Familial Hypercholesterolemia Subjects Research Articles

Regulation of synthesis and cell content of the low-density-lipoprotein receptor protein in cultured fibroblasts from normal and familial hypercholesterolaemic subjects.

Incorporation of [35S]methionine into low-density-lipoprotein (LDL) receptors by normal fibroblasts and those from a homozygous familial hypercholesterolaemic (FH) subject who produced defective but immunoprecipitable receptor proteins of normal size, was compared with the ability of the cells to bind LDL and their content of LDL receptor protein determined using a double-antibody radioimmunoassay. The FH cells produced precursor protein with a longer half-life (3-4 h) than normal cells (40 min), most of which was eventually processed to a mature form of the receptor. Total receptor half-life was similar to normal (approx. 12 h) and LDL binding about 20% of normal. Incubation of normal fibroblasts with lipoprotein-deficient serum (LPDS) led to an increase in the amount of LDL receptor protein in the cells which was closely followed by the increase in their ability to bind LDL. Receptor synthesis increased rapidly at first, but then fell by more than 60% before remaining constant. The peak of synthesis coincided with the greatest rate of increase in receptor content. At equilibrium in LPDS receptor synthesis, LDL binding and receptor protein content were all approximately 3.3-fold higher than in cells maintained in 10% foetal calf serum (FCS). The FH cells also responded to LPDS with a rise and fall in the rate of receptor synthesis. They did not compensate for their inefficiency in producing active receptors with an increase in total receptor content. In LPDS, peak synthesis and maximum receptor content of the FH cells were similar to normal. In FCS receptor synthesis and content were well below maximum so that they did not fully employ even the low capacity for LDL uptake of which they were capable. With both types of cell, inhibition of mevalonic acid and cholesterol synthesis with compactin delayed, but did not prevent the secondary fall in the rate of receptor synthesis, again suggesting a regulatory role for some factor not directly related to cholesterol metabolism.

Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells.

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.

Diversity in expression of heterozygous familial hypercholesterolemia. Characterization of a unique kindred.

Clinical and biochemical characteristics of familial hypercholesterolemia (FH) heterozygotes possessing an abnormally high molecular weight low density lipoprotein receptor (HMWR) are reported. The disorder is transmitted as an autosomal dominant trait and is not distinguishable from classic heterozygous FH on clinical grounds. The average plasma low density lipoprotein (LDL) level is 360 mg/dl and tendon xanthomata and early coronary disease are present. LDL receptor activity is higher than expected. In skin fibroblast cultures two types of functional LDL receptors are present, one with a normal apparent native molecular weight of 140,000, and the other of 176,000. When immobilized on nitrocellulose paper both receptors bind LDL. Maximum 125I-LDL binding capacity of fibroblast monolayers is reduced only 20%, compared with 50% in typical heterozygous FH. Affinity for 125I-LDL is increased and a 38% reduction in the Michaelis constant for LDL is observed. When autologous 125I-LDL was injected intravenously, the fractional catabolic rate of LDL was 205% and the LDL apoprotein B production rate was 328% of that found in a typical heterozygous FH subject. Thus, both in vitro and in vivo testing indicated only a modest deficiency of LDL receptor activity. Kindred members possessing the HMWR had an associated abnormality of cholesterol biosynthesis. Cholesterol balance studies in three individuals with the HMWR trait demonstrated elevated cholesterol biosynthesis of two to three times the mean of normal subjects. These findings suggest that increased LDL production and increased cholesterol production may assume a significant role in the pathologic manifestations of heterozygous FH. Functional abnormalities in LDL receptor activity as measured in fibroblast culture may be relatively small.

