Relation of genetic polymorphisms of apolipoprotein E, angiotensin converting enzyme, apolipoprotein B-100, and glycoprotein IIIa and early-onset coronary heart disease.

OBJECTIVE: Apolipoprotein E (APOE) E4, apolipoprotein B-100 (APOB) Q3611 allele, the angiotensin converting enzyme (ACE) deletion (D) allele and glycoprotein IIIa (GP3A) P33 mutant allele are reported to predispose to early-onset coronary heart disease (CHD). These associations were not all confirmed in more recent studies. To determine the impact of these alleles on CHD, we examined the prevalence of these mutations in patients presenting with early-onset CHD and compared them to those manifesting CHD later in life. The delayed-onset was considered a sign of longevity and would serve as a comparative group to assess prevalence of the biochemical and genetic risk factors. METHODS: 300 patients with a history of myocardial infarction or angina pectoris and angiographically documented CHD were studied. Patients were divided into two groups: group 1 (G1 = 150 patients) presenting with these findings under the age of 50 years; while group 2 (G2 = 150 patients) were patients presenting for the first time over the age of 65 years. Prevalence of the alleles of APOE, APOB, ACE and GP3A was assessed by molecular analysis. An association of any of these genotypes with early onset CHD could lead to a higher prevalence in the younger age group. RESULTS AND CONCLUSIONS: None of the suspected alleles namely APOB Q3611 [G1: 10.7% vs. G2: 9.0%, p = 0.57], ACE D (G1: 52.0% vs. G2: 49.7%, p = 0.57), or the GP3A P33 (G1: 17.3% vs. G2: 15.7%; p = 0.58) showed any significant difference between the two groups. Subjects with APOE E4 were more frequent in the younger age group (G1: 18.3% vs. G2: 13.7%; p = 0.047), while APOE E2 was more frequent in G2 (G2: 10.0% vs. G1: 2.7%; p = 0.0002). Multivariate analysis showed an odds ratio of APOE E2 allele in G1 of 0.27 with a confidence interval of 0.10-0.73.

Elevated LDL triglyceride concentrations in subjects heterozygous for the hepatic lipase S267F variant.

Although naturally occurring loss-of-function mutations in human hepatic lipase (HL) have been described, the biochemical phenotype of heterozygous HL deficiency remains ill defined. This may be due to the relatively small numbers of heterozygous adult carriers of HL mutations in index kindreds. We have identified several new heterozygotes for the catalytically inactive, nonsecreted HL variant S267F in the kindred that was originally ascertained because of hypertriglyceridemia due to the mutant, secreted, circulating apolipoprotein (apo) CII variant apo CII-T. Pairwise comparisons with family controls showed that only the plasma low density lipoprotein triglycerides (LDL TGs) were higher in 11 simple heterozygotes for HL S267F (P=0.002). In contrast, both plasma total TGs and LDL TGs were significantly higher in 12 simple heterozygotes for apo CII-T than in family-matched control subjects (P=0.005 and 0.009, respectively). These findings suggest that the TG content of LDL is increased by heterozygosity for 2 different mutations that affect different proteins involved in lipolysis. However, the mechanisms underlying this compositional change in LDL appear to be different for the 2 mutations, because the total TGs are also elevated in subjects heterozygous for apo CII-T but not in subjects heterozygous for HL S267F.

Human plasma CETP deficiency: identification of a novel mutation in exon 9 of the CETP gene in a Caucasian subject from North America.

