Mass spectral analyses of the two major apolipoproteins of great ape high density lipoproteins

The two major apolipoproteins associated with human and chimpanzee (Pan troglodytes) high density lipoproteins (HDL) are apoA-I and dimeric apoA-II. Although humans are closely related to great apes, apolipoprotein data do not exist for bonobos (Pan paniscus), western lowland gorillas (Gorilla gorilla gorilla) and the Sumatran orangutans (Pongo abelii). In the absence of any data, other great apes simply have been assumed to have dimeric apoA-II while other primates and most other mammals have been shown to have monomeric apoA-II. Using mass spectrometry, we have measured the molecular masses of apoA-I and apoA-II associated with the HDL of these great apes. Each was observed to have dimeric apoA-II. Being phylogenetically related, one would anticipate these apolipoproteins having a high percentage of invariant sequences when compared with human apolipoproteins. However, the orangutan, which diverged from the human lineage between 16 and 21 million years ago, had an apoA-II with the lowest monomeric mass, 8031.3 Da and the highest apoA-I value, 28,311.7 Da, currently reported for various mammals. Interestingly, the gorilla that diverged from the lineage leading to the human–chimpanzee branch after the orangutan had almost identical mass values to those reported for human apoA-I and apoA-II. But chimpanzee and the bonobo that diverged more recently had identical apoA-II mass values that were slightly larger than reported for the human apolipoprotein. The chimpanzee A-I mass values were very close to those of humans; however, the bonobo had values intermediate to the molecular masses of orangutan and the other great apes. With the already existing genomic data for chimpanzee and the recent entries for the orangutan and gorilla, we were able to demonstrate a close agreement between our mass spectral data and the calculated molecular weights determined from the predicted primary sequences of the respective apolipoproteins. Post-translational modification of these apolipoproteins, involving truncation and oxidation of methionine, are also reported.     

Mass spectrometric measurements of the apolipoproteins of bovine (Bos taurus) HDL

As is the case in most mammals, high density lipoproteins (HDL) also comprise the major group of lipid carriers that circulate in bovine (Bos taurus) blood. As a continuation of our proteogenomic studies of mammalian apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with bovine HDL. The major apolipoprotein on the HDL surface is apoA-I, but other apolipoproteins were also detected. Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I, proA-I and A-II, as well as post-translationally modified apoA-I. Analyses of tryptic fragments did reveal the presence of apoA-IV and apoC-III. However, in contrast to our previous studies of other mammalian HDL, we did not detect apoC-I. Interestingly, examination of the current assembly for the bovine genome does not show any evidence for an apoC-I gene.     

Mass spectral measurements of the apoHDL in horse (Equus caballus) cerebrospinal fluid

As a continuation of our proteogenomic studies of equine apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with the HDL in horse cerebrospinal fluid (CSF). Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I and A-II, as well as acylated apoA-I. In comparison with our previously published data on equine plasma apolipoproteins, there appears to be a higher percentage of acylated apoA-I in the CSF than in plasma. As was the case in plasma, apoA-II circulates as a homodimer. These studies also revealed a protein with a mass of 34,468 Da that we are speculating is the value for horse apoE.     

Speciated human high-density lipoprotein protein proximity profiles

It is expected that the attendant structural heterogeneity of human high-density lipoprotein (HDL) complexes is a determinant of its varied metabolic functions. To determine the structural heterogeneity of HDL, we determined major apolipoprotein stoichiometry profiles in human HDL. First, HDL was separated into two main populations, with and without apolipoprotein (apo) A-II, LpA-I and LpA-I/A-II, respectively. Each main population was further separated into six individual subfractions using size exclusion chromatography (SEC). Protein proximity profiles (PPPs) of major apolipoproteins in each individual subfraction was determined by optimally cross-linking apolipoproteins within individual particles with bis(sulfosuccinimidyl) suberate (BS(3)), a bifunctional cross-linker, followed by molecular mass determination by MALDI-MS. The PPPs of LpA-I subfractions indicated that the number of apoA-I molecules increased from two to three to four with an increase in the LpA-I particle size. On the other hand, the entire population of LpA-I/A-II demonstrated the presence of only two proximal apoA-I molecules per particle, while the number of apoA-II molecules varied from one dimeric apoA-II to two and then to three. For most of the PPPs described above, an additional population that contained a single molecule of apoC-III in addition to apoA-I and/or apoA-II was detected. Upon composition analyses of individual subpopulations, LpA-I/A-II exhibited comparable proportions for total protein (～58%), phospholipids (～21%), total cholesterol (～16%), triglycerides (～5%), and free cholesterol (～4%) across subfractions. LpA-I components, on the other hand, showed significant variability. This novel information about HDL subfractions will form a basis for an improved understanding of particle-specific functions of HDL.     