Metabolism of low-density lipoproteins by cultured hepatocytes from normal and homozygous familial hypercholesterolemic subjects

The profoundly elevated concentrations of low-density lipoproteins (LDL) present in homozygous familial hypercholesterolemia lead to symptomatic cardiovascular disease and death by early adulthood. Studies conducted in nonhepatic tissues demonstrated defective cellular recognition and metabolism of LDL in these patients. Since mammalian liver removes at least half of the LDL in the circulation, the metabolism of LDL by cultured hepatocytes isolated from familial hypercholesterolemic homozygotes was compared to hepatcytes from normal individuals. Fibroblast studies demonstrated that the familial hypercholesterolemic subjects studied were LDL receptor-negative (less than 1% normal receptor activity) and LDL receptor-de fective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL ( B max = 2.2 μg LDL/ mg cell protein). Preincubation of normal hepatocytes with 200 μg/ml LDL reduced binding and internalization by approx. 40%. In contrast, 125I-labeled LDL binding and internalization by receptor-negative familial hypercholesterolemic hepatocytes was unaffected by cholesterol loading and considerably lower than normal. This residual LDL uptake could not be ascribed to fluid phase endocytosis as determined by [ 14C]sucrose uptake. The residual LDL binding by familial hypercholesterolemia hepatocytes led to a small increase in hepatocyte cholesterol content which was relatively ineffective in reducing hepatocyte 3-hydroxy-3-methylglutaryl-CoA reductase activity. Receptordefective familial hypercholesterolemia hepatocytes retained some degree of regulatable 125I-labeled LDL uptake, but LDL uptake did not lead to normal hepatocyte cholesterol content or 3-hydroxy-3-methylglutaryl-CoA reductase activity. These combined results indicate that the LDL receptor abnormality present in familial hypercholesterolemia fibroblasts reflects deranged hepatocyte LDL recognition and metabolism. In addition, a low-affinity, nonsaturable uptake process for LDL is present in human liver which does not efficiently modulate hepatocyte cholesterol content or synthesis.

Plasma triglycerides related decrease in high-density lipoprotein cholesterol and its association with myocardial infarction in heterozygous familial hypercholesterolemia

Recent studies suggest that decreased levels of high-density liopoprotein (HDL) may contribute to the risk of premature occlusive atherosclerosis in familial hypercholesterolemia (FH). To investigate further, we have analyzed the concentration as well as distribution of HDL cholesterol in relation to plasma triglycerides and their influence on ischaemic heart disease in FH subjects. The study was carried out in 71 men with heterozygous FH and 46 matched controls. FH subjects were relatively young with a mean age of 38 ± 11 years. Tendon xanthomatas were observed in 57% of the subjects, whereas ischemic heart disease was identified in 33%. Compared to normals, the mean value of HDL cholesterol is significantly reduced by 21% in FH heterozygotes (42 ± 12 v 33 ± 9 mg/dL, P < 0.001). The decrease in HDL cholesterol is highly correlated to the levels of plasma triglycerides ( r = −0.50, P < 0.001) and VLDL cholesterol ( r = −0.53, P < 0.001). Moreover, HDL cholesterol decrease is not associated with elevated levels of LDL cholesterol ( r = −0.20, NS), which is the primary characteristic feature of FH subjects. However, HDL cholesterol decrease is weakly related to total plasma cholesterol concentration ( r = −0.24, P < 0.05). The body weight is also contributory to the reduction of HDL cholesterol ( r = −0.42, P < 0.01), probably due to its strong positive correlation to plasma triglycerides ( r = +0.54, P < 0.001). Grouping of subjects on the basis of triglyceride levels of less than 200 mg/dL (IIa phenotype) and more than 200 mg/dL (IIb phenotype) shows that the concentration of HDL cholesterol undergoes a further significant decrease in the latter group (36 ± 9 v 30 ± 11 mg/dL, P < 0.001). Since the level of LDL cholesterol is similar in both groups (314 ± 68 v 316 ± 76 mg/dL, NS), a further reduction in HDL cholesterol concentration results in an increased LDL/HDL ratio in IIb phenotypes. Although HDL cholesterol is normally distributed in controls and type IIa phenotypes, its distribution is skewed to lower values in type IIb. In addition to similar levels of LDL cholesterol, the presence of tendon xanthomatas is equally observed in both type IIa and type IIb subjects (51.1% and 62.5%, respectively). Similarly, the incidence of angina pectoris (19.2% and 12.5% in type IIa and type IIb, respectively) is also approximately the same in both groups. However, the differences are striking in the incidence of myocardial infarction (MI), which is increased three-fold (25% v 8.5%) in type IIb subjects, as compared to type IIa. These findings indicate that in addition to LDL excess, HDL deficiency associated with elevated plasma triglycerides contributes to the severity of ischemic heart disease, as revealed from the manifestation of MI in some FH heterozygotes.