Human plasma cholesteryl ester transfer protein (CETP) is a 476-residue hydrophobic glycoprotein that catalyzes the heterotransfer of cholesteryl esters and triacylglycerols among lipoproteins: Mutations in the CETP gene have been identified, mostly in the Japanese population. These mutations result in hypercholesterolemia due to the presence of large cholesteryl ester-rich HDL particles, elevated plasma apoA-I and apoE, and reduced apoB levels. Here we report the plasma lipoprotein phenotype and molecular defect in a 57-year-old female Nova Scotian subject lacking Japanese ancestry who is homozygous for a novel mutation in the CETP gene. Her total plasma cholesterol was 7.3 mmol/l with an LDL cholesterol of 2.9 mmol/l and HDL cholesterol of 4.4 mmol/l. She was mildly hypertriglyceridemic (1.6 mmol/l) and had markedly elevated apoA-I (256 mg/dl) and apoE (14.4 mg/dl) with only slightly reduced apo/B levels (94 mg/dl). Her VLDL and LDL were cholesteryl ester-poor (1.8 and 37.2% of lipids, respectively) and triacylglycerol-rich (67.3 and 18.9% of lipids, respectively) while her HDL was cholesteryl ester-rich (40.2-45.7% of lipids) and triacylglycerol-poor (3.3-2.5% of lipids). No plasma CETP activity or mass was detected. Bi-directional DNA sequence analysis of PCR products from all 16 exons showed a single base substitution (C-->T at nucleotide 836 in exon 9 resulting in 268 Arg-->STOP) in both alleles. No other mutation was detected. A single base mismatched, 26 bp reverse PCR primer that produced a single Mae III RFLP site upon amplification of the mutated DNA sequence was designed for rapid population screening. This subject is, we believe, the first Caucasian North American patient reported to have CETP deficiency.

Genetic variation in factor VII associated with variation in plasma lipoprotein(a) concentration.

Cross-sectional and prospective studies have shown that individuals with high plasma lipoprotein(a) [Lp(a)] concentrations are at increased risk for coronary heart disease. Size polymorphism of the apolipoprotein(a) [apo(a)] glycoprotein accounts for approximately 35% of the variation in plasma Lp(a) concentrations. However, there is no convincing evidence for associations between plasma Lp(a) and common genetic variation outside APO(a), the gene that encodes apo(a). We tested for association of common genetic variation of candidate genes in lipid metabolism and also of F7 with variation of plasma Lp(a) concentrations in Alberta Hutterites. Variation at codon 353 of F7 has been associated with variation in the plasma factor VII activity (FVIIc), with the 353Q allele associated with lower FVIIc and the 353R allele associated with higher FVIIc. We found significant associations between variation in plasma concentrations of Lp(a) and both apo(a) isoform size and F7 codon 353 genotype (both P < .0001). The effects on plasma Lp(a) concentration of the alleles at codon 353 were additive. The average effects of the F7 353Q and 353R alleles were, respectively, to decrease by 1.71 micrograms/mL and to increase by 0.301 microgram/mL plasma Lp(a) concentration from the sample mean. This suggests that common genomic variation in F7 is associated with variation in plasma Lp(a) concentration.

Effect of lipid transfer proteins on lipoprotein lipase induced transformation of VLDL and HDL.

Lipoprotein lipase-induced lipolysis of human plasma VLDL usually does not yield a complete conversion of VLDL to LDL due to insufficient loss of surface and core lipids and apolipoprotein E. In order to assess the role of lipid transfer proteins in this process human VLDL and apo E free HDL, in approximately physiologic proportions, and with sufficient albumin to bind all released fatty acids, were subjected to 90% lipolysis of triglycerides in 2 h by lipoprotein lipase in the presence or absence of partially purified human cholesteryl ester and phospholipid transfer proteins. Lipoprotein lipase caused a partial transfer of VLDL unesterified cholesterol (16%) and phospholipid (11%), apo E (19%) and almost complete transfer of apo CII and CIII to HDL. VLDL remnants possessed excess apo E and surface and core lipids when compared to plasma LDL, and densities ranging from that of VLDL/IDL to LDL. With addition of the lipid transfer proteins to the lipolysis incubation there was an increased transfer of phospholipid and unesterified cholesterol (2-fold) and apo E (1.6-fold) to HDL over that for lipoprotein lipase incubations. The source of transferred material was primarily from remnants which isolated in the LDL density range in lipoprotein lipase incubations. This transfer resulted in LDL-like particles which had a smaller particle size but lighter density compared to those in lipoprotein lipase incubation. Transfer of cholesteryl esters to VLDL from HDL in exchange for triglyceride was absent or substantially reduced in incubations containing lipoprotein lipase and lipid transfer proteins compared to incubations with only lipid transfer proteins. It is concluded that during rapid lipolysis lipid transfer proteins promote the loss of phospholipid, unesterified cholesterol and apo E from VLDL remnants but do not promote the transfer of cholesteryl ester from HDL to VLDL.

Secretion and processing of apolipoprotein A-I in the avian sciatic nerve during development.

