Measurment of rhesus monkey (Macaca mulatta) apolipoprotein B in serum by radioimmunoassay: comparison of immunoreactivities of rhesus and human low density lipoproteins.

A sensitive and specific double antibody radio-immunoassay for the major apolipoprotein (apoB) of rhesus (Macaca mulatta) serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. The anti-serum was raised to LDL (d 1.030-1.040 g/ml) and the LDL(2) (d 1.020-1.050 g/ml) was labeled with (125)I by the chloramine-T or iodine monochloride method. The assay, which was sensitive to 0.02-0.5 micro g of LDL(2), had an inter-assay coefficient of variation of 4.5%. This assay was successfully used to measure apoB in the whole serum and low density lipoproteins of control monkeys maintained on a standard Purina monkey chow (PMC) diet and of three groups of monkeys fed atherogenic diets: an "average American diet," a 25% peanut oil and 2% cholesterol-supplemented PMC diet, and a 25% coconut oil and 2% cholesterol-supplemented PMC diet. The control monkeys (n = 13) had a serum cholesterol of 146 +/- 28 mg/dl and an apoB of 50 +/- 18 mg/dl. In the monkeys maintained on the atherogenic diets the serum apoB was elevated: 103 +/- 28 mg/dl (American), 102 +/- 35 mg/dl (peanut oil), and 312 +/- 88 mg/dl (coconut oil). The values for serum total cholesterol were 333 +/- 65 mg/dl (American), 606 +/- 212 mg/dl (peanut oil), and 864 +/- 233 mg/dl (coconut oil) and were elevated relative to controls (P < 0.001). For each of the diets, total serum cholesterol correlated with serum apoB (P < 0.001). The slopes of the regression lines of serum apoB vs. cholesterol for the monkeys on the PMC, American, and coconut oil diets were similar (m = 0.531, 0.401, and 0.359, respectively), but differed from that of monkeys on the peanut oil diet (m = 0.121). The immunoreactivities of rhesus and human LDL were compared using specific antisera raised against these antigens. In homologous assay systems, monkey and human LDL exhibited unique immunological determinants. The same results were obtained with the delipidated preparations of the two LDLs using antisera raised against either monkey or human apoB. Crossover studies using a heterologous tracer with each anti-serum resulted in the selection of a specific population of antibodies directed against antigenic sites shared by these two LDL species.     

Metabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia

Diet-induced hypercholesterolemia in non-human primates results in the production of a low-density lipoprotein (LDL) of abnormal size and composition. This LDL from hypercholesterolemic monkeys has been shown to be more atherogenic than the same amount of LDL from normocholesterolemic animals. Previous studies have demonstrated that hypercholesterolemic LDL is approximately twice as effective as normal LDL in stimulating cholesterol accumulation and esterification in arterial smooth muscle cells in culture. The purpose of the present study was determine whether this effect was secondary to differences in metabolism of the normal and hypercholesterolemic LDL. for this, the metabolism of 125I-labeled normal and hypercholesterolemic LDL from rhesus and cynomolgus monkeys was compared in several lines of skin fibroblasts and smooth muscle cells. Both normal and hypercholesterolemic LDL bound with high affinity to the same cell surface receptor. However, the affinity for binding of hypercholesterolemic LDL was about twice that of normal LDL (apparent dissociation constant for binding, Kd, was 2.63 micrograms protein/ml and 4.35 micrograms protein/ml, respectively). Conversely, only about 50% as many particles of hypercholesterolemic were able to bind to the receptor, compared with normal LDL. Those cells with the greatest capacity to metabolize LD generally accumulated the most cholesterol with either hypercholesterolemic or normal LDL. In all cell lines, nearly twice as much cholesterol accumulated in cells incubated with hypercholesterolemic LDL compared with normal LDL, and this differential could not be explained by differences in metabolism of the two lipoproteins, suggesting that some cholesterol entered the cells independent of the uptake of the intact LDL molecule. LDL receptors appear necessary for this to occur, since no difference in cholesterol accumulation was observed in cells genetically deficient in LDL receptors.     