Familial hypercholesterolemia and HLA antigens.

Hereditary factors play an important role in the etiology and progression of coronary heart disease (CHD), most probably by causing the appearance and the levels of its risk factors. To investigate if there is a correlation between familial hypercholesterolemia and HLA system antigens, 25 subjects of 7 families with familial hypercholesterolemia were compared with 14 normocholesterolemic related subjects. Compared to normocholesterolemic kindreds, the familial hypercholesterolemic subjects have a significant increased HLA-Bw35 antigen and a lack of HLA-A1. Therefore, the correlation found with some HLA system phenotypes improves our knowledge about familial hypercholesterolemia and aids in the identification of subjects affected with this disorder of lipid metabolism, which is one of the most important CHD risk factors.

Increased fibrinogen binding to platelets from patients with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a disease marked by a high incidence of thrombotic episodes and hypersensitivity of the patients' platelets to naturally occurring aggregating agents. Prostaglandin/thromboxane (PG/Tx) formation, adenosine 5'-diphosphate (ADP) secretion, and fibrinogen binding to platelets are all believed to be involved in the mechanisms of platelet aggregation. Therefore, we studied the interrelated roles of these processes in the platelets of nine FH patients and 10 controls. In response to ADP, collagen, or thrombin, FH platelets bound about twice as much 125I-fibrinogen as controls. This ratio did not change after suppression of PG/Tx formation by aspirin. With or without aspirin, FH platelets always aggregated in response to significantly lower concentrations of these agents than did platelets from normal controls. After stimulation with thrombin or collagen, the hyperaggregable platelets from FH patients were shown to bind significantly more fibrinogen than control platelets even when PG/Tx formation was suppressed (aspirin) and secreted ADP was scavenged (apyrase). To determine whether the increased fibrinogen binding observed in FH platelets is due to a qualitative or quantitative abnormality of the platelet receptor, we used a monoclonal antibody (B79.7) that is specific for the receptor. The amount of B79.7 that bound to platelets from control and FH subjects was similar. In addition (as in normal individuals), the antibody inhibited aggregation and fibrinogen binding of FH platelets.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in human fibroblasts by reversible phosphorylation: Modulation of enzymatic activity by low density lipoprotein, sterols, and mevalonolactone

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase exists in interconvertible active and inactive forms in cultured fibroblasts from normal and familial hypercholesterolemic subjects. The inactive form can be activated by endogenous or added phosphoprotein phosphatase. Active or partially active HMG-CoA reductase in cell extracts was inactivated by a ATP-Mg-dependent reductase kinase. Incubation of phosphorylated (inactive) HMG-CoA reductase with purified phosphoprotein phosphatase was associated with dephosphorylation (reactivation) and complete restoration of HMG-CoA reductase activity. Low density lipoprotein, 25-hydroxycholesterol, 7-ketocholesterol, and mevalonolactone suppressed HMG-CoA reductase activity by a short-term mechanism involving reversible phosphorylation. 25-Hydroxycholesterol, which enters cells without the requirement of low density lipoprotein-receptor binding, inhibited the HMG-CoA reductase activity in familial hypercholesterolemic cells by reversible phosphorylation. Measurement of the short-term effects of inhibitors on the rate of cholesterol synthesis from radiolabeled acetate revealed that HMG-CoA reductase phosphorylation was responsible for rapid suppression of sterol synthesis. Reductase kinase activity of cultured fibroblasts was also affected by reversible phosphorylation. The active (phosphorylated) reductase kinase can be inactivated by dephosphorylation with phosphatase. Inactive reductase kinase can be reactivated by phosphorylation with ATP-Mg and a second protein kinase from rat liver, designated reductase kinase kinase. Reductase kinase kinase activity has been shown to be present in the extracts of cultured fibroblasts. The combined results represent the initial demonstration of a short-term regulation of HMG-CoA reductase activity and cholesterol synthesis in normal and receptor-negative cultured fibroblasts involving reversible phosphorylation of both HMG-CoA reductase and reductase kinase.