Apolipoprotein A-I (apo A-I), a major apolipoprotein synthesized by liver and intestine to facilitate transport of plasma lipids as lipoproteins, has been detected also in the avian sciatic nerve. The mRNA and protein levels of apo A-I have been shown to increase during the period of rapid myelination (LeBlanc et al.: J Cell Biol 109:1245-1256, 1989). In order to assess the synthesis of apo A-I protein and the processing of apo A-I isoforms during development, endoneurial slices of avian sciatic nerves from chicks during active myelination at 15 and 17 days embryonic and 1 day posthatch age were incubated with [35]S-methionine. The incubations were fractionated into secreted and intracellular fractions, and incorporation of the label was assessed for apo A-I protein. The pattern of labeling of Po protein, as a marker of myelination, was also determined in the intracellular and compact myelin fractions. Methionine incorporation into Po protein was highest in the intracellular compartment at the 15-day embryonic stage and decreased thereafter, with a corresponding increase in the myelin fraction. During these developmental periods, the levels of nascent apo A-I increased in both the secreted and intracellular fractions. The synthesis of apo A-I specifically increases in the secreted fraction compared with total protein synthesis. The processing of the pro-apo A-I is also developmentally regulated. In the intracellular compartment, there are approximately equal proportions of the acidic and basic isoforms. However, with increasing age, a higher proportion of the apo A-I is secreted as acidic isoforms. It is concluded that the secretion and processing of apo A-I is developmentally regulated in the chick sciatic nerve, in parallel with the process of active myelination.

Influence of lipoprotein lipase and hepatic lipase on the transformation of VLDL and HDL during lipolysis of VLDL.

In order to study the relative effects of lipolytic enzymes on the removal of lipids and apolipoproteins, in particular apolipoprotein (apo) E and cholesteryl ester, from human very low density lipoprotein (VLDL) during its conversion to product lipoproteins, the action of lipoprotein lipase (LPL) and the combined action of lipoprotein lipase and hepatic lipase (HL) were studied in the presence of physiological proportions of high density lipoprotein (HDL) (10 mg protein), VLDL (2 mg protein) and albumin in an amount sufficient for the binding of all released fatty acids. The HDL used in the incubation was free of apo E in order to facilitate assessment of apo E transfer from VLDL to HDL. The redistribution of lipid and apolipoprotein mass and the movement of labeled cholesteryl ester from VLDL to other lipoprotein fractions was assessed by density gradient ultracentrifugation. Following 90%-95% lipolysis of VLDL triglycerides by rat heart LPL in 2 h, there was an almost complete transfer of apo C-II and apo C-III to HDL but only 20% of VLDL apo E was transferred to HDL. There was significant augmentation of HDL unesterified cholesterol and phospholipid mass during LPL action despite a substantial overall phospholipid hydrolysis (30%). The transfer of cholesteryl ester mass to HDL was variable (0%-13%) with a mean transfer of 7% of VLDL cholesteryl ester. Transfer of labeled VLDL cholesteryl ester to HDL was 3%-6%. A considerable amount of the VLDL lipid mass appeared in the light fraction of the low density lipoprotein (LDL) region, but a substantial amount remained in the VLDL/intermediate density lipoprotein (IDL) region. The post-lipolysis particles that were isolated in the VLDL-LDL density range were larger than LDL and contained a high ratio of surface lipids relative to core lipids as compared to plasma LDL. The inclusion of human HL with LPL did not alter the redistribution of apolipoproteins proteins or lipids from VLDL to LDL or to HDL. The major effect of HL, relative to that observed with LPL alone, was a marked hydrolysis of HDL triglycerides (68%). Despite the combined action of LPL and HL on VLDL in the presence of HDL and over 90% lipolysis of triglycerides, a major portion of residual VLDL mass remained in fractions lighter than normal LDL density and retained apo E. It is concluded that lipoprotein lipase of LPL in combination with HL are ineffective in bringing about the complete conversion of plasma VLDL to LDL. Lipoprotein lipase was effective in substantially augmenting the HDL mass including cholesteryl while the major effect of HL was the selective hydrolysis of HDL triglycerides.

Biosynthesis and compartmentalization of Po, apolipoprotein A-I, and lipids in the myelinating chick sciatic nerve.