Receptor-mediated binding and degradation of subfractions of human plasma low-density lipoprotein by cultured fibroblasts

The receptor-mediated metabolism of human plasma low-density lipoprotein (LDL) subfractions was studied. LDL was isolated from healthy donors and further fractionated by density gradient ultracentrifugation into three subfractions: (I) d = 1.031-1.037, (II) d = 1.037-1.041 and (III) d = 1.041-1.047 g/ml, comprising 24 +/- 7%, 46 +/- 8% and 30 +/- 9% of the total LDL protein, respectively. As assessed by electron microscopy and gradient gel electrophoresis, the LDL particle size decreased and the relative protein content increased from fraction I towards fraction III. Fraction II had the highest (Kd 2.6 micrograms/ml) and fraction I the lowest (Kd 5.8 micrograms/ml) binding affinity to LDL receptors of human fibroblasts at 4 degrees C. The rate of receptor-mediated degradation of fraction II was also higher than that of the other two fractions at 37 degrees C. These results suggest that LDL subfractions have different rates of receptor-mediated catabolism depending on particle size or composition, and therefore their metabolic fate and atherogenic properties may also differ.     

Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100

A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody. MB24, which binds an epitope in the amino terminal half of the apoB-100 and identifies both apoB-100 and apoB-48, is conjugated to horseradish peroxidase and is utilized as the indicating antibody. The assay was calibrated with LDL (d 1.030-1.050 g/ml) and the LDL protein was determined by a sodium dodecyl sulfate (SDS) Lowry procedure. The working range of the assay is 0.25-1.25 micrograms/ml. Optimal dilution of whole plasma was found to be 1:2000. In the assay, MB47 bound approximately 97% of the apoB in all low density lipoprotein, and greater than 90% of the apoB in the majority of very low density lipoprotein preparations. Small dense LDL from subjects with familial combined hyperlipidemia (FCHL) and large bouyant LDL from subjects with familial hypercholesterolemia (FH) exhibited binding properties similar to LDL from healthy normolipidemic subjects when tested in the reference ELISA. The intra- and interassay coefficients of variation averaged 2.5% and 6.0%, respectively. Plasma B-100 levels were not influenced by freezing and thawing or storage at 4 degrees C for up to 3 weeks or storage at -70 degrees C for up to 11 months. Excellent agreement was obtained between the reference ELISA and a polyclonal RIA which measures total apoB (r = 0.93, n = 105, mean ELISA B-100 value = 100 mg/dl, mean RIA value = 101 mg/dl, Sy = 9.6). Reference ELISA B-100 values of samples pretreated with bacterial lipase were not significantly increased in most samples with plasma triglyceride levels below 600 mg/dl. To help reduce the large among-laboratories variability of apoB measurements, we recommend that this candidate reference direct binding ELISA be used to assign apoB target values to apoB reference pools.     

Characterization of apolipoprotein B-containing lipoproteins separated by preparative free flow isotachophoresis