Mass spectral analysis of pig (Sus scrofa) apo HDL: Identification of pig apoA-II, a dimeric apolipoprotein

Comparative studies of mammalian high density lipoproteins have clearly indicated that the major apolipoprotein is apoA-I and in some mammals apoA-II is the second major apolipoprotein. However, in pigs, apoA-II has been considered to be either present in trace amounts or absent. Recently, cDNA sequences for pigs A-II have been entered into the database. Translation of these sequences revealed that pig A-II consisted of 77 amino acids and that a cysteine residue was at residue 6. The A-II of three other mammals, chimpanzees, horses and humans, also has a cysteine residue at this position. As a result of a disulfide bond formed between monomers, the A-II in each of these cases circulates as a homodimer. Using electrospray-ionization mass spectrometry (ESI-MS), we obtained molecular mass data demonstrating that dimeric apoA-II is also present in pig plasma. In addition to being the first to report on the presence of apoA-II in pig plasma, we also obtained values for the molecular masses of apoA-I, apoC-III, apoD and serum amyloid A protein.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.     

Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring

Endogenous labeling with stable isotopes is used to study the metabolism of proteins in vivo. However, traditional detection methods such as GC/MS cannot measure tracer enrichment in multiple proteins simultaneously, and multiple reaction monitoring MS cannot measure precisely the low tracer enrichment in slowly turning-over proteins as in HDL. We exploited the versatility of the high-resolution/accurate mass (HR/AM) quadrupole Orbitrap for proteomic analysis of five HDL sizes. We identified 58 proteins in HDL that were shared among three humans and that were organized into five subproteomes according to HDL size. For seven of these proteins, apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE, and apoM, we performed parallel reaction monitoring (PRM) to measure trideuterated leucine tracer enrichment between 0.03 to 1.0% in vivo, as required to study their metabolism. The results were suitable for multicompartmental modeling in all except apoD. These apolipoproteins in each HDL size mainly originated directly from the source compartment, presumably the liver and intestine. Flux of apolipoproteins from smaller to larger HDL or the reverse contributed only slightly to apolipoprotein metabolism. These novel findings on HDL apolipoprotein metabolism demonstrate the analytical breadth and scope of the HR/AM-PRM technology to perform metabolic research.     

The surface properties of apolipoproteins A-I and A-II at the lipid/water interface

