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Very small apolipoprotein A-I-containing particles from human plasma: isolation and quantification by high-performance size-exclusion chromatography.

HDL is a family of particles that share a high hydrated density (d >1.063 kg/L) and the presence of apolipoprotein (apo) [3] A-I as the major structural protein in nearly all particles. Much of the interest in HDL is related to its strong inverse association with the extent and risk of developing atherosclerosis (1). However, because HDL particles are highly polymorphic, differing in size, charge, lipid and apo composition, and metabolic stability, there remains uncertainty as to whether all subfractions contribute equally to that association (2, 3).

Very small, lipid-poor apo A-I-containing lipoprotein (Sm LpA-I) particles, sometimes referred to as "free" or pre-[[beta].sub.1] LpA-I, have been the focus of two contrasting lines of research, one of which centers on their putative role as "nascent" HDL. Perfused organ and tissue culture studies have demonstrated that the liver secretes small particles that contain apo A-I, but no other protein and very little lipid (4-6), which may be similar to the Sm LpA-I particles studied in this report. In vitro studies using cultured cells have suggested that Sm LpA-I particles are highly efficient acceptors of cell membrane lipids, and their concentration appears to be rate limiting for cholesterol efflux from fibroblasts, hepatoma cells, and macrophages (7-11). Lipid acceptance by Sm LpA-I may be direct, by interaction with proteinase-sensitive cell surface microdomains (12-15), or indirect, through the unstirred water layer that surrounds cells (16). Moreover, Sm LpA-I particles filter readily into extracellular fluid (17) and the arterial intima (18, 19), as predicted by their small size and low surface charge, facilitating their acceptance of cholesterol from cells and tissues (20). The acquisition of phospholipid (PL) and unesterifed cholesterol (UC) by Sm LpA-I particles allows their conversion into discoidal HDL on which UC is readily esterified by lecithin:cholesterol acyltransferase (LCAT; phosphatidylcholine-sterol O-acyltransferase, EC, ultimately converting the particles into mature, spheroidal HDL with a neutral lipid core (21-24). Thus, Sm LpA-I may be "nascent" HDL, crucial to both extracellular HDL maturation and net transport of cholesterol from peripheral tissues to the liver, which is generally believed to be a major antiatherogenic function of HDL (25).

A contrasting line of evidence implicates Sm LpA-I as "senescent" HDL. Lipid-poor apo A-I may be generated in plasma spheroidal or discoidal HDL by the actions of lipid transfer proteins (26-29) and/or endothelial lipases (26, 30-33). Studies in perfused organ and intact organism model systems in rats (34, 35), rabbits (36), dogs (37), non-human primates (38), and humans (39-41) have suggested that such Sm LpA-I particles are highly susceptible to rapid and irreversible clearance by renal glomerular filtration. In this way, the clearance of Sm LpA-I particles could mediate a substantial portion of total apo A-I catabolism. Because the overall fractional catabolic rate of apo A-I has been reported by others (42-44) and ourselves (45) to play a key role in determining plasma HDL concentrations, the generation and clearance of the Sm LpA-I particles may help regulate HDL pool size and residence time.

Current methods for quantifying Sm LpA-I concentrations in native plasma usually are slow and technically difficult. Results have been reported using autoradiography (46, 47), chemiluminescence (48), or phosphorimaging (49) of anti-apo A-I Western blots from two-dimensional agarose/native gradient polyacrylamide gel electrophoretic (gPAGE) separations. These methods tend to be only semiquantitative because of intrinsic nonlinearities in solid-phase capture efficiency and immunologic staining intensity of the apo A-I moiety in lipid-poor, Sm LpA-I compared with lipid-rich, large HDL (50-52). A rough quantification of Sm LpA-I in whole plasma can be obtained by agarose electrophoresis followed by immunofixation (53, 54), immunoprecipitation (55-59), or Western blotting (22). Preparative free-flow isotachophoresis has been used by Nowicka et al. (60) to demonstrate the presence in lymph and serum of a rapidly migrating, sudanophilic LpA-I subfraction with potent UC efflux-promoting activity against cultured macrophages, but its identity with the Sm LpA-I described by others has yet to be confirmed. Radial immunodiffusion (56) and ultracentrifugation at d >1.21 kg/L (61) appear to overestimate Sm LpA-I concentrations because of a lack of specificity or excess artifactual dissociation of apo A-I from bulk HDL. Ultrafiltration conveniently separates HDL by size (62, 63) but appears to have very poor resolution and has not been validated for use with lipoproteins. Fielding et al. (8) have developed a monoclonal antibody capable of selectively binding pre-[[beta].sub.1] LpA-I, but they have not reported its use to quantify this lipoprotein subclass.

These methods have been used to measure the distribution of apo A-I in the pre-[beta] or Sm LpA-I fraction in various clinical settings. Generally, the absolute concentration and/or percentage distribution of apo A-I in this fraction is reported to be increased in various pathologic states, including hypertriglyceridemia (46,53,58,59); hypercholesterolemia (46,59); combined dyslipidemia (58); Tangier disease (64); deficiencies of cholesteryl ester transfer protein (65), LCAT (46, 66), and apo C-II (46, 58); coronary artery disease (67); diabetes (46); obesity (68); hepatic cirrhosis (54); and renal insufficiency (57). In contrast, only a few studies of pre-[beta] or Sm LpA-I particles have been done in subjects selected primarily for a broad range of HDL concentrations, with conflicting results. Some have reported an inverse correlation between HDL-cholesterol (HDL-C) and pre-[beta] or Sm LpA-I concentrations (59, 69), whereas others have found a positive correlation (55) or no relationship at all (70). The origin of these confusing differences remains unclear, and our understanding of the clinical significance of these particles remains poor.

Because of the likely importance of Sm LpA-I as either nascent or senescent HDL or both, further study of Sm LpA-I is very important. Such studies would be facilitated by improvements in the methodology for isolating, purifying, and quantifying these particles. Preferably, such methods would be both analytical and preparative, and gentle enough to allow study of Sm LpA-I particles close to their in vivo state. The purpose of the present study was to develop a simple, highly reproducible method for size-based separation and quantification of native apo A-I-containing particles across their entire size distribution, with special emphasis on Sm LpA-I particles. In addition, flexibility with regard to the starting material was desired such that the method could be applicable to whole plasma and to more dilute fluids, such as immunoaffinity-purified lipoprotein fractions, tissue fluids, and cell-culture media. We describe this high-performance size-exclusion chromatographic (HP-SEC) method with preliminary results from its use, and we provide a review of the published literature regarding previously available methods for separation and measurement of Sm LpA-I particles.

Materials and Methods


Male and female subjects with a wide range of HDL-C concentrations were recruited from two sources. Most were patients of one of the authors (E.A.B.) at the Clinical Lipid Center of Wake Forest University, Winston-Salem, NC. Written informed consent was obtained from all of these individuals. A few samples were obtained from patients of other physicians at remote sites; however, because these samples were obtained incidentally to clinically indicated blood drawing, only verbal assent was obtained in those cases. Subject identity was kept confidential, and the Institutional Review Board of Wake Forest College of Medicine approved the study protocol.