Myelin deposition in developing chick sciatic nerve is associated with rapid synthesis of lipids, the major myelin protein Po and apo A-I, a major constituent of plasma lipoproteins. In order to understand possible roles of apo A-I in myelin assembly the synthesis and appearance of Po, apo A-I and lipids was studied in an intracellular fraction, an intralamellar fraction thought to be related to, or derived from, myelin and compact myelin from rapidly myelinating sciatic nerve of 1 day chicks. Incorporation with methionine or pulse-chase experiments indicated that initial synthesis of Po occurs in the intracellular fraction followed by movement to the intralamellar fraction and myelin. Incorporation of labelled oleate into phospholipids suggested that initial synthesis occurs in the intracellular and intralamellar fractions with slow movement to myelin. Incorporation of labelled galactose into cerebrosides suggested that initial synthesis occurs partially in myelin with slow loss from this fraction to the intralamellar fraction. However, incorporation of methionine into apo A-I indicated that initial synthesis occurred in the intracellular fraction with some transfer to the intralamellar fraction and secretion of a major portion into the incubation medium. It is concluded that the subcellular distribution of nascent apo A-I is not well coordinated with the distribution of other nascent constituents of the myelin membrane. The accumulation of nascent Po, phospholipids and cerebrosides in the intralamellar fraction compared to compact myelin suggests that this fraction may play a role as a precursor membrane or as a storage site for assembly of myelin constituents into compact myelin.

Post-translational modifications of apolipoprotein A-I and Po proteins in the avian peripheral nerve.

Apolipoprotein A-I (apo A-I), a soluble lipid transporter, and Po, the major glycoprotein of myelin, are actively synthesized during myelination. To explore the status of post-translational modifications of these proteins in the avian PNS during rapid myelination, endoneurial slices from one day old chick sciatic nerves were incubated with various radioactive precursors that could serve as indicators of such processes. The proteins were isolated from the incubation medium (secreted fraction), the 1% Triton-X-100-soluble intracellular-endoneurial (intracellular) fraction, and myelin-related and purified compact myelin fractions by immunoprecipitation with monospecific anti-apo A-I and or anti-Po antisera. Our results demonstrated that secreted apo A-I is fatty acylated, but not phosphorylated or sulfated. Avian Po protein was phosphorylated by a phorbol ester sensitive protein kinase. Sulfation, as well as fatty acylation, of avian Po protein was observed in organ culture using highly sensitive methods of detection. These results indicate that fatty acylation of secreted apo A-I and phosphorylation, sulfation and fatty acylation of Po have been conserved during evolution, and that these post-translational modifications may play a common function in various species.

Development of a density gradient ultracentrifugation technique for the resolution of plasma lipoproteins which avoids apo E dissociation.

A density gradient ultracentrifugation technique for analyzing and isolating plasma lipoproteins was developed that was simple to set up, allowed for the isolation of the plasma lipoproteins in one centrifugal spin, and avoided the dissociation of apolipoprotein E from high-density lipoprotein (HDL) which can occur when plasma is subjected to ultracentrifugation at high concentrations of salt. The density gradient emphasized the resolution of the HDL density region while still enabling the separation of low-density and very low-density lipoprotein.

Characterization of monoclonal antibodies to apolipoprotein (a) and development of a chemiluminescent assay for phenotyping apolipoprotein (a) isomorphs.