The monolayer system was employed to investigate the relative affinities of apolipoproteins A-I and A-II for the lipid/water interface. The adsorption of reductively 14C-methylated apolipoproteins to phospholipid monolayers spread at the air/water interface was determined by monitoring the surface pressure of the mixed monolayer and the surface concentration of the apoprotein. ApoA-II has a higher affinity than apoA-I for lipid monolayers; for a given initial surface pressure, apoA-II adsorbs more than apoA-I to monolayers of egg phosphatidylcholine (PC), distearoyl-PC and human high-density lipoprotein (HDL3) surface lipids. Comparison of the molecular packing of apolipoproteins A-I and A-II suggests that apoA-II adopts a more condensed conformation at the lipid/water interface compared to apoA-I. The ability of apoA-II to displace apoA-I from egg PC and HDL3 surface lipid monolayers was studied by following the adsorption and desorption of the reductively 14C-methylated apolipoproteins. At saturating subphase concentrations of the apoproteins (3.10(-5) g/100 ml), two molecules of apoA-II absorbed for each molecule of apoA-I displaced. This displacement was accompanied by an increase in surface pressure. An identical stoichiometry for the displacement of apoA-I from HDL particles by apoA-II has been reported by others. At low subphase concentrations of apoproteins (5.10(-6) g/100 ml), the apoA-I/lipid monolayer was not fully compressed and could accommodate the adsorbing apoA-II molecules without displacement of apoA-I molecules. ApoA-I molecules were unable to displace apoA-II from the lipid/water interface. The average residue hydrophobicity of apoA-II is higher than that of apoA-I; this may contribute to the higher affinity of apoA-II for lipids compared to apoA-I. The probable helical regions in apolipoproteins A-I and A-II were located using a secondary structure prediction algorithm. The analysis suggests that the amphiphilic properties of the alpha-helical regions of apoA-I and apoA-II are probably not significantly different. Further understanding of the differences in surface activity of these apolipoproteins will require more knowledge of their secondary and tertiary structures.     

The structure of apolipoprotein A-II in discoidal high density lipoproteins

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.     

Plasma turnover of HDL apoC-I,apoC-III,and apoE in humans: in vivo evidence for a link between HDL apoC-III and apoA-I metabolism

Numerous factors are known to affect the plasma metabolism of HDL, including lipoprotein receptors, lipid transfer protein, lipolytic enzymes and HDL apolipoproteins. In order to better define the role of HDL apolipoproteins in determining plasma HDL concentrations, the aims of the present study were: a) to compare the in vivo rate of plasma turnover of HDL apolipoproteins [i.e., apolipoprotein A-I (apoA-I), apoC-I, apoC-III, and apoE], and b) to investigate to what extent these metabolic parameters are related to plasma HDL levels. We thus studied 16 individuals with HDL cholesterol levels ranging from 0.56-1.66 mmol/l and HDL apoA-I levels ranging from 89-149 mg/dl. Plasma kinetics of HDL apolipoproteins were investigated using a primed constant (12 h) infusion of deuterated leucine. Plasma HDL apolipoprotein levels were 41.8 +/- 1.5, 9.7 +/- 0.5, 4.9 +/- 0.5, and 0.7 +/- 0.1 micromol/l for apoA-I, apoC-I, apoC-III and apoE. Plasma transport rates (TRs) were 388.6 +/- 24.7, 131.5 +/- 12.5, 66.5 +/- 9.1, and 31.4 +/- 3.3 nmol.kg-1.day-1; and residence times (RTs) were 5.1 +/- 0.4, 3.7 +/- 0.3, 3.6 +/- 0.3, and 1.1 +/- 0.1 days, respectively. HDL cholesterol and apoA-I levels were significantly correlated with HDL apoA-I RT (r = 0.69 and r = 0.56), and were not significantly correlated with HDL apoA-I TR. In contrast, HDL apoC-I, apoC-III, and apoB levels were all positively related to their TRs and not their RTs. HDL apoC-III TR was positively correlated with levels of HDL apoC-III (r = 0.73, P < 0.01), and with those of HDL cholesterol and apoA-I (r = 0.54 and r = 0.53, P < 0.05, respectively). HDL apoC-III TR was in turn related to HDL apoA-I RT (r = 0.51, P < 0.05). Together, these results provide in vivo evidence for a link between the metabolism of HDL apoC-III and apoA-I, and suggest a role for apoC-III in the regulation of plasma HDL levels.     