Fasting venous blood was drawn using minimal stasis into vacuum-filled glass tubes containing potassium EDTA (1 g/L final concentration; Vacutainer; Becton Dickinson), and the plasma was separated immediately by low-speed (15008) centrifugation for 30 min at 4[degrees]C. Aprotinin (Sigma) was added to the samples (1 mL of a 27 000 kilounits/mL stock solution per 1 mL of plasma), which were then assayed on the same day (held in wet ice) or stored at -70[degrees]C in multiple aliquots.


A standard lipid profile was obtained by measuring total cholesterol, triglycerides (TGs), and HDL-C by Lipid Research Clinics protocols (71) in the Lipid Laboratory of Wake Forest University College of Medicine, Winston-Salem, NC, (Director, Dr. R.W. St. Clair), which is standardized by the CDC (Atlanta, GA). LDL-cholesterol was calculated by the equation of Friedewald et al. (72).


HP-SEC was performed at ambient temperature using a 30 cm x 10 mm Superdex 200 HR 10/30 column (globular protein inclusion range, 10-600 kDa) connected in series to a 30 cm x 10 mm Superdex 75 HR 10/30 column (inclusion range, 3-70 kDa; Pharmacia Biotech). After a brief (0.5 min) clarification at 12 000 rpm in a Microfuge E (Beckman Instruments), whole plasma samples were chromatographed at 0.5 mL/min on an Apple Macintosh-driven HPLC system (Rabbit 110B pumps controlled by Dynamax software; Rainin Instruments) and a buffer system composed of degassed Tris-buffered saline (TBS; 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCI) supplemented with 1 g/L sodium EDTA and 1 g/L Na[N.sub.3]. A 20-100 [micro]L aliquot of whole plasma was injected per run, and protein elution was monitored at 280 nm with a Knauer variable wavelength ultraviolet detector. After the void volume (~14 mL) was discarded, 64 fractions, ~0.25 mL each, were collected using the drop detection mode on a Model FC203 fraction collector (Gilson) directly into polystyrene microtiter plates (Nunc) for lipid assays or into 12 x 75 mm borosilicate glass test tubes for apolipoprotein RIAs. The fractional distribution of apo A-I in the Sm LpA-I particles was calculated by summing the apo A-I mass in peak III (see Results below) and dividing by the apo A-I mass in all 64 fractions. The absolute plasma Sm LpA-I concentration was calculated as the product of the percentage of apo A-I in the Sm LpA-I particles and the plasma total apo A-I concentration (in mg/L) divided by 100. The apparent molecular size of the various HDL subfractions was determined by comparing their elution volumes with those of the proteins in commercially available gel filtration protein calibration mixtures (protein size range, 29-669 kDa; Sigma Chemical Co.). For this purpose, a plot of log molecular mass vs fraction number was constructed and the points fitted using curvilinear least-squares regression analysis.


The apo A-I and apo A-II concentrations in column fractions and whole plasma were measured by a RIA method developed by one of the authors (M.N.N.), using commercial, monospecific polyclonal goat antibodies against purified human apos A-I and A-II (INCStar Corp), as described previously (73).


Total cholesterol, UC, TGs, and choline-containing PLs were measured in column fractions by enzymatic colorimetry using commercially available Trinder-class assays (Boehringer Mannheim and Wako Chemicals USA) and a microtiter plate reader (Titertek Multiskan II; Lab-systems), as described previously (74).


Undiluted plasma samples (1-3 [micro]L) were electrophoresed in 25 cm x 12 cm x 0.5 mm slab gels composed of 1% low electroendosmosis agarose (Bio-Rad Laboratories) without albumin for 1-3 h at 150 V in a barbital/EDTA buffer system (50 mmol/L barbital, 20 mmol/L barbituric acid, 1 mmol/L sodium EDTA, pH 8.5) using water-cooled (4[degrees]C) flat-bed electrophoresis chamber (Multiphor II and Multitemp III; Pharmacia Biotech). After electrophoresis, the agarose gels were pressure-blotted for 15 min under a 1-kg load onto 0.45 [micro]m pore size nitrocellulose membranes (BA85; Schleicher & Schnell), which had been soaked previously in 2.5 mL/L glutaraldehyde, 50 mmol/L phosphate buffer, pH 7.4, to allow covalent immobilization of apos on to the solid-phase matrix. After blocking of unoccupied sites with 25 g/L nonfat milk (Carnation) in TBS for 1 h at room temperature, apo A-I was identified by reaction with 1:100 (by volume) rabbit anti-human apo A-I IgG-peroxidase conjugate (BioDesign) and visualized using a mixture of 0.54 g/L diaminobenzidine tetrahydrochloride, 3 g/L Ni[Cl.sub.2], and 1 mL/L [H.sub.2][O.sub.2] in TBS.

Preparative agarose electrophoresis was performed with a 2-mm thick horizontal gel, run as the analytical gels above but with multiple 1-cm wide lanes for each sample. After electrophoresis, the gels were sliced transversely into 22 narrow strips (from origin to ~125% of the maximum [R.sub.f] of a bromphenol-blue-stained plasma reference marker) from which the proteins were extracted by brief ultracentrifugation (360 000g for 15 min at 4[degrees]C). Recovery of apo A-I from the agarose gel slices (assessed using [sup.125]I-labeled Sm LpA-I) was >95%.


A nonsieving charge-based separation at pH 8.6 in 1% agarose constituted the first dimension. This was followed by a second dimension using the same agarose impregnated with 5 mL/L goat polyclonal anti-apo A-I serum (INCStar), 1 mL/L Tween 20, and 30 g/L polyethylene glycol 8000 to measure apo A-I concentration, as described previously (73).


Isopycnic ultracentrifugation was performed on 3-4 mL of plasma by standard methods using KBr at a solvent density of 1.21 kg/L in 6-mL polyallomer Quick Seal tubes at 280 000g for 48 h at 4[degrees]C in a 40.3 Ti rotor (Beckman Instruments). The tubes were sliced one-third of the distance from the top, and the supernatant (d <1.21 kg/L) and infranatant (d >1.21 kg/L) fractions were assayed for apo A-I mass by RIA as described above. HDL subfraction distribution was also studied by density gradient ultracentrifugation using a Beckman SW41 swinging bucket rotor at 230 000g for 24 h at 4[degrees]C, utilizing the method of Kelley and Kruski (75). After centrifugation, 22 fractions (0.5-mL each) were harvested from the top of the tubes by pumping Fluorinert FC-40 (Sigma) through the bottom; the fractions were then gamma counted directly (radiotracer experiments) or assayed for apo A-I mass by RIA.


HDL was prepared by immunoaffinity column chromatography as described previously (76). Briefly, fresh whole fasting human plasma was exposed to an anti-apo B-100 column to remove VLDL and LDL particles. This was followed by an anti-apo A-I column to trap the HDL, which was then eluted with 0.1 mol/L glycine-HCI, pH 2.5, and neutralized immediately to pH 7.4 by the addition of Tris base.


Correlations between two independent variables were calculated by Pearson least-squares regression analysis using the Statview 512+ program (Brainpower). In comparisons where curvilinear relationships were apparent, [log.sub.10] transformation was used to linearize the data before calculating r values. We assessed differences between grouped variables using the Student t-test.