Preparative free flow isotachophoresis (ITP) was used for the fractionation of apoB-containing lipoproteins (d less than 1.063 g/ml) from fasting and postprandial sera derived from normolipidemic individuals. According to their net electric mobility, four major particle groups (I-IV) have been recognized. The fast-migrating particles in group I, which correspond predominantly to very low density lipoproteins (VLDL), are rich in triglycerides, free cholesterol, phosphatidylcholine, and apoE and C apolipoproteins. This group expresses nonspecific binding to fibroblasts but binds to HepG2 cells with high affinity (KD = 3.6 micrograms/ml, Bmax = 37 ng) to a single class of binding sites. The particles migrating in group II, which are related to intermediate density lipoproteins (IDL), are richer in cholesteryl esters and apoB than those in group I. They interact specifically with a single site on fibroblasts (KD = 7.8 micrograms/ml, Bmax = 54 ng) while on HepG2 cells two binding sites, one with a higher (KD = 3.5 micrograms/ml, Bmax = 22 ng) and one with a lower affinity component (KD = 16.9 micrograms/ml, Bmax = 53 ng), are involved. The particles migrating in groups III and IV correspond to low density lipoproteins (LDL). The protein moiety of both fractions consists almost exclusively of apoB. Group III represents cholesteryl ester-rich LDL particles, while the particles in group IV contain smaller amounts of cholesteryl esters. The lipoproteins of both groups are ligands for apoB,E-receptors. However, the particles in group IV interact with fibroblasts with the highest affinity (KD = 2.3 micrograms/ml, Bmax = 58 ng) and with the biphasic HepG2 cell binding sites with the lowest affinity of all analyzed groups (KD1 = 11.2 micrograms/ml, Bmax1 = 58 ng, KD2 = 68 micrograms/ml, Bmax2 = 170 ng). When apoB-containing lipoproteins were isolated from postprandial sera of the same individuals, significant changes in the lipid composition were observed only in particle groups I and II, where the triglyceride and phospholipid content was enhanced. Group I particles from postprandial serum bind to HepG2 cells with a higher affinity (KD = 2.5 micrograms/ml) than group I particles from fasting serum. Postprandial group II particles bind with the same affinity to the biphasic HepG2 cell receptor as fasting group II particles, while the affinities of postprandial group III (KD1 = 4.1 micrograms/ml, KD1 = 47 micrograms/ml) and group IV particles (KD1 = 3.9 micrograms/ml, KD2 = 38 micrograms/ml) to the high affinity binding site of the biphasic receptor are enhanced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

In vivo metabolism of LDL subfractions in patients with heterozygous FH on statin therapy: rebound analysis of LDL subfractions after LDL apheresis

LDL can be subfractionated into buoyant (1.020-1.029 g/ml(-1)), intermediate (1.030-1.040 g/ml(-1)), and dense (1.041-1.066 g/ml(-1)) LDLs. We studied the rebound of these LDL-subfractions after LDL apheresis in seven patients with heterozygous familial hypercholesterolemia (FH) regularly treated by apheresis (58 +/- 9 years, LDL-cholesterol = 342 +/- 87 mg/dl(-1), triglycerides = 109 +/- 39 mg/dl(-1)) and high-dose statins. Apolipoprotein B (apoB) concentrations were measured in LDL subfractions immediately after and on days 1, 2, 3, 5, and 7 after apheresis. Compartmental models were developed to test three hypotheses: 1) that dense LDLs are derived from the delipidation of buoyant and intermediate LDLs (model A); 2) that dense LDLs are generated directly from LDL-precursors (model B); or 3) that a model combining both pathways (model C) is necessary to describe the metabolism of dense LDLs. In all models, it was assumed that apoB production and fractional catabolic rate (FCR) did not change with apheresis. Apheresis decreased buoyant, intermediate, and dense LDL-apoB by 60 +/- 12%, 67 +/- 5%, and 69 +/- 11%, respectively. Models B and C, but not model A, described the rebound data. The model with the greatest biological plausibility (model C) was used to estimate metabolic parameters. FCR was 1.05 +/- 0.86 d(-1), 0.48 +/- 0.11 d(-1), and 0.69 +/- 0.24 d(-1) for buoyant, intermediate, and dense LDLs, respectively. Dense LDL production was 17.3 +/- 0.2 mg/kg(-1)/d(-1), 58% of which was derived directly from LDL precursors (VLDL, IDL, or direct secretion), while 42% was derived from buoyant and intermediate LDLs. Thus, our data indicate that in statin-treated patients with heterozygous FH dense LDLs originate from two sources. Whether this is also valid in other metabolic situations (with predominant small, dense LDLs) remains to be determined.     