The effect of portacaval shunt on hepatic lipoprotein metabolism in familial hypercholesterolemia

The hyperlipidemia observed in familial hypercholesterolemia can be reduced by portacaval anastomosis. We report the effects of a portacaval shunt on hepatic morphology and biosynthetic pathways crucial to hepatic cholesterol homeostasis in homozygous receptor-negative familial hypercholesterolemia. Portacaval anastomosis was associated with a dramatic change in hepatocyte morphology, 28% reduction in plasma low-density lipoprotein concentration, and a decrease in hepatic total and free cholesterol content by 27 and 75%, respectively. Furthermore, the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase was decreased by 56%. Finally, the reduced binding of low-density lipoproteins to hepatic membranes preoperatively was increased following the portacaval shunt. These combined results indicate that the changes in circulating lipoprotein concentrations observed after portacaval shunt are due to alterations in the metabolic consequences of the defective recognition of lowdensity lipoproteins by the liver of familial hypercholesterolemic subjects.

Differential effect of platelet inhibitors in normal and in hypercholesterolaemic subjects.

Dibutyryl cyclic AMP, forskolin, dipyridamole and butyl imidazole inhibited platelet aggregation (induced by ADP or collagen) in washed platelets more than in platelet-rich plasma preparations. Aspirin, indomethacin and epoprostenol (prostacyclin, PGI2) showed no preferential inhibition of these platelet preparations. When platelet-rich plasma from either normal or familial hypercholesterolaemic (FH) subjects was used, aspirin, indomethacin and dipyridamole (but not forskolin) inhibited platelet aggregation in normal subjects more than in FH patients. When low doses of aspirin (75 mg daily for 7 days) or dipyridamole (250 mg, single dose) were administered in vivo, platelet aggregation was inhibited more in the normal subjects in comparison to the patient group.

Sterol synthesis in vitro in freshly isolated blood mononuclear leukocytes from familial hypercholesterolemic patients treated with probucol

Sterol synthesis rates were measured in freshly isolated blood mononuclear leukocytes obtained from familial hypercholesterolemic patients undergoing treatment with either probucol alone or probucol plus cholestyramine. Subjects with heterozygous familial hypercholesterolemia on probucol had a significant 31% reduction in mononuclear cell sterol synthesis rates as compared to control patients; sterol synthesis in cells from homozygous familial hypercholesterolemic patients on probucol did not differ from that in control subjects. Addition of Cholestyramine to probucol therapy in heterozygous familial hypercholesterolemic patients caused an increase in sterol synthesis rates equal to but not greater than control values, thus negating the decreased mononuclear leukocyte sterol synthesis associated with probucol administration alone. Probucol treatment effectively decreased plasma cholesterol levels in both homozygous and heterozygous familial hypercholesterolemic subjects; however, the data suggest that the drug may exert different effects on sterol synthesis in peripheral tissues depending upon the presence or absence of cellular receptors for low-density lipoproteins.

Native and modified low-density-lipoprotein interaction with human platelets in normal and homozygous familial-hypercholesterolaemic subjects.