When fresh human plasma samples were chromatographed over the Superdex 200-Superdex 75 tandem gel permeation columns, we invariably identified at least three distinct peaks of apo A-I. Results from three representative subjects (subjects 2, 8, and 31) with high, normal, and extremely low HDL concentrations are shown in Fig. 1. The first peak, representing the largest apo A-I-containing species, eluted at the void volume in fractions 2-6 (same position as dextran blue 1000) and contained particles of ~1000 kDa (diameter ~15 nm); this peak accounted for 0.2-3.0% of the total apo A-I recovered from the column. The second peak, which eluted in column fractions 9-34, included particles of 100-500 kDa (diameter ~7.5-12 nm), the size of typical, spheroidal HDL, and contained 80-95% of the total apo A-I. The third peak, which eluted in column fractions 35-49, included Sm LpA-I particles 40-60 kDa in size (diameter ~5.8-6.3 nm.), and contained 3-15% of the total apo A-I. No immunoreactive apo A-I was detected in fractions that corresponded to intact monomeric polypeptide (~28.5 kDa) or fragments thereof. A nadir between the major and Sm LpA-I peaks was consistently observed near the peak elution position of albumin (67 kDa), but in most of the specimens we tested, the apo A-I content of these fractions in the trough region never reached zero. This fact probably was not attributable simply to a lack of resolution of the second and third peaks but rather to the presence of a separate group of apo A-I-containing particles of a size similar to that of albumin (see Fig. 4). The ~1000-kDa particles eluting in the void volume most likely represented pre-[[beta].sub.2] and [[beta].sub.3] LpA-I particles, but we could not discount the possibility that a small proportion of apo A-I bound to triglyceride-rich, apo B-containing species also was present in this region.


Our initial attempts to purify Sm LpA-I particles by HP-SEC using single columns of Pharmacia Superose 6, Superose 12, Superdex 200, or Superdex 75 were unsuccessful, despite the fact that their selectivity curves for a mixture of well-defined marker proteins (29-669 kDa) predicted a good separation of Sm LpA-I particles from bulk HDL (data not shown). Only with a combination of one Superdex 200 plus one Superdex 75 column could we achieve satisfactory resolution between these two subfractions; attaching more than two Superdex columns in series gave excellent resolution of large and small HDL subspecies but also produced long run times, high backpressures, and broad peak widths with grossly diluted fractions. Although switching the order of the two Superdex columns had no discernible effect on the subclass resolution, peak dispersion, or particle retention times, we standardized using the Superdex 200-Superdex 75 orientation to minimize any potential diffusion-related band broadening that might occur for solutes in the small molecular size range because this was the region of our primary interest.


We investigated the recovery of HDL subclasses from the Superdex columns in two ways: (a) by chromatographing a known volume of plasma, directly assaying the eluted fractions for apo A-I (by RIA) or total cholesterol (by enzymatic colorimetry), and then comparing the summed lipid/protein mass in all fractions with that in an equivalent volume of the starting material; or (b) by running known quantities of [sup.125]I-labeled lipoprotein fractions (d = 1.063-1.21 kg/L) and then determining the recovery of radioactive counts in all eluted fractions. With both methods, the recovery of protein or lipid mass or radioactivity was consistently in excess of 92%. HP-SEC columns that had not been used previously or columns that had been freshly cleaned of bound material, however, had much lower apo A-I recoveries (<85% in the first run), presumably because of adsorptive losses to nonspecific binding sites on the column walls and stationary phase (77). For this reason, we routinely "presaturated" new and previously cleaned columns with ~10 mL of pooled plasma under a low flow rate (0.1 mL/min) before use in quantitative assays of HDL subclass distribution.

When the sample load applied to the columns was varied in the range 16-124 [micro]L, there was a linear response, with the absolute mass of apo A-I eluting in the Sm LpA-I fraction (r = 0.922; P <0.001). Volumes >125 [micro]L produced a marked deterioration in resolution of Sm LpA-I from larger HDL, with a gradual coalescing of the two peaks into one broader, larger-sized peak (data not shown). We believe, based on these facts, that exceeding the columns' capacity with albumin (the major plasma protein and the one closest in molecular size to Sm LpA-I) deprives Sm LpA-I particles of that proportion of the gel's interstices that they would usually occupy, artifactually displacing them toward the higher molecular size separation region. Using ultracentrifugally isolated HDL, immunoaffinity-purified lipoprotein fractions, or human peripheral lymph, we found no artifactual band broadening or peak shifts with loads as large as 500 [micro]L. HP-SEC quantification of Sm LpA-I concentration in very small volumes of biologic fluids (10-25 [micro]L) was accomplished by adjusting the dynamic measurement range of the apo A-I RIA (using more dilute primary antibody and less [sup.125]I-labeled tracer mass per tube) and by collecting larger fraction sizes at the expense of reduced resolution (with tube changeovers coinciding with the nadir between peaks II and III).

In addition to their adhesiveness to column supports and matrices, HP-SEC-purified Sm LpA-I particles, once eluted off the columns, continued to be extremely "adherent" toward all tested surfaces, including borosilicate glass, dimethyldichlorosilane-treated glass, virgin polystyrene, polypropylene, and Teflon. Only by preconditioning with bovine albumin (50 [micro]L of a 50 g/L solution in TBS per tube) or detergents [1 mL/L Triton X-100, Tween-20, Nonidet P-40, or sodium dodecyl sulfate (SDS)] were we able to harvest Sm LpA-I particles into glass test tubes and then withdraw aliquots quantitatively for subsequent split-sample analyses.

Compared with other HP-SEC matrices such as Hi-Load Sephacryl S100HR and Superose 12, the Superdex columns we used (which are made up of a composite or cross-linked agarose plus dextran) have a less deformable and more chemically resistant gel matrix and thus are good for >100 separations before requiring cleaning or replacement of end frits. However, because their life span could be compromised by specimens with particulate matter (e.g., cryoglobulin precipitates that usually develop in samples stored for extended periods on wet ice) or signs of bacterial burden, we routinely centrifuged all samples at 130 000g for 0.5 min immediately before injection.

When we used a 0.5 mL/min flow rate and collected 64 fractions (fraction volume, 250 [micro]L), a complete fractionation of the entire apo A-I particle size spectrum could be achieved in ~60 min. Because there was no need for fraction collection for the first 28 min, which is also sufficient washout time for elution of very small molecular mass contaminants from a previous run, an overlapping injection protocol at hourly intervals could be used to maximize throughput. Typically, six specimens could be manually chromatographed in 1 working day on a single HPLC system, and the overall turnaround time from HP-SEC to data reduction was <1.5 days.


We determined the within-batch precision of our method by performing six repeated HP-SEC runs during a single day, using one batch of freshly isolated EDTA-anticoagulated plasma from a single healthy subject (total apo A-I concentration, 1020 mg/L). The following percentages of apo A-I in Sm LpA-I were found: 5.19%, 5.79%, 4.56%, 4.48%, 4.52%, and 5.19% (mean, 4.95%; CV, 11%).

Paired aliquots of three different plasma samples (total apo A-I concentration, 560, 990, and 1880 mg/L) were chromatographed before and after rapid freezing at -70[degrees]C and thawing 4-6 h later and were assayed immediately for Sm LpA-I particles by HP-SEC. There was no detectable effect of the single freezing and thawing procedure on the percentage of apo A-I in Sm LpA-I (mean [+ or -] SE, 4.7% [+ or -] 0.25% before vs 4.95% [+ or -] 0.18% after freezing; P, not significant by paired t-test). Storage of plasma at -70[degrees]C for up to 8 months also failed to show any appreciable trend over time in the percentage of Sm LpA-I distribution (5.02%, 4.77%, 5.54%, and 4.94% for single assays of one pool frozen for 4 h, 2 months, 4.5 months, and 8 months, respectively).