Native low-density lipoproteins stimulate leukotriene B4 production by human monocyte-derived macrophages.

We have evaluated the effect of native low-density lipoproteins (LDL) on the production of leukotriene B4 (LTB4), a potent inflammatory and chemotactic factor, by human monocyte-derived macrophages. The capacity of LDL (d, 1.024-1.050 g/ml) to increase LTB4 secretion was dose-dependent with an optimal response at 100 micrograms LDL protein/ml, representing an approx. 7.5-fold stimulation over basal levels at 10 days of culture; the half-maximal response occurred at 20 micrograms/ml. The effect of LDL on LTB4 production was rapid (within 15 min) and was maintained for at least 21 h. The generation of LTB4 in response to LDL was partially inhibited (approx. 70% inhibition) by EDTA (5 mM) and by a monoclonal antibody (IgG-C7; 160 micrograms/ml) directed against the binding site of the cellular LDL receptor. In addition, the effects of native LDL and acetylated LDL were additive. These findings suggest that the specific interaction of LDL with its high affinity receptor represents a major component in the stimulation of the production of LTB4 by human monocyte-derived macrophages.     

Study of abnormal plasma low-density lipoprotein in rhesus monkeys with diet-induced hyperlipidemia.

Male rhesus monkeys were divided into three groups: five were fed a regular primate chow diet and were used as controls; four received an "average" American diet; and five a special low-fat primate chow diet supplemented with 25% coconut oil and 2% cholesterol. In all of these animals, the plasma low-density lipoproteins (LDL) were isolated by ultracentrifugal flotation between densities of 1.019 and 1.050 g/ml. The LDL of the five control monkeys had variable molecular weights, with a mean value of 3.12 +/- 0.21 X 10(6) (range: 2.92 X 10(6) to 3.45 X 10(6)), and an average partial specific volume of 0.969 +/- 0.003 ml/g; both were assessed by flotation equilibrium analysis in the analytical ultracentrifuge. In the individual animals, however, the physical properties of LDL were invariant with time. The administration of either an "average" American diet or a coconut oil-cholesterol diet was accompanied by hypercholesterolemia associated with changes in LDL which were characterized by increases in molecular weight to 3.52 +/- 0.21 X 10(6) (average of nine monkeys) and in partial specific volume to 0.973 +/- 0.002 ml/g. These changes were particularly evident when the molecular weight of LDL from monkeys in the normolipidemic state was compared with that obtained from the same monkeys during the hyperlipidemic state. Chemical analyses revealed that the particles from the hyperlipidemic animals had a relatively higher cholesteryl ester content, a slight increase in phospholipids, and a marked decrease to nearly complete absence of triglycerides. The other lipoprotein components, protein, carbohydrate, free cholesterol, and fatty acids, did not vary significantly from those of control LDL. It is concluded that the administration of atherogenic diets causes structural changes in LDL which appear to be accounted for, at least in part, by changes in the composition of the lipid moiety. The changes in physical and chemical properties noted in the LDL of rhesus monkeys with experimentally induced hypercholesterolemia contrast with the apparent structurally normal LDL from rhesus monkeys with spontaneous hypercholesterolemia reported previously.     

Serum thyroxine and triiodothyronine radioimmunoassay values in the normal ferret

The European ferret, Mustela putorius furo, has become increasingly popular as an animal model in biomedical research. However, certain important normal clinical data have not been established for the ferret. In this study, serum thyroxine (T4) and 3,3',5-triiodothyronine (T3) values were obtained from ferrets by the use of commercial radioimmunoassays. Sera from 44 animals, 31 males (27 intact and 4 castrated) and 13 females (10 intact and 3 spayed) were assayed. Serum T4 values ranged from 1.01-8.29 micrograms/dl for males (mean = 3.24 +/- 1.65 micrograms/dl), and 0.71-3.43 micrograms/dl for females (mean = 1.87 +/- 0.79 micrograms/dl). Serum T4 values of adult female ferrets, juvenile ferrets (less than 1 year old) of either sex, and castrated males were similar to the normal T4 values of the cat, 1.20-3.80 micrograms/dl. Intact adult male ferrets had higher serum T4 values which were more comparable to those of the normal dog 1.52-3.60 micrograms/dl. Serum T3 values ranged from 0.45-0.78 ng/ml for males (mean = 0.58 +/- 0.09 ng/ml), and 0.29-0.73 ng/ml for females (mean = 0.53 +/- 0.13 ng/ml). These values are comparable to those of dogs and cats which are 0.50-1.50 ng/ml.     

The intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts

Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol.     

Effects of lead on luteal function in rhesus monkeys

Exposure to lead in the workplace or home environment has been implicated as a cause of decreased fertility in women. In a previous study, as part of our effort to determine effects of lead in primates, female rhesus monkeys were exposed to lead acetate in drinking water (n = 10) or provided water with no added lead (n = 7) for 33 mo. Lead was administered at levels between 2 and 8 mg/kg/day, with doses adjusted to keep blood lead values near a target of 70 micrograms/dl (observed mean +/- SEM = 68.9 +/- 6.54 micrograms/dl). Blood lead concentrations in control animals were less than 10 micrograms/dl. No significant differences were detected between control and experimental animals in body weight, hematocrit, or general health. Female monkeys receiving lead exhibited longer and more variable menstrual cycles and shorter menstrual flow. In the present study, circulating amounts of progesterone (P4) were determined to evaluate luteal function during the final 7 mo of treatment with lead. Several characteristics were altered as a result of lead treatment: circulating amounts of P4 were reduced as indicated by relative units of area under the concentration-time curve, maximal amounts of P4 were reduced, and P4 levels were greater than 1 ng/ml on fewer days. There were no significant differences between groups in mean percent of anovulatory cycles. Therefore, although chronic treatment with the levels of lead used in this study did not prevent ovulation, luteal function was suppressed. These results extend previous observations of adverse effects of lead on ovarian activity and fertility in monkeys.     

Characterization of low density and high density lipoprotein receptors in the rat corpus luteum and regulation by gonadotropin

Freshly prepared plasma membranes from rat corpora lutea were examined for the presence of low density lipoprotein (LDL) and high density lipoprotein (HDL) receptors by determining the specific binding of 125I-LDL and 125I-HDL. These membranes have two types of binding site for 125I-LDL, one with high affinity (Kd = 7.7 micrograms of LDL protein/ml), the other with low affinity (Kd = 213 micrograms of LDL protein/ml) and one type of binding site for 125I-HDL with Kd = 17.8 micrograms of HDL protein/ml. LDL receptor is sensitive to pronase and trypsin; HDL receptor, however, is resistant. The binding reaction was further characterized with respect to effect of time and temperature of incubation, requirement of divalent metal ion, influence of ionic strength, and binding specificity. In vivo pretreatment of rats with human choriogonadotropin (hCG) resulted in induction of both LDL and HDL receptors in a dose- and time-dependent manner when compared with saline-injected controls. The induction of lipoprotein receptors by hCG treatment is target organ-specific since the increase was seen only in the ovarian tissue. Membranes prepared from liver, kidney, and heart did not show an increase in lipoprotein receptors after hCG injection. An examination of the equilibrium dissociation constants for 125I-LDL and 125I-HDL binding after hCG administration revealed that the increase in binding activity was due to an increase in the number of binding sites rather than to a change in the binding affinity. In conclusion, rat corpus luteum possesses specific receptors for both LDL and HDL and these receptors are regulated by gonadotropins.     

