresponding to plasma HDL reacted with anti-HDL serum. In the lipoprotein fraction with the mobility of HDL, the proportion of particles larger than catalase was greater in lymph than in plasma. It is suggested that the shift in size distribution towards larger HDL particles in lymph compared with plasma is due to uptake of cholesterol from extravascular tissue by HDL particles after they have reached the interstitial fluid from the plasma, rather than to preferential movement of larger particles across the capillary walls.

Evidence for the presence of tissue-free cholesterol in low density and high density lipoproteins of human peripheral lymph.

The specific radioactivity of free and esterified cholesterol in the lipoproteins of human peripheral lymph was measured in 4 normal human subjects at various intervals after labelling the tissue cholesterol by a single intravenous injection of [14C]cholesterol. The free : esterified cholesterol specific-activity ratios in lymph LDL and HDL at short and long intervals after labelling in vivo suggest that both lipoproteins were capable of acting as acceptors for tissue-free cholesterol, but that in 3 of the 4 subjects the predominant acceptor was HDL.

Restriction fragment length polymorphisms in the apo B gene in relation to coronary artery disease.

We have determined the frequencies of the alleles at the EcoRI (E), XbaI (X) and PvuII (P) polymorphic restriction sites in the apo B gene in 124 white men with coronary artery disease (CAD) and in 146 white men free from CAD. The frequencies of the E- (restriction site absent) and X- alleles were both significantly higher in normocholesterolaemic men with CAD than in those without CAD, but the frequency of the P+ allele (restriction site present) was similar in the 2 groups. The frequency of the E- allele was significantly higher in CAD men with hypertriglyceridaemia than in normal men without hypertriglyceridaemia. In the normocholesterolaemic men without CAD, the mean serum cholesterol concentration was higher in those with genotype X++ than in those with genotype X--. Mean serum LDL-apo B and LDL-cholesterol concentrations did not differ significantly between men with different XbaI or EcoRI genotypes. Serum apo A-I levels differed significantly between normal men with different XbaI genotypes. Serum HDL-cholesterol levels differed significantly between CAD men with different XbaI genotypes. These results suggest that in white men the E- and X- alleles are in linkage disequilibrium with a nearby allele that is causally related to CAD. It is also possible that the amino acid substitution at position 4154 in apo B, brought about by the nucleotide change responsible for the EcoRI polymorphism, has a direct effect on the atherogenicity of LDL.

Effective reduction of plasma LDL levels by LDL apheresis in familial defective apolipoprotein B-100.

The clinical response to long-term reduction of the plasma LDL cholesterol concentration was studied in a man with severe coronary artery disease associated with familial defective apolipoprotein B-100 (FDB). Plasma exchange repeated at 2-week intervals, combined with lipid-lowering drugs, led to remission of angina and improved exercise test performance. A similar clinical response was achieved after LDL apheresis with dextran sulphate columns repeated once every 2 weeks in combination with drug treatment. The reduction in plasma LDL cholesterol level brought about by LDL apheresis was at least as marked in the FDB patient as in 5 patients with familial hypercholesterolaemia. We conclude that FDB patients with coronary artery disease may derive clinical benefit from prolonged reduction of their plasma cholesterol levels and that LDL containing apo B-100 in which arginine at position 3500 is replaced by glutamine is removed from plasma by dextran sulphate columns as efficiently as is normal LDL.

Lipoprotein(a) in subjects with familial defective apolipoprotein B100.

The plasma lipoprotein(a) (Lp(a)) concentration and apolipoprotein(a) (apo(a)) phenotype were determined in the members of two families affected with familial defective apo B100 (FDB), resulting from the Arg3500----Gln mutation in apo B that disrupts binding to LDL receptors. Eleven different phenotypic species of apo A were identified, five of which were present in both families. Although there was a general increase in Lp(a) concentration as the size of the predominant apo(a) component decreased, there was considerable variability and in three clear instances the concentration of an inherited phenotypic species was atypically low. In five cases where a direct comparison could be made, the plasma Lp(a) concentration was significantly higher in heterozygous FDB subjects than in their non-FDB siblings or close relatives with the same phenotype. However, in vitro competition studies using purified Lp(a) that had been reduced with dithiothreitol to remove the apo(a) component, indicated that the Lp(a) from FDB heterozygotes contained a smaller proportion of defective particles than their LDL. Lp(a) particles containing normal and binding-defective apo B were present at approximately the same concentration, suggesting that the increase in Lp(a) concentration observed in FDB subjects could not be explained by the inability of the particles containing the defective apo B100 to be cleared through LDL-receptor mediated processes.