Apolipoprotein specificity for lipid efflux by the human ABCAI transporter

ABCAI, a member of the ATP binding cassette family, mediates the efflux of excess cellular lipid to HDL and is defective in Tangier disease. The apolipoprotein acceptor specificity for lipid efflux by ABCAI was examined in stably transfected Hela cells, expressing a human ABCAI-GFP fusion protein. ApoA-I and all of the other exchangeable apolipoproteins tested (apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, apoE) showed greater than a threefold increase in cholesterol and phospholipid efflux from ABCAI-GFP transfected cells compared to control cells. Expression of ABCAI in Hela cells also resulted in a marked increase in specific binding of both apoA-I (Kd = 0.60 microg/mL) and apoA-II (Kd = 0.58 microg/mL) to a common binding site. In summary, ABCAI-mediated cellular binding of apolipoproteins and lipid efflux is not specific for only apoA-I but can also occur with other apolipoproteins that contain multiple amphipathic helical domains.     

Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci

We have tested for evidence of linkage between the genetic loci determining concentrations and composition of plasma high density lipoproteins (HDL) with the genes for the major apolipoproteins and enzymes participating in lipoprotein metabolism. These genes include those encoding various apolipoproteins (apo), including apoA-I, apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, apoE, and apo(a), cholesteryl ester transfer protein (CETP), HDL-binding protein, lipoprotein lipase, and the low density lipoprotein (LDL) receptor. Polymorphisms of these genes, and nearby highly polymorphic simple sequence repeat markers, were examined by quantitative sib-pair linkage analysis in 30 coronary artery disease families consisting of a total of 366 individuals. Evidence for linkage was observed between a marker locus D16S313 linked to the CETP locus and a locus determining plasma HDL-cholesterol concentration (P = 0.002), and the genetic locus for apoA-II and a locus determining the levels of the major apolipoproteins of HDL, apoA-I and apoA-II (P = 0.009 and 0.02, respectively). HDL level was also influenced by the variation at the apo(a) locus on chromosome 6 (P = 0.02). Thus, these data indicate the simultaneous involvement of at least two different genetic loci in the determination of the levels of HDL and its associated lipoproteins.     

Role of LCAT in HDL remodeling: investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (-77% and -13%), and LCAT activities (-99% and -39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated prebeta-1 and alpha-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with alpha-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, prebeta-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with prebeta-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of prebeta-1 into alpha-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.     

Expression of human apolipoprotein A-II and its effect on high density lipoproteins in transgenic mice.

Apolipoproteins A-I and A-II comprise approximately 70 and 20%, respectively, of the total protein content of HDL. Evidence suggests that apoA-I plays a central role in determining the structure and plasma concentration of HDL, while the role of apoA-II is uncertain. To help define the function of apoA-II and determine what effect increasing its plasma concentration has on HDL, transgenic mice expressing human apoA-II and both human apoA-I and human apoA-II were produced. Human apoA-II mRNA is expressed exclusively in the livers of transgenic animals, and the protein exists as a dimer as it does in humans. High level expression of human apoA-II did not increase HDL concentrations or decrease plasma concentrations of murine apoA-I and apoA-II in contrast to what was observed in mice overexpressing human apoA-I. The primary effect of overexpressing human apoA-II was the appearance of small HDL particles composed exclusively of human apoA-II. HDL from mice transgenic for both human apoA-I and human apoA-II displayed a unique size distribution when compared with either apoA-I or apoA-II transgenic mice and contain particles with both these human apolipoproteins. These results in mice, indicating that human apoA-II participates in determining HDL size, parallel results from human studies.     

Isolation and identification of HDL particles of low molecular weight

Small particles of high density lipoproteins (HDL) were isolated from fresh, fasting human plasma and from the ultracentrifugally isolated high density lipoprotein fraction by means of ultrafiltration through membranes of molecular weight cutoff of 70,000. These particles were found to contain cholesterol, phospholipids, and apolipoproteins A-I and A-II; moreover, they floated at a density of 1.21 kg/l. They contained 67.5% of their mass as protein and the rest as lipid. Two populations of small HDL particles were identified: one containing apolipoprotein A-I alone [(A-I)HDL] and the other containing both apolipoproteins A-I and A-II [A-I + A-II)HDL]. The molar ratio of apoA-I to apoA-II in the latter subclass isolated from plasma or HDL was 1:1. The molecular weights of these subpopulations were determined by nondenaturing gradient polyacrylamide gel electrophoresis and found to be 70,000; 1.5% of the plasma apoA-I was recovered in the plasma ultrafiltrate.     

