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We previously showed that plasminogen (Plg) isolated from the plasma of normal human subjects contains 1-2 moles of oxidized phosphatidylcholine (oxPtdPC) adducts/mole of protein. Moreover, we suggested that these species are generated at the hepatic site and speculated that they may play a role in the reported cardiovascular pathogenicity of Plg. We aimed to determine whether mouse Plg also harbors linked oxPtdPCs and whether these molecules are metabolized by lipoprotein-associated phospholipase A(2)/PAF acetylhydrolase (Lp-PLA(2)/PAF-AH), an enzyme specific for hydrolysis of oxPtdPCs. We determined the total concentration of Plg in plasma samples from control (WT) and Lp-PLA(2)-deficient (KO) mice, we isolated Plg, and assessed its content of oxPtdPCs by immunoblot analyses. We also evaluated whether human recombinant Lp-PLA(2) metabolized Plg-linked oxPtdPCs in vivo and in vitro. WT and KO mice expressed comparable levels (14.4-15.8 mg/dL) of plasma Plg, as determined by ELISA. We observed no differences in the content of oxPtdPC in Plg isolated from the two mouse strains and in parallel no changes in oxPtdPC content in mouse Plg following incubation with pure recombinant Lp-PLA(2). Plg from mouse plasma contains oxPtdPC adducts that are not affected by the action of Lp-PLA(2), suggesting that linkage to Plg protects oxPtdPCs from metabolism during their transport in the plasma. This modification may have important physio-pathological implications related to the function of Plg, oxPtdPCs, or both.

Human apolipoprotein(a) (apo(a)), synthesized in the liver, contains oxidized phosphatidylcholine (oxPtdPC) adducts probably generated at the hepatic site. Since plasminogen (Plg), also synthesized in the liver, is genetically related and structurally homologous to apo(a), we wanted to determine whether it contains oxPtdPCs and their location. We used Plg isolated from fresh or frozen normal human plasma and several commercial preparations. Some were freed of non-covalently bound lipids by organic solvent extraction. By immunoblot analyses all products reacted against T15, a natural IgM monoclonal antibody specific for phosphorylcholine -containing oxidized phospholipids (ox-PLs). This immunoreactivity was retained in urokinase type plasminogen activator -generated plasmin and was abrogated in Plg previously digested with lipoprotein-associated phospholipase A2 (Lp-PLA2), a reaction that generated that predominantly C16:0 lysophosphatidylcholine species as determined by mass spectrometry. Lysoderivatives were also generated upon the cleavage by Lp-PLA2 of a model ox-PL chemically linked to a lysine-containing pentapeptide. From inorganic phosphorous analyses, we found 2 moles of oxPtdPC/mole of Plg distributed between the kringles 1-4 and mini-Plg domain. OxPtdPCs were also present in the Plg isolated from the serum-free medium of cultured human HepG2 cells. In conclusion, our results provide strong evidence that naturally occurring Plg contains oxPtdPC probably linked by a Schiff base and also suggest that the linkage occurs at the hepatic site. Given the emerging evidence for the cardiovascular pathogenicity of oxPtdPCs we speculate that they may impart athero-thrombogenic properties to Plg under inflammatory conditions.

Plasma lipoprotein(a) (Lp[a]) level is an independent risk factor of cardiovascular disease that is under strong genetic control. We conducted a genome-wide association study of plasma Lp(a) in 386 members of a founder population that adheres to a communal lifestyle, proscribes cigarette smoking, and prepares and eats meals communally. We identified associations with 77 single nucleotide polymorphisms (SNPs) spanning 12.5 Mb on chromosome 6q26-q27 that met criteria for genome-wide significance (Por= 1.3 x 10(-7)) and were within or flanking nine genes, including LPA. We show that variation in at least six genes in addition to LPA are significantly associated with Lp(a) levels independent of each other and of the kringle IV repeat polymorphism in the LPA gene. One novel SNP in intron 37 of the LPA gene was also associated with Lp(a) levels and carotid artery disease number in unrelated Caucasians (P = 7.3 x 10(-12) and 0.024, respectively), also independent of kringle IV number. This study suggests a complex genetic architecture of Lp(a) levels that may involve multiple loci on chromosome 6q26-q27.