Because Sm LpA-I particles appear to constitute the vast majority of HDL with pre-[beta] mobility by agarose electrophoresis, the amount of apo A-I in Sm LpA-I particles by our HP-SEC method should be roughly equivalent to its amount in pre-[beta]-migrating species by agarose electrophoresis. Fig. 2 shows the results of distribution of the concentrations of apo A-I in Sm LpA-I and pre-[beta] LpA-I particles from split samples (HDL-C, 0.57-2.07 mmol/L). We found a good correlation (r = 0.98; P <0.0001) between the two measurements.

Sm LpA-I particles also appear to be lipid-poor, protein-rich particles (25, 50, 58, 61, 77, 78) (see Fig. 6) and have a greater buoyant density (d >1.21 kg/L) than most lipid-rich, spheroidal HDL (d <1.21 kg/L) (49). Thus, it was of interest to compare the percentage of total apo A-I in Sm LpA-I particles obtained by our HP-SEC method with the percentage in the d >1.21 kg/L plasma fraction after preparative ultracentrifugation. As can be seen in Fig. 3, these two measures of apo A-I particle distribution were significantly correlated (r = 0.86; P <0.0001), although results obtained by ultracentrifugation were approximately twofold greater than those by HP-SEC. In experiments where HP-SEC-purified and radioiodinated Sm LpA-I was subjected to ultracentrifugation (n = 3 preparations), <15% of counts were recoverable in fractions with solvent densities <1.21 kg/L (data not shown).

It has been shown that Sm LpA-I particles or pre-[beta]-migrating HDL may be highly unstable in plasma at 37[degrees]C. In short-term incubations in the presence of LCAT, they are quantitatively remodeled into larger spheroidal HDL, whereas in long-term incubations, they are generated in excess through the effects of lipid transfers and/or particle fusion (21-33, 48, 64, 66, 79). Using our HP-SEC method, we have confirmed the unstable character of Sm LpA-I particles by incubating fresh whole plasma at 37[degrees]C in vitro for various periods of time. As can be seen in Fig. 4 (representative of four normolipidemic plasmas), after 2 h at 37[degrees]C, the Sm LpA-I concentration decreased by ~45% to 16 mg/L, from a starting concentration of 36 mg/L, whereas after 18 h, the concentration had increased by ~350%, to 127 mg/L. Note that it was also possible with our method to demonstrate significant remodeling of apo A-I in other HDL subspecies upon in vitro incubation; band broadening of the major HDL peak occurred after 2 h, and generation of very large particles (~1000 kDa) was evident after 18 h.




To examine the relationship between particle size and agarose electrophoretic mobility, we loaded fresh whole plasma on the Superdex gel filtration system and then subjected selected column fractions (collected in tubes preconditioned with bovine serum albumin) to one-dimensional agarose gel electrophoresis, followed by covalent immobilization on nitrocellulose membranes and immunodetection with peroxidase-conjugated polyclonal anti-apo A-I antibodies. As can be seen in Fig. 5 (representative result of a hyperalphalipoproteinemic plasma specimen; total apo A-I concentration, 2710 mg/L), fractions corresponding to the major apo A-I peak (fractions 10-30) consisted predominantly of [alpha]-mobile species, whereas fractions corresponding to the third apo A-I peak (Sm LpA-I, fractions 37-41) had pre-[beta] electrophoretic mobility.

To further explore the relationship between agarose electrophoretic mobility and particle size, we performed the converse of the above experiment by examining the size distribution of apo A-I-containing particles contained within the pre-[beta] mobile zone of a one-dimensional, preparative agarose gel loaded with [sup.125]I-labeled immunoaffinity-purified total HDL. The predominant moiety present in the pre-[beta] band was Sm LpA-I (>87% of the total counts), with only a minor contribution from very large apo A-I-containing species, most likely contamination by pre-[[beta].sub.2] and pre-[[beta].sub.3] LpA-I particles or conceivably aggregated Sm LpA-I particles (data not shown).

Sm LpA-I particles appear to be neither created nor destroyed during the process of HP-SEC: repeat chromatography of the major HDL peak (peak II) failed to generate Sm LpA-I particles (peak III), and repeat chromatography of previously isolated Sm LpA-I particles did not produce appreciably larger apo A-I particles. The concentration of Sm LpA-I particles is not changed when aliquots of a sample are run at varying chromatographic flow rates over a 20-fold range (0.05-1 mL/min), suggesting that the moderate hydraulic pressures (<3 MPa) created at 0.5 mL/min, our optimal flow rate for apo A-I, do not substantially alter HDL subpopulation integrity (data not shown).



The lipid and apo composition of Superdex HP-SEC column fractions from a subject with a high HDL-C concentration (4.2 mmol/L; representative of the results from two such subjects) are shown in Fig. 6. The bottom panel of Fig. 6 shows the typical three apo A-I-containing peaks (the center peak being partially split, possibly corresponding to the size difference between [HDL.sub.2] and [HDL.sub.3]). In contrast, immunoreactive apo A-II appears only in the first two peaks and is undetectable in Sm LpA-I. The choline-containing PLs also elute as three distinct peaks (Fig. 6, center panel), but the smallest peak elutes before the Sm LpA-I peak (Fig. 6, magnified inset on right), coinciding with the nadir between the second and third apo A-I-containing subspecies. The column fractions in this trough region contain apo A-I-bearing particles of very slow electrophoretic mobility, as shown in Fig. 4, and the calculated mean PL:apo A-I molar ratio in this region is very high at 200:1. In contrast to this PL-rich region, the later-eluting Sm LpA-I region has far less PL, with a mean PL:apo A-I molar ratio of ~2:1. This value was confirmed by gas-liquid chromatography of pooled fractions of this region, apo A-I peak III (Wilson, unpublished data). The profiles in Fig. 6 also show that Sm LpA-I particles have little or no TGs, UC, or cholesteryl ester (CE). Autoradiography of reducing and nonreducing SDS-polyacrylamide gels loaded with radio-iodinated Sm LpA-I revealed that only a single band of molecular mass ~28 kDa was visible (data not shown).


To test for the possible physiologic relevance of Sm LpA-I particles, the apo A-I mass in these particles was measured by HP-SEC as described above in plasma samples taken from 30 human subjects free from thyroid, renal, and hepatic disease and not taking medications known to alter lipoprotein concentrations. HDL-C concentrations in these specimens were spread over a 10-fold range, 0.363.83 mmol/L (Table 1). The percentage of apo A-I in Sm LpA-I particles correlated strongly and linearly with fasting plasma TG concentrations (r = 0.581; P <0.0005; Fig. 6), and inversely (hyperbolically) with plasma total apo A-I (r = -0.551; P = 0.0013 for log-transformed apo A-I) and HDL-C (r = 0.532; P = 0.0017 for log-transformed HDL-C). The latter two relationships, however, were dependent on inclusion of data from two severely hypoalphalipoproteinemic subjects (HDL-C concentrations, 0.03 and 0.13 mmol/L) in whom the fraction of total apo A-I in the Sm LpA-I particles was markedly increased to 25% and 37%, respectively. In the combined data set (n = 32), the absolute concentration of apo A-I in Sm LpA-I particles was positively and linearly correlated with plasma total apo A-I (r = 0.856; P <0.0001) and HDL-C (r = 0.816; P <0.0001) but negatively (hyperbolically) with plasma TGs (r = 0.45; P <0.001).