Iodide-induced hypothyroidism in a patient with anorexia nervosa

A 39-year-old woman who had been suffering from anorexia nervosa was found to have hypothyroidism. Serum T4, free T4, T3, free T3 and TSH were 3.19 micrograms/dl, 0.5 ng/dl, 15.3 ng/dl, 1.2 pg/ml and 162.1 microU/ml, respectively. On careful questioning, she was found to have taken an iodine-rich diet. The serum iodine concentration was 122 micrograms/dl (normal: 4-9 micrograms/dl) and urinary iodide excretion was 13.05 mg/day (normal: less than 2 mg). After withdrawal of the iodine-rich diet, her serum T4 gradually increased and TSH returned to the normal range. She was diagnosed as having iodide-induced hypothyroidism. However, no significant elevation of serum T3 or free T3 was observed. Serum T4, free T4, T3, free T3 and TSH were 7.85 micrograms/dl, 0.8 ng/dl, 13.6 ng/dl, 4.3 pg/ml and 6.02 microU/ml, respectively. The iodide-perchlorate discharge test result was negative. These findings suggest that there exists some unknown mechanism by which a patient with anorexia nervosa may be sensitive to excess iodide. Furthermore, it is of interest to note that in a recovery phase from the hypothyroid state, normalization of serum T4 rather than T3 is well-correlated to TSH secretion.     

Characterization of human apolipoprotein B100 oligosaccharides in LDL subfractions derived from normal and hyperlipidemic plasma: deficiency of alpha-N-acetylneuraminyllactosyl-ceramide in light and small dense LDL particles

The carbohydrate composition of apolipoprotein (apo) B100, particularly its degree of sialylation, may contribute to the atherogenic properties of low-density lipoprotein (LDL). We analyzed LDL apoB100 glycans derived from normolipidemic, hypercholesterolemic, and hypertriglyceridemic diabetic subjects. Using exoglycosidase carbohydrate sequencing and matrix-assisted laser desorption/ionization mass spectrometry to analyze fluorescently labeled oligosaccharides, we report evidence for several carbohydrates not previously identified on apoB100, including truncated complex biantennary N-glycans and hybrid N-glycans. The distribution and diversity of the apoB100 glycans isolated from all individuals was highly conserved. The N-glycan composition of apoB100 derived from five LDL subpopulations (LDL1, d = 1.018-1.023; LDL2, d = 1.023-1.030; LDL3, d = 1.030-1.040; LDL4, d = 1.040-1.051; LDL5, d = 1.051-1.065 g/ml) did not vary in normolipidemic or hypercholesterolemic subjects. Furthermore, we found no evidence for "desialylated" apoB100 glycans in any of the samples analyzed. Analysis of the most abundant LDL ganglioside, alpha-N-acetylneuraminyllactosyl-ceramide, revealed a deficiency in small dense LDL and in the most buoyant subpopulation. These data provide a novel explanation for the apparent deficiency of sialic acid in small dense LDL and indicate that the global apoB100 N-glycan composition is invariable in the patient groups studied.     

Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred

Lecithin-cholesterol acyltransferase mass levels and activity and apolipoproteins A-I, A-II, B and D were measured in a Japanese family who have a familial lecithin-cholesterol acyltransferase deficiency. This analysis was performed to gain insight into the molecular basis of the enzyme deficiency and to compare findings in this family with other families with familial lecithin-cholesterol acyltransferase deficiency. The mass of the enzyme in plasma was determined by a sensitive double antibody radioimmunoassay, and enzyme activity was measured by using a common synthetic substrate comprised of phosphatidylcholine, cholesterol and apolipoprotein A-I liposomes prepared by a cholate dialysis procedure. The lecithin-cholesterol acyltransferase-deficient subject had an enzyme mass level that was 35% of normal (2.04 micrograms/ml, as compared with an average normal level of 5.76 +/- 0.95 micrograms/ml in 19 Japanese subjects) and an enzyme activity of less than 0.1% of normal (0.07 nmol/h per ml, as compared with normal levels of 100 nmol/h per ml). This subject also had lower levels of apolipoproteins: apolipoprotein A-I was 53 mg/dl (42% of normal), apolipoprotein A-II was 10.6 mg/dl (31% of normal), apolipoprotein B was 68 mg/dl (68% of normal), and apolipoprotein D was 3.6 mg/dl (60% of normal). The three obligate heterozygotes had enzyme mass levels ranging from 65% to 100% of normal and enzyme activity levels ranging from 23% to 65% of normal (23.4, 56.8, and 64.7 nmol/h per ml, respectively). The proband's sister had an enzyme mass level of 6.55 micrograms/ml (114% of normal) and an enzyme activity of only 64.8 nmol/h per ml (65% of normal), suggesting that she was also a heterozygote for lecithin-cholesterol acyltransferase deficiency. The obligate heterozygotes and the sister had normal apolipoprotein levels. We conclude that the lecithin-cholesterol acyltransferase deficiency in this family is due to the production of a defective enzyme that is expressed in the homozygote as well as in the heterozygotes, and, further, that this family's mutation differs from that reported earlier for other Japanese lecithin-cholesterol acyltransferase-deficient families.     