Lipoprotein (a) [Lp(a)], a cardiovascular risk factor, is a low-density lipoprotein (LDL) variant shown to bind to oxidized phospholipids (oxPLs); however, its binding mode and origin have not been clearly established. We isolated both LDL and Lp(a) from the plasma of a population of high-Lp(a) subjects and in each Lp(a) particle separated apolipoprotein(a) [apo(a)], from the LDL component, Lp(a−). These products were assayed by an ELISA using monoclonal antibody T15 with a known specificity for oxPLs. In each subject, the T15 reactivity was confined to apo(a). Moreover, the amount of oxPL bound to apo(a) was unaffected by plasma Lp(a) levels and apo(a) size polymorphism. We have previously shown that kringle V (KV) is the site of oxPL linkage in human apo(a). In this work, we expressed in human embryonic kidney cells a KV-containing recombinant that, when purified from the medium, contained oxPLs. In summary, in human plasma Lp(a), the oxPLs are located in apo(a) and not in the circulating LDLs, suggesting a cellular origin. This latter concept is supported by the studies in which an expressed KV-containing apo(a) microdomain exhibited oxPL reactivity. Thus, apo(a) can undergo potentially pathogenic posttranslational modifications in a cellular environment able to generate oxPL.—Edelstein, C., Philips, B., Pfaffinger, D., Scanu, A. M. The oxidized phospholipids linked to human apolipoprotein(a) do not derive from circulating low-density lipoproteins and are probably of cellular origin.

Lipoprotein(a) [Lp(a)] represents a class of lipoprotein particles having as a protein moiety apoB-100 linked by a single disulfide bond to apolipoprotein(a) [apo(a)], a multikringle structure with a high degree of homology with plasminogen. A recognized feature of Lp(a) is its instability on storage caused by attendant protein and lipid modifications that affect the structural, functional, and immunological properties of this lipoprotein. Here we present data showing that, under appropriate conditions of cryopreservation, Lp(a) retains the properties of the freshly isolated product, and we provide examples supporting the stability of this cryopreserved product as a primary standard in immunoassay settings and in cell culture systems.

We previously observed that rhesus monkey lipoprotein(a) [Lp(a)], is lysine-binding defective (Lys-) and attributed this deficiency to the presence of Arg72 in the lysine-binding site (LBS) of kringle IV-10 of apolipoprotein(a) [apo(a)] [Scanu, A.M., Miles, L.A., Fless, G.M., Pfaffinger, D., Eisenbart, J., Jackson, E., Hoover-Plow, J.L., Brunck, T., & Plow, E.F. (1993) J. Clin. Invest. 91, 283-291]. We also identified human mutants having Arg72 instead of Trp72 (wild type) in the LBS of kringle IV-10 [Scanu, A M., Pfaffinger, D., lEE, J.C., & Hinman, J. (1994) Biochim. Biophys. Acta 1227, 41-45]. Unique to the human mutant phenotype were the very low levels of plasma Lp(a), suggesting structural differences between human and rhesus apo(a) and a possible divergent mode of Lp(a) assembly. In order to explore the possibility of a relationship between apo(a) LBS and Lp(a) assembly, we developed a novel method for isolating wild-type and mutant apo(a) phenotypes in a free form by subjecting each parent Lp(a) to mild reductive conditions using 2 mM dithioerythritol (DTE) and 100 mM of the lysine analogue, epsilon-aminocaproic acid (EACA). The application of this method to the study of wild-type and mutant apo(a) species showed that regardless of the source of Lp(a), i.e., positive lysine binding (Lys+) or negative lysine binding (Lys-), all of the isolated free apo(a)s were Lys+. Moreover, incubation of free apo(a)s with their autologous human or rhesus low-density lipoproteins (LDL) generated Lp(a) complexes which were structurally and functionally indistinguishable from their parent native Lp(a). In each instance, the reassembly process was inhibited by the presence of either EACA or proline. These two reagents had a minimal effect on either Lp(a) or reassembled Lp(a) [RLp(a)]. Free apo(a) bound to apoB100 of very low density lipoproteins (VLDL) to form a triglyceride-rich Lp(a). These results show that (1) both human and rhesus Lp(a) are amenable to dissassembly and reassembly, (2) the presence of Arg72 in the LBS of kringle IV-10 is not involved, at least directly, in this process, (3) its cleavage from apoB100 opens up in apo(a) a domain that is both EACA and proline sensitive and involved in Lp(a) assembly, and (4) the apoB100 of VLDL is also competent to bind apo(a). Our observations also suggest that the difference in plasma Lp(a) levels between the rhesus and the human mutant, both having Arg72 in the LBS of apo(a) kringle IV-10, is not related to the assembly process, but more likely to a divergence in production/secretion rates between the two apo(a) phenotypes.