This is the first report of a simple and reproducible method for the preparative isolation of HDL subpopulations from fresh or frozen plasma across their full particle size spectrum. We have quantified the mass of apo A-I in the smallest, most lipid-poor particles, which we term Sm LpA-I, and we have validated this method in comparison with other available techniques. We have also provided preliminary data regarding the possible physiologic relevance of the proportional and absolute distribution of apo A-I in Sm LpA-I particles.


What are the relative merits of using HP-SEC for HDL subfraction characterization over other previously established methods? Although two-dimensional agarose/native gPAGE provides excellent size and charge resolution, it is not a preparative technique, which precludes its utility in detailed analytical work and in the preparation of HDL subspecies for in vitro and in vivo metabolic studies. In practical terms, this method can be costly with regard to reagents and equipment, is technically demanding to perform, and often has relatively long turnaround times of 2 or more days. Moreover, accurate quantification of all LpA-I subclasses is rarely possible because of nonlinearities in electrotransfer, solid-phase capture, and immunologic detection of particles with widely dissimilar charges, conformations, and concentrations (50-52). Quantification of apo A-I in the pre-[beta] mobile zone in one-dimensional agarose gels by various methods [e.g., crossed immunoelectrophoresis (CIEP) (56-59) or immunofixation (53, 54)] will be inaccurate depending on the degree of contamination with larger apo A-I-containing particles of equivalent electrophoretic migration [pre-[[beta].sub.2] and pre-[[beta].sub.3] LpA-I particles, and apo A-I associated with TG-rich, apo B-containing lipoproteins (TGRLs)]. Analogously, the reliability of Sm LpA-I measurements in whole plasma by ultrafiltration-based strategies will be affected by the accuracy and dispersion of pores in the ultrafilter membranes used; currently available supports do not provide discrete molecular size cutoffs but rather a gaussian distribution of porosities, deviating by up to 20% from the nominal value (Nanjee and Brinton, unpublished results, and manufacturers' literature), and they also lack physical and chemical inertness. Quantification of Sm LpA-I particles in plasma by centrifugal ultrafiltration incorporating isotope dilution (62, 69) can overcome some of these deficiencies, but care must be taken to prevent introduction of physicochemical disparities in the exogenous Sm LpA-I tracer during the in vitro radiolabeling procedure, and to ensure that there is no isotopic exchange/fusion of the tracer particles with other LpA-I pools coexistent in the test specimens. Physical separation of Sm LpA-I particles from bulk HDL is not routinely carried out before assay by radial immunodiffusion, which may lead to a tendency to overestimate the concentrations of the Sm LpA-I particles (56).


Several groups have used low-pressure, gravitationally assisted gel filtration chromatography to study HDL particle size distribution (46, 77, 80-83), whereas a few recent studies have used newer, more reproducible, fast-flow HP-SEC columns driven by medium-pressure liquid chromatographic equipment (26, 64, 79, 84, 85). Our report differs from all of these previous reports in three ways. First, by combining two composite-material, low nonspecific-binding stationary phases with relatively steep selectivity curves for solute separation in the size ranges of both large, spheroidal HDL (mainly a function of Superdex 200) and Sm LpA-I (mainly accomplished by Superdex 75) within a single tandem unit, we have succeeded in obtaining good resolution, high recovery, and reproducible separation of multiple HDL species in a single chromatographic step. Second, our method permits the use of whole plasma as the starting material, which avoids artifacts known to be induced by ultracentrifugation and possibly by immunoaffinity-based procedures that have traditionally been used to isolate apo A-I-containing particles from complex lipoprotein mixtures. Thus, our HP-SEC method is ideally suited for analysis of Sm LpA-I particles in biologic fluids under conditions that mimic the in vivo situation. Finally, by demonstrating that these particles are essentially devoid of apo A-II, UC, CE, and TGs, our study extends the observations of others who have characterized Sm LpA-I particles solely by their overall protein mass (ultraviolet absorption) (64, 79, 84) or apo A-I content (26, 77, 80, 81, 83, 85).

Our HP-SEC method utilizes commercially available, prepacked, multiple-use columns that are eluted with a simple aqueous isocratic mobile phase under precisely controlled flow rates. In combination with the advantages afforded by the inherent sensitivity and broad measurement range of RIA, it can attain high precision and throughput and can be readily adapted for automation by the use of unattended robotic sample injectors plus liquid-transfer devices. The equipment required (single channel HPLC solvent delivery module attached to a fraction collector, centrifuge, and gamma scintillation counter) is available in most biochemistry laboratories, and one pair of Superdex HP-SEC columns potentially can be used for several hundred chromatographic runs.


Because the size-separation step in our HP-SEC method is carried out at ambient temperature under moderate hydraulic pressures (but not exceeding 3 MPa) and the various plasma components incur a considerable dilution relative to the starting material (10- to 50-fold), it may be argued that the Sm LpA-I particles we have observed are created artifactually in vitro by the breakdown of large HDL particles by passive (physicochemical) (32, 33) and/or active (enzymatic) (21-31, 48, 64, 66, 79, 84) remodeling events. Conceivably, our finding of diverse percentages of apo A-I in Sm LpA-I particles between different subjects might be explained simply by variations in the in vitro dissociation of apo A-I. Several lines of evidence, however, argue against such an artifact: (a) Rechromatography of previously isolated large HDL does not produce a Sm LpA-I peak. (b) There is no measurable change in apo A-I particle size distribution when the HP-SEC flow rate (hence fluid pressure plus separation time) is changed over a 20-fold range. (c) If HP-SEC causes apo A-I stripping by processes such as selective adsorption on to the stationary phase, then Sm LpA-I particles should form a trailing edge to the major, spheroidal HDL peak, whereas in fact we observe a discrete, symmetrical peak. (d) The percentage of apo A-I mass in Sm LpA-I particles is highly concordant with that of pre-[beta] LpA-I particles by CIEP (see Fig. 2), a method radically different from our HP-SEC technique. (e) Perhaps most convincingly, in vitro incubation of human plasma from healthy individuals at 37[degrees]C for 2-4 h ablates the Sm LpA-I peak (see Fig. 4), an effect that can be blocked by chemical inhibitors of cholesterol esterification. Thus, Sm LpA-I particles are not invariably created during HP-SEC, and thus are invariably detected, but rather they appear only under conditions in which they would be expected to exist before chromatography. We cannot entirely rule out the occurrence of enzymatic or other changes in HDL architecture during the HP-SEC procedure, but these processes probably take place at slower rates at ambient temperature and are largely restricted by the rapid separation of the various substrate, cofactor, and donor/ acceptor species that occur during permeation of the plasma sample into the Superdex matrix. If desirable, the possibility of enzymatic degeneration of the HDL during the HP-SEC procedure can be further minimized by carrying out HP-SEC at 4[degrees]C or by pretreating the test samples with (immuno)chemical inhibitors of LCAT, lipase, and/or lipid transfer protein activity.