Apo(a) is linked to Lp(a) through non-covalent interactions and disulfide bond with apo B. Monoclonal antibodies were raised to reduced and carboxymethylated apo(a) in order to study apo(a) interaction with apo B and to develop a sensitive immunoassay for apo(a) and Lp(a). Nine antibodies were characterized for overlapping epitopes and for single or multiple binding sites on native Lp(a) or denatured apo(a). All monoclonal antibodies bound to Lp(a) and denatured apo(a) when these preparations were absorbed on polystyrene. In contrast, three antibodies (3D1, 4B4 and 6H9) failed to react with Lp(a) in solution, in a competitive displacement assay. This observation indicates that these epitopes are masked in native Lp(a). Cross-reactivity with plasminogen was noted for only one monoclonal antibody (4B4). An assay of competitive binding to immobilized Lp(a) or apo(a) revealed that four distinct groups of epitopes were recognized by the monoclonal antibodies: (A) 1G7, 3A5 partially overlapping with 8B6, (B) 5C4, 5B10 partially overlapping with 7C1, (C) 3D1 overlapping with 6H9, and (D) 4B4. A double antibody sandwich assay, using homologous and heterologous combinations of monoclonal antibodies, showed that monoclonal antibodies 1G7, 3A5 and 8B6 of group A, and 5C4 and 5B10 of group B recognized multiple epitopes on Lp(a) while all other antibodies (3D1, 6H9, 4B4) recognized single epitopes. Based on reports of others for the sequence of apo(a), deduced from the cDNA of the human apo(a) gene, it is proposed that monoclonal antibodies which recognize multiple epitopes are directed toward the repetitive kringle 4-like domains of apo(a) while those recognizing single epitopes are probably directed to the kringle 5 or the protease-like domain of apo(a). Monoclonal antibodies which recognized repetitive epitopes were used for the development of a highly sensitive chemiluminescent immunoblotting system for detection of apo(a) isomorphs after resolving plasma protein by polyacrylamide (4%) gel electrophoresis in the presence of sodium dodecyl sulfate. Seven relatively common isomorphs were identified and readily resolved as a mixture. The detection limit was 5-10 pg for each apo(a) isomorph. The high sensitivity allowed for the detection of isomorphs present in over 99% of plasma samples despite a wide range of ratios of apo(a) isomorphs.

Gas chromatographic profiles of plasma total lipids as indicators of dietary history. Correlation with carbohydrate and alcohol intake based on 24-h dietary recall.

Quantitative gas chromatographic estimates of the major lipid classes and molecular species in fasting plasma were correlated with total carbohydrate, starch, fibre, sucrose and alcohol intake based on 24-h dietary recall. Spearman coefficients (rs) and tests of significance (P) were obtained for groups of 775 males and 471 females aged 20-59 years from a Toronto-McMaster Lipid Research Clinics Population Study. The most significant correlations varying from rs 0.1 to 0.2 and P 0.001 to 0.0005 (n = 400-773) were between increased intake of alcohol and increased ratios of C50/C54 triacylglycerols, C34/C36 phosphatidylcholines and phosphatidylcholine/free cholesterol (PC/FC) of plasma. Increase in total dietary carbohydrate, starch and fibre correlated with decreasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios (rs = -0.1-0.2; P less than 0.002-0.04; n = 400-773). In contrast, consumption of high levels of alcohol was associated with increasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios. A high intake of alcohol (50-150 ml per day) distinguished itself from other simple carbohydrate-induced lipid profiles by its marked effect on increased C50/C52 triacylglycerol and PC/FC ratio.

Interaction between variant apolipoproteins C-II and E that affects plasma lipoprotein concentrations.

The genes for apolipoprotein (apo) C-II, a cofactor for activation of lipoprotein lipase, and apo E, a ligand for receptor-mediated uptake of triglyceride-rich lipoproteins, are physically linked on chromosome 19q13.1. In a large Caribbean Caucasian family, several individuals had clinical features of the complete absence of lipoprotein lipase activity and were homozygous for a DNA frameshift mutation of apo C-II, imparting functional inactivity to the mutant protein. Plasma from heterozygous carriers of this mutation, when compared with plasma from relatives who were noncarriers, had significantly diminished capacity to activate lipoprotein lipase in vitro. We also observed in heterozygotes for this mutation a wide range of serum lipid and lipoprotein levels. When age and sex were taken into account, the presence of a single apo E allele encoding the E4 isoform occurring in individuals with a single mutant apo C-II allele was strongly associated with higher levels of cholesterol, triglycerides, very low density lipoprotein cholesterol, and non-high density lipoprotein cholesterol when compared with those of relatives who carried neither or only one variant allele. This suggests that a single genetic mutation that usually has a recessive effect on lipoprotein metabolism can have an interactive effect on lipid phenotype when it is coinherited with a single mutation at another gene whose product affects the same metabolic pathway.

Gas chromatographic profiles of plasma total lipids as indicators of dietary history. Correlation with fat intake based on 24-h dietary recall.