Human lipoprotein(a) or Lp(a) binds, like plasminogen, to lysine Sepharose. However, contrary to plasminogen in which kringles 1 and 4 have been implicated, the binding site or sites on apo(a), the specific glycoprotein of Lp(a), have not been determined. For the first time we now report the occurrence of a human Lp(a) that has a mutant form of apo(a) where Arg has replaced Trp in position 72 of kringle 4-37 and is unable to bind to lysine Sepharose. This observation suggests that Trp72 of apo(a) kringle 4-37 may play a dominant role in lysine binding. Lysine binding has been associated with the thrombogenic potential of Lp(a). Thus, the Trp72 → Arg mutation may render Lp(a) ‘benign’ from the cardiovascular viewpoint.

A VLDL-like particle containing the apo B100-apo(a) complex was isolated from the post-prandial plasma of subjects fed a fat meal enriched in saturated fatty acids. The abundance of this lipoprotein particle, that we call TG-Lp(a), varied among subjects but not in the same subject. TG-Lp(a), but not the classic Lp(a), contained apo E; this apolipoprotein may cause divergence in cellular uptake and degradation between these two classes of lipoproteins.

Rhesus lipoprotein(a) (Lp[a]) binds less efficiently than human Lp(a) to lysine-Sepharose and to cultured U937 cells. Studies using elastase-derived plasminogen fragments indicated that neither kringle 5 nor the protease domain of Lp(a) are required in these interactions pointing at an involvement of the K4 region. Comparative structural analyses of both the human and simian apo(a) K4 domain, together with molecular modeling studies, supported the conclusion that K4(37) plays a dominant role in the lysine binding function of apo(a) and that the presence of arginine 72 rather than tryptophan in this kringle can account for the functional deficiency observed with rhesus Lp(a). These in vitro results suggest that rhesus Lp(a) may be less thrombogenic than human Lp(a).

Samples of human plasma having lipoprotein(a) (Lp[a]) protein levels between 5 and 15 mg/dl and a single apolipoprotein(a) (apo[a]) isoform were incubated in vitro at pH 7.7 with various concentrations (1-20 mM) of N-acetylcysteine, homocysteine, 2-mercaptoethanol (2ME), and dithiothreitol (DTT) for 1 hour at 37 degrees C under a nitrogen atmosphere. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analyses using a polyclonal antibody specific for apo(a) showed a progressive decrease in apo(a) immunoreactivity as a function of reductant concentration. This decrease of apo(a) immunoreactivity was corroborated by enzyme-linked immunosorbent assay (ELISA) using anti-apo(a) as the capture antibody and either anti-apo B or anti-apo(a) as the developing antibody. In turn, there was no significant decrease in the immunoreactivity of apo B-100, as assessed by ELISA using anti-apo B as both the capture and the detecting antibody. In the case of high concentrations of DTT the plasma samples had to be diluted to prevent gel formation on addition of the reductant. A progressive drop in immunoreactivity as a function of reagent concentration was also observed in pure preparations of Lp(a) incubated with the reducing agents at pH 7.7. At equivalent stoichiometries the changes were more marked than those observed with whole plasma, suggesting a quenching effect by the plasma proteins on the activity of the reductants. The changes in immunoreactivity were attended by dissociation of apo(a) from Lp(a) as assessed by Western blotting. This dissociation, which we interpret as the result of cleavage of the interchain disulfide bond(s), was complete at 5 mM DTT and 100 mM 2ME.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma Lp[a] levels and apo[a] isoform distribution among lipoproteins isolated by density gradient ultracentrifugation were studied in subjects with one-band or two-band apo[a] phenotypes as assessed by gradient gel electrophoresis before and after an oral fat load. There were no significant differences in the ultracentrifugal profile between fasting plasma and postprandial plasma that was freed of triglyceride-rich particles (TRP). One-band phenotypes exhibited a single symmetrical peak in the density gradient, whereas two-band phenotypes exhibited a multi-modal distribution. Low molecular weight apo[a] isoforms were preferentially associated with low density Lp[a] whereas high molecular weight apo[a] isoforms were found with high density Lp[a] particles. Feeding a high fat meal caused no significant increase in the total plasma level of Lp[a]. However, the isolated TRP contained the apoB-100-apo[a] complex in a quantity that represented only about 1% of its total amount in the fasting plasma. In all cases the apo[a] isoforms present in TRP were also present in the fasting plasma; however, in the two-band apo[a] phenotypes the ratio of the slow over the fast migrating band was in all cases about eightfold higher in TRP than in the fasting plasma. These observations indicate that postprandially a small percentage of apoB-100-apo[a] associates with TRP and suggest that this complex may derive from de novo synthesis rather than from a pre-existing Lp[a] plasma pool. The liver would be the source of the complex due to the presence in the latter of apoB-100.