The binding of low-density lipoproteins (LDL) as well as LDL modified by cyclohexanedione (CHD-LDL) to gel-filtered platelets (GFP) and its effect on platelet function were studied in normal and in homozygous familial hypercholesterolaemic (HFH) subjects. Only normal-derived LDL could significantly compete with normal 125I-labelled LDL for binding to normal platelets. When GFP from normal subjects were incubated with normal LDL at concentrations of 25-200 micrograms of protein/ml, platelet aggregation in the presence of thrombin (0.5 i.u./ml) was increased by 65-186%. CHD-LDL, at similar concentrations, caused the opposite effect and decreased platelet aggregation by 26-47%. Both LDL and CHD-LDL (100 micrograms/ml) from HFH patients, when incubated with normal GFP, caused a significant reduction in platelet aggregation (33 and 50% respectively). When HFH-derived platelets were used, both patient LDL and CHD-LDL (but not the normal lipoprotein) could markedly compete with the patient 125I-labelled LDL for binding to the platelets. LDL and CHD-LDL (100 micrograms/ml) from normal subjects decreased aggregation of HFH-platelets by 52 and 85% respectively, while corresponding concentrations of LDL derived from HFH subjects (HFH-LDL) and CHD-LDL derived from HFH subjects (CHD-HFH-LDL) increased platelet aggregation by 165 and 65% respectively. The present results support the following conclusions: platelet activation by LDL in normal subjects is through the arginine-rich apoprotein-binding site; more than one binding site for LDL exists on platelets; under certain circumstances, LDL binding can cause a reduction in platelet activity; specificity for LDL binding to the platelets resides in different regions of the lipoprotein in HFH and in normal subjects. We have thus suggested a model for LDL-platelet interaction in normal and in HFH subjects.

The absolute rate of cholesterol biosynthesis in monocyte-macrophages from normal and familial hypercholesterolaemic subjects.

The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.

Characterization of hepatic low density lipoprotein binding and cholesterol metabolism in normal and homozygous familial hypercholesterolemic subjects.

Patients with familial hypercholesterolemia have elevated levels of plasma low density lipoproteins (LDL), increased hepatic synthesis of apolipoprotein B-containing lipoproteins, defective binding of low density lipoproteins to fibroblasts, and premature atherosclerosis. The role of a hepatic low density lipoprotein receptor in normal man and its importance in the pathogenesis of familial hypercholesterolemia have not been previously determined. In the present study, direct comparison was made of the binding of LDL to hepatic membranes from normal and receptor-negative homozygous familial hypercholesterolemic subjects. The effects of calcium, EDTA, and temperature on the binding of lipoproteins to the hepatic membranes were also evaluated. At 4 degrees C, no significant difference in specific binding of LDL to hepatic membranes from normal and familial hypercholesterolemic subjects was observed. At 37 degrees C, both total and specific binding of LDL were significantly reduced in patients with familial hypercholesterolemia. Hepatic membrane binding of LDL from the two patients homozygous for receptor-negative familial hypercholesterolemia was 53 and 59% of normal. The activity of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase was normal; however, the total hepatic cholesterol and cholesteryl ester content was significantly increased from 53 to 129%. These results indicate that patients with familial hypercholesterolemia have a defect in the interaction of hepatic membranes with low density lipoproteins. This defect may lead to accelerated atherosclerosis by decreasing the cellular catabolism of LDL and enhancing the production of LDL, which is characteristic of patients homozygous for familial hypercholesterolemia.

Sucrose polyester: substitution for dietary fats in hypocaloric diets in the treatment of familial hypercholesterolemia

In five obese women heterozygous for familial hypercholesterolemia, we assessed the combination of weight loss and sucrose polyester (SPE) in lowering low-density lipoprotein cholesterol (LDLC). After a 10-day basal hypocaloric (1426 cal/day), 270 mg cholesterol, P/S 1.2:1 diet, an average of 36 g of dietary fat/day was replaced by 36 g of an 80/20 SPE-hydrogenated palm oil mixture, providing 30 g SPE for 30 days; during the SPE substitution period mean dietary cholesterol and P/S were unchanged, mean caloric intake was 1104 cal/day. During the hypocaloric basal diet, mean weight fell 1.2 kg, p < 0.02, total plasma cholesterol fell 8% from 358 ± 46 to 330 ± 47 mg/dl, p < 0.01, LDLC fell 4% from 264 ± 37 to 254 ± 44 mg/dl, p > 0.1, and mean high-density lipoprotein cholesterol fell 11%, from 52 ± 4 to 46 ± 4, p < 0.05. Over the 30-day SPE substitution, mean cholesterol fell 20% from 330 ± 47 at the end of the basal diet to 265 ± 42 mg/dl, p < 0.001; mean LDLC fell 23%, from 254 ± 44 to 195 ± 41 mg/dl (p < 0.01); weight fell 4%, p < 0.01, from 91 ± 7 to 87 ± 7 kg, and mean high-density lipoprotein cholesterol fell 11% from 46 ± 4 to 41 ± 2, p < 0.05. Hypocaloric removal of dietary fat by SPE, an artificial fat with culinary properties of conventional dietary fats, effectively reduces LDLC (by 23%) in familial hypercholesterolemia subjects, with additive effects of SPE and weight loss.