Several published reports have studied Sm LpA-I size by a variety of separation methods. Although they differ somewhat in results, they generally agree with the findings of the current report. Vezina et al. (78) isolated Sm LpA-I particles in normal serum by nondenaturing gPAGE and detected one minor species of ~56 kDa. Using a similar technique, Ishida et al. (46) detected two major components 67 and 75 kDa in size in dyslipidemic plasmas. Castro and Fielding (7), Francone et al. (47), and Asztalos et al. (49) combined this method with agarose gel electrophoresis into a two-dimensional separation procedure to demonstrate the existence of a group of particles of 71 kDa mean molecular mass (5.4-5.8 nm Stokes diameter). Several workers have utilized SEC, alone or in combination with other methods, to characterize Sm LpA-I particles. Schonfeld et al. (77) subjected whole plasma to low-resolution gel filtration chromatography through Sephadex G100 and observed a 50-kDa particle. Kunitake et al. (55) used Sephacryl S300 chromatography to estimate the size of pre-[beta] LpA-I particles (previously isolated by starch-block electrophoresis), and found a value of 80 kDa. Neary and Gowland (41) reported an apo A-I-containing species of 43 kDa when plasma was denatured in 9 mol/L urea and then passed through Sephacryl S200. Melchior and Castle (81) fractionated conditioned media from primary cultures of cynomolgus monkey hepatocytes using 0.5 mol/L Biogel A and found that approximately one-third of the total apo A-I eluted in a peak of 50 kDa. When the HDLZ ultracentrifugal subfraction of plasma was reacted with purified phosphatidyl transfer protein, Sm LpA-I particles of ~45 kDa were generated that could be detected using Superose 6 HP-SEC (84). To characterize conditioned media from macrophages exposed to human plasma, Huang et al. (64) used a combination of immunoaffinity chromatography and HP-SEC through Sephacryl S100HR, and observed a discrete ultraviolet-absorbing species that eluted at 43-67 kDa (6.0-7.0 nm Stokes diameter), which was apparently larger than delipidated, monomeric apo A-I (28 kDa). Using HP-SEC of immunoaffinity-purified lipoproteins through Superose 12, Hennessey et al. (79) detected a minor, 105-kDa apo A-I-containing peak in the LpA-I subfraction of plasma that was absent in the LpA-I + A-II subfraction.


Published data on the chemical composition of Sm LpA-I particles are somewhat more conflicting than reports of their size. Daerr et al. (58) could not detect any lipids in the pre-[beta]-migrating fraction, which prompted them to name the fraction "free" apo A-I. Atmeh (63) isolated small HDL from fresh whole plasma by ultrafiltration through 70-kDa cutoff cellulose acetate membranes and found that they consisted of 67.5% protein by mass and 32.5% lipid (2.7% UC, 14.2% CE, 12.7% PL, and 3.0% TGs). The presence of nonpolar lipids strongly indicates contamination with spheroidal HDL and implies the presence of pores larger than 70 kDa-equivalents. Benvenga (83) isolated a particle with a mean molecular size of 68 kDa by a combination of immunoaffinity, ion-exchange, and gel filtration chromatography, and reported that it was 83% protein by weight, with an apo A-I:PL:CE molar ratio of 2:11:5. Using two-dimensional agarose/gPAGE, Castro and Fielding (7) calculated that pre-[[beta].sub.1] LpA-I particles have an apo A-I:UC:PL molar ratio of 1.2:13.9:40.9. On the other hand, Kunitake et al. (55) reported that Sm LpA-I particles are >90% protein by weight, with the remaining mass in small amounts of UC, CE, and PL, but no TGs. Liang et al. (86) used Superose 6 HP-SEC and ultrafiltration to recover Sm LpA-I particles that had dissociated from bulk spheroidal HDL under the influence of cholesteryl-ester transfer protein and calculated that each molecule of apo A-I was associated with, at most, only one molecule each of PL and UC; no additional apos were present. Labeur et al. (87) demonstrated that when reconstituted discoidal LpA-I particles, doubly labeled in the outer lipid bilayer with fluorescent PL and in the core with fluorescent CE, were exposed to excess exogenous apo A-II in vitro, there was generation of Sm LpA-I particles with significant amounts of PL but no CE.

In the present work, we measured the chemical composition of several preparations of Sm LpA-I particles from several different normolipidemic plasma specimens by HP-SEC (see Fig. 5 for an example) and found an apo A-I:PL molar ratio of ~1:2, without evident variability among subjects. There was negligible contribution from UC, CE, and TGs. In addition, apos A-II, A-IV, C, and E were undetectable in this HDL subfraction as assessed by specific RIAs or autoradiography of SDS-gPAGE gels loaded with a radioiodinated Sm LpA-I fraction, in accord with previous reports (5, 22, 55, 56, 70) with one exception (63). Because the low-end detection limits of the enzymatic colorimetric lipid assays that we used were in the range 0.5-1 nmol/well, it may be argued that our lipid estimates are biased toward the low side; however, we have confirmed the almost total absence of UC and CE and the very low mass of PLs in Sm LpA-I particles by making measurements in pooled and 5- to 10-fold concentrated HP-SEC fractions, as well as by utilizing more sensitive lipid measurement techniques such as gas-liquid chromatography and fluorometry of organic solvent extracts. We currently are undertaking additional studies using HP-SEC and capillary electrophoresis to determine whether pyrene-labeled PL can partition into Sm LpA-I particles and permit their quantification in native plasma and extravascular fluids. That the tissue origin of Sm LpA-I particles is likely to be a critical determinant of their chemical composition is suggested by the work of Jaspard et al. (88), who reported significant amounts of CE and TGs (and therefore a hydrophobic core) in human follicular fluid pre-[[beta].sub.1] HDL.

On the basis of reports that apo A-I tends to self-associate in aqueous solution (89) and according to our Superdex HP-SEC estimates of 40-60 kDa apparent molecular mass for Sm LpA-I particles, we speculate that on average, these particles consist of two copies of apo A-I monomeric peptide with their hydrophobic surfaces in apposition, and with approximately four molecules of PL intercalated. Clearly, this species lacks sufficient PL to be discoidal. The slightly larger, pre-[beta] LpA-I particles that coelute in the albumin size range have many more PLs per particle, possibly enough to be discoidal (compare Figs. 5 and 6). From our pilot in vitro incubation studies, we further speculate that Sm LpA-I particles may be physiologic precursors of the larger, PL-rich species, and that the relatively low abundance of the latter (as assessed by apo A-I content) may suggest that the slower, rate-limiting step of its maturation is the acquisition of PLs rather than acquisition and esterification of UC. If this is true, then the former process, the "phospholipidation" of lipid-poor Sm LpA-I particles in the intravascular and/or tissue fluid compartments, may be a key target for future investigation (90, 91).