We previously described a family of rhesus monkeys in which three out of six members had a spontaneous hypercholesterolemia related to a decrease in number of low density lipoprotein receptors (LDL-R) (Scanu et al. 1988. J. Lipid Res. 29: 1671-1681). During the current work an additional female normocholesterolemic offspring was generated from the mating of the original dam and sire. Moreover, from the breeding of one of the affected male offspring with six unrelated normocholesterolemic female monkeys, eight offspring were generated of which three were hypercholesterolemic on a cholesterol-free diet and exhibited the same degree of LDL-R deficiency as shown by studies in skin fibroblast cultures. All of the animals studied had levels of plasma lipoprotein[a] protein ranging between 1.0 mg/dl and 57.5 mg/dl that were only weakly correlated with total plasma cholesterol, LDL cholesterol, and apoB. LDL-R deficiency correlated with plasma LDL but not Lp[a]. A 7 week fat challenge (16.5% lard, 0.64% cholesterol) that raised the plasma LDL levels markedly had no effect on plasma Lp[a]. Animals with the single band apo[a] phenotype moving on SDS-PAGE faster than apoB-100 exhibited a tendency for high plasma Lp[a] levels which, however, varied widely. Wide variations in Lp[a] levels were also noted with the other apo[a] phenotypes. Taken together our results demonstrate a successful transmission to second generation animals of the LDL-R deficiency phenotype and provide evidence that this phenotype correlates well with plasma LDL levels but not Lp[a]. Our data also suggest that the apo[a] gene is only partially involved in the regulation of the plasma Lp[a] levels.

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

Abstract —Lipoprotein(a) [Lp(a)] represents a class of low density lipoprotein (LDL) particles that have as a protein moiety apolipoprotein B-100–linked covalently to a single molecule of apolipoprotein(a) [apo(a)], a specific multikringle protein of the plasminogen family. Lp(a) is polymorphic in density because of either the density heterogeneity of constitutive LDL, apo(a) size, or both. Authentic LDL also represents a set of heterogeneous particles whose density is affected by metabolic events. Whether in vivo these events may also affect Lp(a) density is not clearly established. To this effect, we studied 75 subjects with plasma Lp(a) protein levels between 7 and 50 mg/dL and containing a single apo(a) size isoform. We used density gradient ultracentrifugation to simultaneously monitor the changes in the peak density of LDL and Lp(a) at entry and during the course of treatments directed at reducing plasma triglyceride levels. In each case, we found that at entry, Lp(a) peak density was correlated with LDL peak density ( r =0.71, P r =0.94, P

Lp(a) and plasminogen share lysine binding property1. For plasminogen this binding function is localized in kringles 1 and 4, whereas for apo(a) the kringle (or kringles) responsible for this function has not been unequivocally identified2. However, there is evidence to suggest that kringle 4-37 contains the necessary structural elements for lysine binding3,4. Experimental support for this prediction has come from studies in rhesus monkeys showing that their apo(a) kringle 4-37 has a mutation associated with a lysine binding deficient Lp(a)5. This mutation is located in the nonpolar trough of the lysine binding pocket that comprises 7 amino acids (two anionic: asp55, asp57; two cationic: arg35, arg71 and three nonpolar: trp62, trp72 and phe64). According to the crystallographic studies on plasminogen kringle 4 by Tulinsky6, trp72 plays a key role in lysine binding. Since lysine binding is related to fibrin binding and to the antifibrinolytic potential of Lp(a), a mutation at this level may render this lipoprotein comparatively less thrombogenic5.