Impaired plasma cholesteryl ester transfer with accumulation of larger high density lipoproteins in some families of baboons (Papio sp.)

Baboons from some families have a higher concentration of plasma high density lipoproteins (HDL) on a chow diet and accumulate large HDL (HDL1) when challenged with a high cholesterol and high saturated fat (HCHF) diet. HDL1 from high HDL1 animals contained more (1.5-fold) cholesteryl ester than HDL (HDL2 + HDL3) from high or low HDL1 animals. HDL from high HDL1 baboons had lower triglyceride content than that from low HDL1 baboons. HDL3 or HDL labeled with [3H]cholesteryl linoleate was incubated with entire lipoprotein fraction (d less than 1.21 g/ml) or very low density lipoprotein + low density lipoprotein (VLDL + LDL) (d less than 1.045 g/ml) and with lipoprotein-deficient serum (LPDS), and the radioactive cholesteryl ester and mass floating at d 1.045 g/ml (VLDL + LDL) after the incubation was measured. The transfer of cholesteryl esters from either HDL or HDL3, prepared from plasma of high HDL1 animals fed chow or the HCHF diet, was slower than the transfer from either HDL or HDL3 of low HDL1 animals, regardless of the source of transfer activity or the ratio of LDL:HDL-protein used in the assay. Addition of HDL from high HDL1 baboons into an assay mixture of plasma components from low HDL1 baboons decreased the transfer of cholesteryl ester radioactivity and mass from HDL to VLDL and LDL. In addition to HDL, a fraction of intermediate density lipoprotein (IDL) and denser HDL were also effective in inhibiting the transfer. These observations suggest that accumulation of HDL1 in high HDL1 baboons fed an HCHF diet is associated with a slower transfer of cholesteryl esters from HDL to LDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-mediated uptake of hypertriglyceridemic very low density lipoproteins by normal human fibroblasts