The discrepancies between our values for Sm LpA-I size, chemical composition, and fractional distribution in normolipidemic plasma and those in the published literature may stem from several factors: (a) the properties and extent of preanalytic manipulation of the starting material (e.g., plasma, serum, immunoaffinity-purified or ultracentrifuged HDL); (b) differences in the methods used for the isolation of Sm LpA-I particles because their identity is based on different physicochemical principles (size and/or charge); and (c) the detection limits and specificity of the techniques used for apo and lipid characterization and quantification. Using a CIEP assay, Daerr et al. (58) were unable to detect any apo A-I unassociated with typical pre-[beta]-migrating HDL in 16 normolipidemic sera because their assay was not sensitive enough to detect pre-[beta] LpA-I particles if present in concentrations <10% of the total apo A-I mass. Low sensitivity and possible losses from multiple preanalytical steps could possibly explain the inability of Kunitake et al. (55) to detect pre-[beta] LpA-I particles in patients with very low HDL. Castro and Fielding (7), Ishida et al. (46), and Francone et al. (47) estimated that Sm LpA-I particles are 67-75 kDa in size, but this could be a slight overestimation because the 4-27% gPAGE used by these authors may not have been run to equilibrium. We also have had difficulty in accurately determining the hydrated Stokes diameter of Sm LpA-I particles by two-dimensional agarose/gPAGE using commercial 8-25% Pharmacia Phastgels or in-house fabricated 4-30% gels; when we loaded radioiodinated or unlabeled immunoaffinity-purified total LpA-I particles, the smallest-sized apo A-I-containing particles migrated off the bottom of the gels even when run to less than one-half the 3000 V-h necessary for achieving electrophoretic equilibrium (Nanjee and Brinton, unpublished data).


The third PL peak coelutes with albumin and with the minimum apo A-I fraction content, but the identities of the particles represented by this peak are unclear. The PL:apo A-I molar ratio in this region is 200:1, consistent with that of apo A-I:PL discs. Such discs, however, would likely be as large as the largest spheroidal HDL particles, whereas the elution position of this PL peak indicates a much smaller particle size. Furthermore, the apo A-I in these fractions has an unusually slow pre-[beta] mobility on agarose electrophoresis, as shown in Fig. 4. Thus, the apo A-I in these fractions appears to belong to a nondiscoidal particle that is not likely to account for much of the PL peak. Some PL might be bound to another apo, such as apo A-IV, of which there is a minor peak in this region (Nanjee and Brinton, unpublished results), but such particles also are likely to contain little PL. Another possible explanation relates to the fact that the PL peak coincides with the peak of plasma albumin that is known to bind large quantities of lysophospholipid, and the latter is measured as PL by our method. Thus, the majority of the third PL peak may be lysophospholipid bound to albumin, rather than PL bound to apolipoproteins.


In the present study we analyzed plasmas from 30 normolipidemic men and women and found a mean of 7.5% (SE, 0.6%; range, 2-15%) of total plasma apo A-I transported in the Sm LpA-I fraction (see Table 1). This amount is similar to those obtained by other workers, using a variety of different analytical techniques: method and mean values (or range), one-dimensional agarose, 4% (46); two-dimensional agarose/gPAGE, 4% (70) and 5% (67); ultrafiltration, 7% (62); CIEP, 6% (59); and HP-SEC, 5-6% (85). However, substantially lower and higher values have also been reported: CIEP, undetectable (58); one-dimensional agarose, 2% (48); ultrafiltration, 2% (63); ultracentrifugation, 8-10% (61); one-dimensional gPAGE, 12% (52); one-dimensional starch block, 14% (55); and radial immunodiffusion, 10-30% (56).

In the present study we found a statistically significant relationship between the percentage of total apo A-I in Sm LpA-I particles and fasting plasma TG concentrations in a group of healthy men and women. These findings are in accord with those of Schonfeld et al. (77), who demonstrated that against a background of hypertriglyceridemia, whether attributable to overproduction or undercatabolism of TGRL, Sm LpA-I particles can increase to up to 40% of total plasma apo A-I. Atmeh and Robenek (52) also found statistically significant positive correlations between these two variables in normolipidemic and dyslipidemic subjects. In one study, the highest Sm LpA-I concentrations, both in absolute and proportional terms, were found in patients with type III and type V dyslipidemia (59). Which is the cause and which is the effect in this relationship is unclear. Previously, our group has demonstrated that the most striking response to an intravenous infusion of delipidated apo A-I into healthy humans is an increase in TGRL concentrations (73), and a similar result was found by Ha et al. (92) in rats given either rat or human apo A-I. The TG-raising effect, at least partially, could reflect inhibition of endothelial TG lipases by lipid-poor apo A-I (93-95). On the other hand, in vitro studies have shown that Sm LpA-I particles can be generated by the actions of lipases on large, TG-enriched HDL (26, 30, 31) and possibly also in vivo during lipolysis of TGRL after a fat-rich meal (23, 52, 58). A simple interpretation of the dynamic that exists between Sm LpA-I and TGRL is further confounded by the observations of Neary et al. (23) that concentrations of Sm LpA-I particles decline rapidly after intravenous administration of Intralipid to humans and that greater decrements in concentrations of Sm LpA-I particles occur during in vitro incubation of postprandial serum compared with fasting serum. In any event, it is clear that a more thorough characterization of the properties of Sm LpA-I particles in their homogeneous state, through preparative isolation using a method such as ours, and their interaction with other LPs in simple in vitro model systems, should precede testing of their possible impact on TGRL metabolism in vivo.

Our study joins four previous reports (55, 59, 69, 70) of the relative particle size distribution of plasma apo A-I-containing LPs in a group of healthy subjects selected for a broad range of steady-state HDL concentrations. We found a significant inverse (hyperbolic) relationship between the proportion of apo A-I in Sm LpA-I particles and the concentrations of HDL-C and total apo A-I (positive relationships with absolute Sm LpA-I concentrations; see Fig. 7), although these depended largely on the inclusion of two subjects with extreme HDL deficiency. Our data concur with the results of several other groups (68, 70, 86) and imply that the fractional distribution of apo A-I within Sm LpA-I particles is of physiologic significance for HDL metabolism. The possibility that a preponderance of Sm LpA-I particles may somehow be a cause of low HDL-C and apo A-I concentrations was suggested by our previous work (45) in which excess apo A-I in the d >1.21 kg/L fraction and a low HDL-C:(apo A-I + apo A-II) ratio correlated well with an increased apo A-I fractional catabolic rate. Animal studies by Horowitz et al. (39) have also provided evidence that small dense HDL particles may be cleared rapidly by glomerular filtration. In keeping with these findings, Neary and Gowland (41) found a close relationship between Sm LpA-I concentrations and renal glomerular filtration rate in humans, and a rapid decrease to normal Sm LpA-I concentrations in patients given kidney transplants. In our HP-SEC study, we found no significant correlation between absolute Sm LpA-I concentrations and total apo A-I or HDL-C concentrations. Using two-dimensional agarose/gPAGE, Miida et al. (96) also could find no relationship between pre-[[beta].sub.1] LpA-I concentrations and [HDL.sub.2], [HDL.sub.3] or LpA-I concentrations in normolipidemic and dyslipidemic subjects. This clearly suggests that the ratio of Sm LpA-I concentration to bulk HDL is not always a constant function and, therefore, that Sm LpA-I concentrations can not be predicted by simpler markers.