Fasting plasma total lipid profiles were determined by high-temperature gas chromatography on a total of 1246 free living urban subjects, ages 20-59 years, from the Toronto-McMaster Lipid Research Clinic Population Study. Quantitative estimates of the major molecular species, lipid classes and lipid class ratios were correlated with a total of twelve dietary lipid components, including total saturated and unsaturated fats. oleic and linoleic acids, and cholesterol, to give appropriate Spearman coefficients (rS) and tests of significance (P) for groups of 775 males and 471 females. The intake of the various nutrients was derived from a 24-h dietary recall. The most significant correlations varying from rs +/- 0.1-0.4 and P less than 0.0001-0.0005 were between the intake of total fat, individual saturated and unsaturated fats, and the ratios of C50/C54 triacylglycerols and the C34/C36 phosphatidylcholines, which reflected the nature and quantity of the dietary fat consumed. Increases in dietary cholesterol and saturated fat produced small increases in plasma cholesterol and saturated triacylglycerols, while unsaturated dietary fat produced small decreases in saturated and increases in unsaturated plasma triacylglycerols. These changes in the plasma lipid parameters are consistent with those observed previously in much more limited dietary experiments with accurately known composition of ingested fats. It is, therefore, concluded that direct gas chromatographic profiling of plasma total lipids provides a simple and rapid method of verifying the overall correctness of the dietary recall.

The role of lipoproteins and apolipoproteins in prediction of coronary heart disease risk.

Recent consensus conferences are recommending dietary intervention to lower serum cholesterol in a substantial portion of the population. These recommendations are based on analyses of serum cholesterol in clinical research laboratories which were under rigorous quality control. Sources of biological and analytical variation in serum cholesterol, which may be greater in the routine clinical laboratory or cholesterol screening programme than in a research laboratory, must be recognized by the physician and health care workers for effective use of the diagnostic information and monitoring of treatment procedures in a potentially large segment of the population. The estimation of lipoprotein cholesterol concentrations now offers a more precise measure of risk and should be considered for individuals at modest risk of premature cardiovascular disease on the basis of total cholesterol with a strong family history of cardiovascular disease but mild serum cholesterol elevations (5.2-6.2 mmol/L). The combined estimation of low and high density lipoproteins provides a more accurate estimate of cardiovascular disease risk than total serum cholesterol if the methodology for their estimation is under rigorous quality control. There is strong evidence that genetic variation in apolipoprotein structure provides a significant contribution to variation in serum cholesterol values. The two most common allelic apolipoprotein variants, apo E and apo (a), cause significant effects on serum cholesterol for a substantial portion of the population. Furthermore apo (a), which shows extensive sequence homology with plasminogen, may promote the deposition of lipoprotein (a) in the artery and interfere with fibrinolytic processes following inappropriate thrombus formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the effects of tamoxifen and of a tamoxifen analogue that does not bind the estrogen receptor on serum lipid profiles in the cockerel.

Administration of the nonsteroidal antiestrogen tamoxifen to cockerels results in dose- and time-dependent decreases in the levels of free and esterified cholesterol, phospholipids, and triglycerides in serum and in very low density and low density lipoprotein fractions. Similar changes can be elicited using a tamoxifen analogue, N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine.HCl (DPPE). Like tamoxifen, this compound is capable of binding antiestrogen binding sites and exhibits a relative binding affinity of 90% compared with tamoxifen (Ki approximately 4-5 nM). Unlike tamoxifen, DPPE shows no measureable affinity for the cockerel liver nuclear estrogen receptor. Further, DPPE exhibits no estrogen agonist or antagonist activity as measured at the level of synthesis of apolipoprotein II of very low density lipoprotein by liver, synthesis of ovalbumin by oviduct, or growth of the oviduct. Although it is possible that the lipid-lowering effects of tamoxifen result from the opposition of endogenous estrogen action in the cockerel, the similarity of the effects of tamoxifen and DPPE on the lipid profiles suggests common mechanisms that do not involve the estrogen receptor.

The apolipoprotein A-I gene is actively expressed in the rapidly myelinating avian peripheral nerve.