Interaction of the apoproteins of very low density and high density lipoproteins with synthetic phospholipids.

The interaction of synthetic dimyristoyl phosphatidylcholine (lecithin) liposomes with isolated apoC-I and apoC-III proteins from very low density lipoproteins has been studied by microcalorimetry. Complex formation is a highly exothermal process characterized by a maximal enthalpy of -130 kcal/mol (-544 kJ) apoC-III-1 and -65 kcal/mol apoC-I proteins (-272 kJ). The complex composition determined after its isolation by ultracentrifugal flotation agrees with the value derived from the enthalpy binding curves. The binding of a constant amount of dimyristoyl lecithin to apoprotein mixtures containing various proportions of apoA-I and apoC-III failed to demonstrate the existence of any preferential association between the two apoproteins, in contrast with results obtained previously with apoA-I/apoA-II protein mixtures. Finally the various contributions to the enthalpy of binding such as that arising from an increase in apoprotein helicity have been evaluated. A classification of the apolipoproteins according to their lipid-binding affinity is proposed as: apoA-II congruent to apoC-III greater than apoC-I greater than apoA-I proteins.     

Site-specific methionine sulfoxide formation is the structural basis of chromatographic heterogeneity of apolipoproteins A-I, C-II, and C-III.

ApoA-I and apoC-II are eluted in two isoforms and apoC-III2 is eluted in three isoforms by reversed phase high performance liquid chromatography (HPLC). The structural basis of these nongenetic heterogeneities was unravelled using HPLC of proteolytic peptides and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In apoA-I, the chromatographic microheterogeneity was caused by the formation of methionine sulfoxides (MetSO). However, only residues Met112 and Met148 were found oxidized, whereas Met86 was unaffected and also resistant towards artificial oxidation. To assess whether and to what extent amino acid substitutions in apoA-I might affect methionine sulfoxidation, the tryptic peptides of 13 different mutant apoA-I proteins from 24 heterozygous apoA-I variant carriers were analyzed by HPLC. In normal apoA-I, the ratios MetSO112/Met112 and MetSO148/Met148 were highly variable. By contrast, the relative ratio of oxidation of methionine residues 112 and 148 was constant. The amino acid changes Lys107----Met, Lys107----O, Glu139----Gly, Glu147----Val, and Pro165----Arg resulted in the preferential oxidation of Met112, and Asp103----Asn resulted in a preferential oxidation of Met148; whereas Pro3----Arg, Pro3----His, Pro4----Arg, Asp89----Glu, Ala158----Asp, Glu198----Lys, and Asp213----Gly had no impact. ApoC-II and apoC-III isoforms differed by the oxidation of the two methionine residues in these proteins. Whereas in apoC-II both methionine residues were oxidized in parallel, in apoC-III the two methionine residues differed in their susceptibility towards oxidation. We conclude that the formation of MetSO depends on the molecular microenvironment within a protein.     

ProteinChip technology: a new and facile method for the identification and measurement of high-density lipoproteins apoA-I and apoA-II and their glycosylated products in patients with diabetes and cardiovascular disease

This paper describes a ProteinChip technology for the identification and quantification of apolipoprotein profiles in crude biological samples. Expression levels of apoA-I and apoA-II and their glycosylated products were accomplished using single 1 microL plasma samples. In the present studies, strong anionic and weak cationic exchanger ProteinChips (SAX2 and WCX2 chip surfaces) were tested, and the WCX2 chip was found to be selective for specific apolipoproteins. Using the WCX2 chip and analysis via surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), apoA-I and apoA-II were separated as sharp peaks at 28 and 17 kD and did not overlap with other serum protein peaks. Since these assays can be completed on a large number of clinical samples in approximately 1 h, further development of this technique will facilitate both epidemiological studies and therapeutic trials in assessing the role of the apolipoproteins and their glycosylated products in atherosclerosis.