Our previous studies showed that very low density lipoproteins, Sf 60-400 (VLDL), from hypertriglyceridemia subjects, but not VLDL from normolipemic subjects, suppress HMG-CoA reductase activity in normal human fibroblasts. To determine if this functional abnormality of hypertriglyceridemic VLDL resulted from differences in uptake of the VLDL by the low density lipoprotein (LDL) receptor pathway, we isolated VLDL subclasses from the d less than 1.006 g/ml fraction of normal and hypertriglyceridemic plasma by flotation through a discontinuous salt gradient for direct and competitive binding studies in cultured human fibroblasts. VLDL from the plasma of subjects with hypertriglyceridemia types 4 and 5 were at least as effective as normal LDL in competing for 125I-labeled LDL binding, uptake, and degradation when compared either on the basis of protein content or on a particle basis. By contrast, normolipemic Sf 60-400 VLDL were ineffective in competing with the degradation of 125I-labeled LDL, and Sf 20-60 VLDL (VLDL3) were less effective in reducing specific 125I-labeled LDL degradation than were LDL, consistent with their effects on HMG-CoA reductase activity. In direct binding studies, radiolabeled VLDL from hypertriglyceridemic but not normolipemic subjects were bound, internalized, and degraded with high affinity and specificity by normal fibroblasts. Uptake and degradation of iodinated hypertriglyceridemic VLDL Sf 100-400 showed a saturable dependence on VLDL concentration. Specific degradation plateaued at approximately 25 micrograms VLDL protein/ml, with a half maximal value at 6 micrograms/ml. The most effective competitor of hypertriglyceridemic VLDL uptake and degradation was hypertriglyceridemic VLDL itself. LDL were effective only at high concentrations. Uptake of normal VLDL by normal cells was a linear rather than saturable function of VLDL concentration. By contrast, cellular uptake of the smaller normal VLDL3 was greater than uptake of larger VLDL and showed saturation dependence. After incubation of normal VLDL with 125I-labeled apoprotein E, reisolated 125I-E-VLDL were as effective as LDL in suppression of HMG-CoA reductase activity, suggesting that apoE is involved in receptor-mediated uptake of large suppressive VLDL. We conclude that 1) hypertriglyceridemic VLDL Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by the high affinity LDL receptor-mediated pathway; 2) by contrast, normal VLDL, Sf 60-400 are bound, internalized, and degraded by normal fibroblasts primarily by nonspecific, nonsaturable routes; and 3) of the normal VLDL subclasses, only the smallest Sf 20-60 fraction is bound and internalized via the LDL pathway.     

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 hepatocytes 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-defective (18% normal receptor activity). Cholesterol-depleted hepatocytes from normal subjects bound and internalized 125I-labeled LDL (Bmax = 2.2 micrograms LDL/mg cell protein). Preincubation of normal hepatocytes with 200 micrograms/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. Receptor-defective 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.     

Genetically determined hypercholesterolemia in a rhesus monkey family due to a deficiency of the LDL receptor

A family of rhesus monkeys comprising a sire, a dam, and four male offspring were fed a cholesterol-free Purina Chow diet for several months. The sire, 431-J, and two of the offspring, B-8204 and B-8806, had persistent plasma cholesterol levels in the range of 100-130 mg/dl, whereas the dam, 766-I, and the two other offspring, B-1000 and B-7643, exhibited a marked hypercholesterolemia in the 250-300 mg/dl range associated with an elevation of plasma LDL and apoB. When fed for 12 weeks a diet containing 12.5% lard and 0.25% cholesterol, sire, dam, B-1000 and B-7643 exhibited a marked hypercholesterolemia (500-800 mg/dl range), whereas B-8204 and B-8806 developed only a modest hypercholesterolemia (200-250 mg/dl). All animals were Lp[a]+. Skin fibroblasts from each animal and from control cells were grown in 10% fetal calf serum, transferred to 10% lipoprotein-deficient serum for 48 hr, and then incubated at 4 degrees C or 37 degrees C with 125I-labeled Lp[a]-free LDL. The fibroblasts from dam and offspring B-1000 and B-7643 bound and internalized 125I-labeled LDL less efficiently than control cells. Mathematical analyses of the 4 degrees C binding data indicated that there were no significant differences in LDL binding affinity between test and control cells suggesting that cells from the animals with a spontaneous hypercholesterolemia had a decreased number of LDL receptors. This conclusion was supported by the results of ligand and immunoblot analyses carried out on cell lysates separated by gradient gel electrophoresis. We conclude that a genetically determined LDL receptor deficiency was responsible, in part, for the spontaneous hypercholesterolemia observed in three out of the six family members and that this deficiency accounted for the hyperresponsiveness to a dietary fat and cholesterol challenge by the dam and the two offspring, B-1000 and B-7643. The hyperresponsiveness noted in the sire that had no evidence for LDL-receptor deficiency illustrates that factors other than the LDL receptor were responsible for the hypercholesterolemia attending the fat challenge.