The expression of the apolipoprotein A-I (apo A-I) gene was investigated in the myelinating sciatic nerve. Hybridization analysis with an apo A-I cDNA probe obtained from a cDNA library of mRNA isolated from rapidly myelinating chick sciatic nerve indicated that apo A-I coding transcripts increase during development in the chick sciatic nerve in parallel with the increase of myelin lamellae. Substantial apo A-I-like immunoreactivity in chick sciatic nerve homogenates was detected by Western blotting. The amount of antigen increased from the 15-d embryonic stage to 1 d posthatch and then decreased. Two subcellular fractions corresponding to the cytoplasmic compartments were particularly enriched in apo A-I. apo A-I immunoreactivity was also found in highly purified myelin preparations. Immunohistochemical staining provided further evidence for the presence of apo A-I in the endoneurial compartment of the sciatic nerve. Electron microscopic examination of these fractions after negative staining showed the presence of spherical and disc-shaped particles resembling high density lipoproteins. The presence of apo A-I, cholesterol esters, phospholipids, and triacylglycerols in ultracentrifugal fractions corresponding to serum lipoproteins and the behavior of apo A-I on nondenaturing gradient gels implied that apo A-I was associated with lipid. Studies with short-term organ cultures of sciatic nerves from 1-d chicks strengthened the evidence for local synthesis and secretion of apo A-I and apo A-I-containing lipoproteins by this tissue. These results establish that the apo A-I gene is actively expressed in developing sciatic nerve during the period of rapid myelination. These findings support the hypothesis that apo A-I synthesized within the nerve participates in the local transport of lipids used in myelin biosynthesis.

Redistribution of cholesterol and beta-sitosterol between intralipid and rat plasma lipoproteins and red blood cells in vivo.

Male Wistar rats were injected intravenously with 2 mL of Intralipid containing 7.5 x 10(5) counts per minute (cpm) [14C]cholesterol and 7.5 x 10(5) cpm beta-[3H]sitosterol. Blood was withdrawn immediately and at 5, 10, 20, 60, 120, and 1440 min after injection from different animals. Plasma and red cells were separated and washed by conventional centrifugation, while lipoprotein density classes corresponding to chylomicrons, very low (VLDL), low (LDL), and high density lipoproteins (HDL) were isolated by ultracentrifugation. Total lipid and sterol compositions were determined by thin-layer chromatography in combination with gas-liquid chromatography, whereas radioactivity was measured by scintillation counting. The ratio of [14C]cholesterol/beta-[3H]sitosterol rose from 1 to 3.65 in the plasma VLDL fraction, whereas that in the LDL and HDL fractions were equilibrated at about 2, following an initial transient increase in favour of cholesterol. The appearance and disappearance of the radioactivity from LDL and HDL fractions exhibited precursor-product relationship owing probably to the conversion of the Intralipid into an intermediate lipoprotein-X-like particle, which possesses a density similar to that of LDL. The radioactive cholesterol and beta-sitosterol were incorporated into the red blood cell membranes at nearly similar initial rates, while at later times the incorporation of cholesterol was much preferred.

The linkage with apolipoprotein (a) in lipoprotein (a) modifies the immunochemical and functional properties of apolipoprotein B.

Lipoprotein (a) [Lp(a)] was isolated from several donors and its apolipoprotein (a) [apo(a)] dissociated by a reductive treatment, generating the apo(a)-free form of Lp(a) [Lp(a--)] that contains apolipoprotein B (apo B) as its sole protein. Using anti-apo B monoclonal antibodies, the properties of apo B in Lp(a), Lp(a--), and autologous low-density lipoprotein (LDL) were compared. Marked differences in apo B immunoreactivity were found between these lipoproteins, due to the presence of apo(a) in Lp(a). Apo(a) enhanced the expression of two epitopes in the amino-terminal part of apo B while it diminished the immunoreactivity of three other epitopes in the LDL receptor binding domain. Accordingly, the binding of the lipoproteins to the LDL receptor was also decreased in the presence of apo(a). In a different experimental system, the incubation of antibodies that react with 27 distinct epitopes distributed along the whole length of apo B sequence with plastic-bound Lp(a) and Lp(a--) failed to reveal any epitope of apo B that is sterically hindered by the presence of apo(a). Our results demonstrate that the presence of apo(a) modified the organization and function of apo B in Lp(a) particles. The data presented indicate that most likely the modification is not due to a steric hindrance but that some more profound conformational changes are involved. We suggest that the formation of the disulfide bridge between apo B and apo(a) in Lp(a) alters the system of disulfide bonds present in apo B and thereby modifies apo B structure.