We have been able to amplify the lysine binding pocket region of human apo(a) kringle type 5 starting from the DNA isolated from peripheral blood lymphocytes. This development now permits the identification of Lp(a) mutants that by lacking their ability to bind to lysine/fibrin would have a lesser thrombogenic potential.

The protein moiety of Lp[a] consisting of apoB and apo[a] covalently linked to each other, once freed of lipids by delipidation at pH 8.0 with mixtures of diethyl ether and ethanol, is freely water-soluble at pH values above 6.4. This is in contrast to apoB which, if prepared by similar delipidation techniques, is only soluble at alkaline pH, indicating that the coupling of the carbohydrate-rich apo[a] to apoB confers water solubility to this apolipoprotein that it does not possess on its own. When probed in a sandwich ELISA with antibodies specific to apo[a], the results suggest that some apo[a] epitopes in Lp[a] are masked by lipid but are freely accessible to antibodies in the lipid-free apoB-apo[a] complex. Examination of apoB-apo[a] with an ELISA specific for apoB showed a decreased and altered immunoreactivity of apoB when compared to either low density lipoprotein (LDL) or Lp[a]. These results are consistent with a model in which the hydrophobic lipid binding domains of apoB in apoB-apo[a] self-associate and are shielded from the aqueous environment by the hydrophilic portions of apoB and by an envelope of apo[a]. The apoB-apo[a] complex has lipophilic properties as shown by its interaction with the phospholipid-stabilized triglyceride emulsion, Intralipid. In addition, it has an avidity for all types of lipoproteins although displaying a preference for triglyceride-rich particles. In the presence of plasma, the interaction of apoB-apo[a] with all lipoproteins is reduced. Neither iodinated apo[a] nor iodinated Lp[a] nor LDL had an affinity for lipoproteins, suggesting that the lipophilic properties of apoB-apo[a] are probably due to apoB since apo[a] is rather hydrophilic and is unable to bind to lipids. Thus, the apoB-apo[a] complex has amphipathic properties with apo[a] providing the hydrophilic capacity to interact with the aqueous environment and apoB providing the hydrophobic interactions necessary to bind lipids.

We have recently identified a family of rhesus monkeys with members exhibiting a spontaneous hypercholesterolemia associated with a low density lipoprotein receptor (LDLR) deficiency. By using the polymerase chain reaction, we now show that the affected monkeys are heterozygous for a nonsense mutation in exon 6 of the LDLR gene. This mutation changes the sequence of the codon for amino acid 284 (tryptophan) from TGG to TAG, thereby generating a nonsense codon potentially resulting in a truncated 283-amino acid protein, which needs documentation, however. This G----A mutation also creates a site for the restriction endonuclease Spe I. Using this site as a marker for this nonsense mutation, we have shown that the mutation is present in all of the affected members of the pedigree and absent in unaffected members and that the mutation segregates with the phenotype of spontaneous hypercholesterolemia through three generations. Quantitative analyses of RNA obtained from liver biopsies show that the abundance of the LDLR RNA is also reduced by about 50%. Thus, we have identified a primate model for human familial hypercholesterolemia which will be useful for studying the relationship between the LDLR and lipoprotein metabolism and for assessing the efficacy of diets and drugs in the treatment of human familial hypercholesterolemia.

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.