Changes in the metabolism of modified and unmodified low-density lipoproteins during the maturation of cultured blood monocyte-macrophages from normal and homozygous familial hypercholesterolaemic subjects.

Blood monocyte-macrophages from homozygous familial hypercholesterolaemic (FH) patients showed the same pattern of morphological and biochemical changes during maturation in vitro as cells from normal subjects. The protein content of both types of cell increased eightfold during the first two weeks of culture while the ratio of free cholesterol to protein in the cells remained constant. The specific activities of lysosomal enzymes increased between days 2 and 5 and again between days 7 and 12. The specific activity of 5′-nucleotidase increased rapidly during the first two days and then fell. Degradation of low-density lipoprotein (LDL) by cells from FH patients was not saturable and, when expressed per mg cell protein, gradually declined as the cells matured. Normal cells maintained in medium containing whole serum degraded LDL by an additional, saturable process that was, at all times, decreased if the cells were preincubated in medium with a high concentration of LDL and increased after preincubation with lipoprotein-deficient serum. Saturable degradation of LDL, expressed per mg cell DNA, was highest between the fifth and twelfth days of culture, which was the period of greatest cell growth. The results suggest that normal cells incubated in 20% whole serum expressed LDL receptors to obtain the cholesterol required for their growth, but that the lack of these receptors in cells from FH patients did not prevent normal development. Saturable degradation of acetylated LDL, or LDL complexed with dextran sulphate, was the same in cells from FH patients as in cells from normal subjects. The degradation of each was lower than that of unmodified LDL for the first 5 days in culture, but rapidly increased between days 5 and 9. This increase corresponded with the development of the phagocytic properties of the cells, as indicated by the release of lysosomal enzymes during incubation with opsonized zymosan.

Changes in plasma lipid and lipoprotein fractions after alteration in dietary cholesterol, polyunsaturated, saturated, and total fat in free-living normal and hypercholesterolemic children

To assess the effects of dietary cholesterol and the amount and type of fat on plasma lipid and lipoproteins, nutrient intakes were altered sequentially over 15 months in 11 normal children and 12 children with heterozygous familial hypercholesterolemia. After a 3-month base-line assessment period, on an ad libitum diet, the following diets were given sequentially for three months each: dietary cholesterol greater than 450 mg/day, total fat less than 35% of total calories, and polyunsaturated fat to saturated fat ratio (P/S) greater than 1.5 (diet 1); dietary cholesterol less than 160 mg/day, total fat less than 35% total calories and P/S greater than 1.5 (diet 2); dietary cholesterol less than 160 mg/day, total fat 40% total calories P/S = 1 (diet 3), and dietary cholesterol greater than 450 mg/day total fat greater than 40% total calories, P/S less than 0.04 (diet 4). In normal and familial hypercholesterolemic children the high dietary P/S ratio lowered total and low-density lipoprotein cholesterol in the presence of high dietary cholesterol; sharp reductions in dietary cholesterol lowered the total and low-density lipoprotein cholesterol slightly in familial hypercholesterolemia subjects when P/S was high. High-density lipoprotein cholesterol was not affected by large changes in dietary cholesterol or amount or type of fat. Sustained dietary alteration which significantly lowers total and low-density lipoprotein cholesterol with commercially available products is achievable and practical in free-living children.

Degradation by cultured monocyte-derived macrophages from normal and familial hypercholesterolaemic subjects of modified and unmodified low-density lipoproteins.