In split-sample comparisons of apo A-I distribution by HP-SEC and preparative ultracentrifugation, the percentage of apo A-I in the d >1.21 kg/L fraction usually exceeded that measured in the Sm LpA-I particles by chromatography (see Fig. 3). This likely reflects the reported tendency of apo A-I to dissociate from spheroidal HDL under the influence of prolonged high gravitational burden in the presence of extreme salt concentrations (39, 49, 97, 98). Because the excess percentage of apo A-I in the d >1.21 kg/L fraction vs that in chromatographically isolated Sm LpA-I particles increased with increasing percentage of either, the apparent artifact of ultracentrifugal dissociation of apo A-I may reflect as yet poorly understood factors that reduce apo A-I binding affinity to bulk HDL in subjects with low HDL concentrations.

In summary, we have developed an efficient and reproducible method for preparative separation of Sm LpA-I particles from human plasma. We present evidence that the method is valid and does not induce significant artifacts. Both the composition of the Sm LpA-I particles and the correlation of the percentage of total plasma apo A-I in these particles with physiological markers of HDL metabolism suggest that Sm LpA-I particles may constitute key nascent and/or senescent HDL species. In either case, further study of Sm LpA-I particles is likely to be important in elucidating the metabolism and functions of HDL in its crucial role in antiatherogenesis.

This work was supported in part by a General Clinical Research Center Grant (M01 RR07122) from NIH to Bowman Gray School of Medicine, Wake Forest University, a Clinical Investigator Award (HL-02034) from NIH, a Grant-in-Aid (96013360) from the American Heart Association, and a Merit Review Award from the Department of Veterans' Affairs (all to E.A.B.). We thank Professors William R. Hazzard, Lawrence L. Rudel, and Norman E. Miller for numerous helpful discussions, and Ellen Burleson for expert technical assistance.

Received August 30, 1999; accepted November 23, 1999.


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[3] Nonstandard abbreviations: ape, apolipoprotein; Sm LpA-I, small apo A-I-containing lipoprotein; PL, phospholipid; UC, unesterified cholesterol; LCAT, lecithin:cholesterol acyltransferase; gPAGE, gradient polyacrylamide gel electrophoresis; HDL-C, HDL-cholesterol; HP-SEC, high-performance size-exclusion chromatography; TG, triglyceride; TBS, Tris-buffered saline; SDS, sodium dodecyl sulfate; CE, cholesteryl ester; ClEP, crossed immunoelectrophoresis; and TGRL, triglyceride-rich lipoprotein.


[1] Department of Cardiovascular Biochemistry, St. Bartholomew's and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1 M 6BQ, United Kingdom.

[2] Section of Metabolism, Endocrinology, and Nutrition, 111E Carl T. Hayden VA Medical Center, 650 East Indian School Road, Phoenix, AZ 85012-1892.

* Author for correspondence. Fax 602-200-6004; e-mail eliot.brinton@
Table 1. Demographic and lipoprotein data.

 Age, TC, (b)
Subject (a) Sex years mmol/L

 1 F 52 7.4
 2 F 47 7.3
 3 F NA 7.9
 4 F 57 6.5
 5 F 55 5.7
 6 F 76 5.9
 7 M 54 5.6
 8 F 62 12.2
 9 F 77 5.4
 10 F 92 2.0
 11 M 43 4.5
 12 F 17 7.2
 13 M 32 3.8
 14 M 52 4.7
 15 M 43 5.3
 16 F 63 6.0
 17 M 56 6.2
 18 M 76 3.8
 19 M 42 4.7
 20 F 46 5.3
 21 M 60 4.8
 22 M 52 3.6
 23 M 41 4.2
 24 M 56 4.4
 25 M 60 3.9
 26 M 38 4.7
 27 M 30 4.4
 28 F NA 2.2
 29 M 49 4.5
 30 M 39 4.3
 31 M 30 4.5
 32 M 37 2.1
 Mean 51 5.2
 SE 3 0.3

Subject (a) mmol/L mmol/L mmol/L

 1 3.3 0.6 3.83
 2 3.7 0.6 3.34
 3 4.5 0.4 3.24
 4 3.0 0.7 3.11
 5 2.5 0.5 2.95
 6 3.4 0.6 2.31
 7 3.0 1.3 2.10
 8 10.5 0.9 1.24
 9 3.7 1.0 1.17
 10 0.8 0.5 0.96
 11 3.0 1.2 0.88
 12 6.0 1.0 0.78
 13 2.5 1.2 0.78
 14 3.6 0.7 0.75
 15 3.8 1.6 0.73
 16 3.4 4.1 0.70
 17 4.5 2.3 0.67
 18 2.4 1.6 0.65
 19 3.3 1.8 0.62
 20 3.7 2.1 0.62
 21 NA 6.1 0.57
 22 1.3 3.7 0.57
 23 2.4 2.7 0.54
 24 1.8 4.5 0.52
 25 1.6 3.8 0.49
 26 NA 5.1 0.49
 27 2.4 3.3 0.49
 28 1.5 0.8 0.39
 29 3.0 2.5 0.36
 30 NA 6.2 0.36
 31 NA 9.7 0.13
 32 0.5 3.4 0.03
 Mean 3.2 2.4 1.14
 SE 0.4 0.4 0.19

 apo A-I, apo A-I in apo A-I in d
Subject (a) mg/L SmLpA-I, (c) % >1.21 kg/L, (d) %

 1 2710 9 NA
 2 1780 3 5
 3 2620 7 7
 4 1930 13 6
 5 2110 9 9
 6 1730 6 NA
 7 1590 6 8
 8 1000 7 16
 9 1090 9 NA
 10 610 2 10
 11 NA 5 NA
 12 610 7 19
 13 820 6 13
 14 600 11 16
 15 600 6 19
 16 720 13 47
 17 640 15 NA
 18 520 6 NA
 19 580 7 NA
 20 670 5 17
 21 790 7 22
 22 690 6 NA
 23 520 7 NA
 24 650 9 NA
 25 620 15 28
 26 530 5 NA
 27 530 6 NA
 28 350 3 18
 29 400 9 20
 30 550 7 21
 31 140 37 86
 32 30 25 100
 Mean 930 9 24
 SE 120 1 6

 apo A-I in apo A-I in
 SmLpA-I, d >1.21 kg/L,
Subject (a) mg/L mg/L

 1 244 NA
 2 53 89
 3 183 183
 4 251 116
 5 190 190
 6 104 NA
 7 95 127
 8 70 160
 9 98 NA
 10 12 61
 11 NA NA
 12 43 116
 13 49 107
 14 66 96
 15 36 114
 16 94 338
 17 96 NA
 18 31 NA
 19 41 NA
 20 34 114
 21 55 174
 22 41 NA
 23 36 NA
 24 59 NA
 25 93 174
 26 27 NA
 27 32 NA
 28 10 63
 29 36 80
 30 38 115
 31 52 120
 32 8 30
 Mean 73 128
 SE 11 15

(a) Subjects listed in order of descending HDL-C concentration in
fasting plasma.

(b) TC, total cholesterol; LDL-C, LDL-cholesterol; NA, not available.

(c) Sm LpA-I denotes small apo A-1-containing particles from peak
III from HP-SEC.

(d) d >1.21 kg/L denotes apo A-I in ultracentrifugal fraction after
spinning against solvent with a density of 1.21 kg/L.
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Title Annotation:Lipids, Lipoproteins and Cardiovascular Risk Factors
Author:Nanjee, M. Nazeem; Brinton, Eliot A.
Publication:Clinical Chemistry
Date:Feb 1, 2000
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