Two density gradient ultracentrifugation methods, Redgrave et al. (1975. Anal. Biochem. 65: 42–49) and Nilsson et al. (1981. Anal. Biochem. 110: 342–348), currently used for the separation and analysis of plasma lipoproteins were compared with respect to their resolving power and capacity to obtain pure products as a function of time of ultracentrifugation using the same rotor (Beckman SW-40), speed (150,000 g), and temperature (14 degrees C). The effects of sucrose and salts were also investigated. The Redgrave gradient insured the separation of the major classes of plasma lipoproteins after 24 hr of centrifugation; however, equilibrium conditions were only reached after 48 hr, at which time the lipoproteins were contaminated by albumin. When the effluents from each rotor tube were continuously monitored at 280 nm, each lipoprotein band gave values that were higher than those from mass analyses. This was due to a light scattering effect, the extent of which was dependent on the concentration of lipoproteins and salts. Sucrose prevented the scattering effect and was found to bind irreversibly to the apolipoproteins. In contrast, after 66 hr centrifugation, the lipoproteins obtained from the Nilsson gradient exhibited a close correspondence between protein mass and absorbance values at 280 nm, had no scattering effect, and were uncontaminated by albumin. The difference in spectroscopic behavior between the Redgrave and the Nilsson procedures was attributed to three factors: 1) the presence of sucrose in the latter gradient and incorporation of this sugar into lipoproteins as assessed by mass and radioactivity measurements; 2), the salt density to which the serum samples were exposed to at the beginning of the ultracentrifugation; and 3) the final lipoprotein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid (less than 1 hour) and sensitive technique was developed for the isolation of apolipoprotein E from the other main components of human plasma very low density lipoproteins using molecular sieve high performance liquid chromatography with an approximate 80% recovery. The properties of the pure apoprotein were those reported in the literature for products isolated by conventional chromatographic and/or electrophoretic procedures.

Lipoprotein (a) (Lp(a)) contains equimolar amou- nts of apoB-100 and apolipoprotein (a) (apo(a)). Both proteins are amenable to degradation in vivo by mecha- nisms yet to be clearly defined. In this study, we examined the in vitro susceptibility of LDL and Lp(a), obtained from the same donor, to oxidation by either Cu 2 1 or the combined Crotalus adamanteus phospholipase A 2 and soy- bean lipoxygenase system, monitoring the course of the re- action by the generation of conjugated dienes and fatty acids. In some experiments, treatment with leukocyte elastase (LE) or matrix metalloproteinase 12 (MMP-12) was admin- istered before and after the oxidative step. In the case of Lp(a) we found that with both oxidizing systems, condi- tions that caused the breakdown of apoB-100 did not degrade apo(a) although oxidation-mediated changes were detected in the latter by intrinsic tryptophan fluorescence spectro- scopy. Similar results were obtained with a reassembled Lp(a) obtained by incubating free apo(a) with LDL. Both apo(a) and apoB-100 were cleaved by LE and MMP-12 but the enzymatic cleavage was more marked when the pre- oxidized proteins were used as a substrate. Taken together, our in vitro studies indicate that apo(a) but not apoB-100 resists oxidative fragmentation, whereas both proteins are cleaved by enzymes of the serine and metalloproteinase families. We speculate that the fragments of apo(a) observed in vivo may be preferentially generated by proteolytic rather than oxidative events, whereas apoB-100 can be degraded by vivo studies (3, 8, 9). Regarding the apoB-100 constituent of authentic LDL, its breakdown has been shown to occur in "maximally" oxidized particles (10) and also following the action of biologically occurring enzymes of the elastase and metalloproteinase families (3, 9). In the case of apoB-100 as a constituent of Lp(a), information about the mechanisms underlying its degradation as a function of oxidative versus proteolytic events is comparatively lim- ited, and this also applies to apo(a). At present it is unclear whether the LDL constituent of Lp(a) is susceptible to oxi- dative and proteolytic changes as is authentic LDL and whether apo(a), shown to be cleaved by proteolytic en- zymes, can also be degraded by oxidative events. We rea- soned that an understanding of these processes in vitro would provide a useful background for investigating the mechanism(s) of formation of Lp(a) fragments in vivo. To this end, in the current study we used in vitro systems to study the effect of oxidative and proteolytic conditions on purified preparations of human LDL, Lp(a), and free apo(a), all obtained from the same subject. We show here that oxidative events that in Lp(a) cause the breakdown of apoB-100 do not affect the cleavage of apo(a), whereas both proteins are readily cleaved by the action of proteolytic en- zymes of the serine and metalloproteinase families.