Human blood monocyte-derived macrophages that had been cultured for 7 days in the presence of 20% whole human serum exhibited saturable degradation of low-density lipoprotein (LDL). This degradation could be abolished by pre-incubating the cells with a high concentration of LDL in the medium and increased by pre-incubating the cells in medium containing lipoprotein-deficient serum. Cells obtained from the blood of homozygous familial-hypercholesterolaemic (FH) patients only exhibited a low rate of non-saturable degradation of LDL, even when pre-incubated without lipoproteins. Thus the saturable degradation of LDL by normal cells was mediated by the LDL receptors that are defective in FH patients and little LDL was taken up and degraded through any of the other endocytotic processes present in macrophages. Degradation by normal cells pre-incubated with lipoprotein-deficient serum had a higher apparent affinity for LDL than that of cells maintained in whole serum, which suggests that incubation with lipoprotein-deficient serum may not only induce the formation of LDL receptors but may also have a direct effect on the receptors themselves. Monocyte-derived macrophages from normal and FH subjects showed similar saturable degradation of acetylated LDL and also of LDL complexed with dextran sulphate. Maximal degradation of each was in the same range as the degradation of unmodified LDL by normal cells, and was not increased if the cells were pre-incubated with lipoprotein-deficient serum.

Increased urinary excretion of glycosphingolipids in familial hypercholesterolemia.

The content of glycosphingolipids (GSL) was studied in the urinary sediments (24-hr specimens) from seven normal subjects, a patient with Fabry's disease, and five homozygotes with familial hypercholesterolemia (FH). Normal urinary sediments contained very small amounts of GalCer, GlcCer, GaOse(2)Cer, LacCer, GbOse(3)Cer, and GbOse(4)Cer. In Fabry urinary sediment, the levels (nmole glucose/24 hr) of GaOse(2)Cer and of GbOse(3)Cer were 389 and 550, respectively. In urinary sediments from the FH subjects, the mean contents (nmol glucose/mg protein per 24 hr) of GlcCer, GalCer, and LacCer were 2.7, 1.9, and 15.8 times higher, respectively, than in normals. The mean contents ( micro g/mg protein per 24 hr) of total cholesterol and phospholipid in the urinary sediment of FH (1.1 and 224, respectively) and normals (0.8 and 220) were similar. The mean contents of GlcCer, GalCer, and LacCer, expressed in terms of the cholesterol content of urinary sediment (nmol glucose/ micro g cholesterol per 24 hr), were increased 3.4-, 1.6-, and 5.4-fold, respectively, in the FH homozygotes. Of the five FH homozygotes, only one, who had undergone a portacaval shunt and was also receiving lipid-lowering therapy, had a normal value of LacCer. The other four FH homozygotes had levels of LacCer that were 3- to 55-fold higher (nmol glucose/mg protein per 24 hr) and 5.5- to 7.3-fold higher (nmol glucose/ micro g cholesterol per 24 hr) than the mean of the normals. One homozygote underwent plasma exchange therapy that reduced both the baseline urinary (nmol glucose/24 hr) and plasma (nmol/100 ml) LacCer levels from 86 to 7 and from 1491 to 852, respectively. Eleven days after plasma exchange, the urinary LacCer levels approached pre-exchange levels (59 nmol glucose/24 hr). The data indicate that there is an abnormality of GSL metabolism associated with familial hyper-cholesterolemia and that the urinary excretion of GSL can be modified by plasma exchange therapy.-Chatterjee, S., C. S. Sekerke, and P. O. Kwiterovich, Jr. Increased urinary excretion of glycosphingolipids in familial hypercholesterolemia.

The effect of human plasma on platelet function in familial hypercholesterolemia.

Using a platelet-rich plasma (PRP) preparation, platelets from subjects with familial hypercholesterolemia (FH) were found to be more reactive to the aggregating agents epinephrine, ADP and thrombin than platelets obtained from normal individuals. Gel-filtered platelets (GFP) i.e. platelets free of any plasma constituents also showed increased activation as determined by platelet aggregation and 14C-serotonin release. On incubating washed platelets from normal subjects with plasma obtained from FH subjects the platelet response to aggregating agents was significantly increased. Incubation of washed platelets from the FH patients with normal plasma, however, resulted in a significant decrease in platelet activity. Our data suggest that the increased platelet activation in FH patients is the result of a change in the platelets induced by abnormal plasma constituents.