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Articles |
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.
a Author for correspondence. Fax 602-200-6004; e-mail eliot.brinton{at}med.va.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We developed a preparative method for separating Sm LpA-I particles from human plasma by high-performance size-exclusion chromatography (HP-SEC), using two gel permeation columns (Superdex 200 and Superdex 75) in series and measuring apo A-I content in column fractions in 30 subjects with HDL-cholesterol (HDL-C) concentrations of 0.4–3.83 mmol/L.
Results: Three major sizes of apo A-I-containing particles were detected: an ~15-nm diameter (~700 kDa) species; a 7.5–12 nm (100–450 kDa) species; and a 5.8–6.3 nm species (40–60 kDa, Sm LpA-I particles), containing 0.2–3%, 80–96%, and 2–15% of plasma total apo A-I, respectively. Two subjects with severe HDL deficiency had increased relative apo A-I content in Sm LpA-I: 25% and 37%, respectively. The percentage of apo A-I in Sm LpA-I correlated positively with fasting plasma triglyceride concentrations (r = 0.581; P <0.0005) and inversely with total apo A-I (r = -0.551; P <0.0013) and HDL-C concentrations (r = -0.532; P <0.0017), although the latter two relationships were largely attributable to extremely hypoalphalipoproteinemic subjects. The percentage of apo A-I in Sm LpA-I correlated with that in pre-ß-migrating species by crossed immunoelectrophoresis (r = 0.98; P <0.0001; n = 24) and with that in the d >1.21 kg/L fraction by ultracentrifugation (r = 0.86; P <0.001; n = 20). Sm LpA-I particles, on average, appear to contain two apo A-I and four phospholipid molecules but little or no apo A-II, triglyceride, or cholesterol.
Conclusions: We present a new HP-SEC method for size separation of native HDL particles from plasma, including Sm Lp A-I, which may play important roles in the metabolism of HDL and in its contribution(s) to protection against atherosclerosis. This method provides a basis for further studies of the structure and function of Sm Lp A-I.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Very small, lipid-poor apo A-I-containing lipoprotein (Sm LpA-I) particles, sometimes referred to as "free" or pre-ß1 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)(5)(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)(8)(9)(10)(11). Lipid acceptance by Sm LpA-I may be direct, by interaction with proteinase-sensitive cell surface microdomains (12)(13)(14)(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 2.3.1.43), ultimately converting the particles into mature, spheroidal HDL with a neutral lipid core (21)(22)(23)(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)(27)(28)(29) and/or endothelial lipases (26)(30)(31)(32)(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)(40)(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)(43)(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)(51)(52). A rough quantification of Sm LpA-I in whole plasma can be obtained by agarose electrophoresis followed by immunofixation (53)(54), immunoprecipitation (55)(56)(57)(58)(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-ß1 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-ß 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-ß 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-ß 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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sample handling
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 (1500g) centrifugation
for 30 min at 4 °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 °C in multiple aliquots.
plasma lipid profiles
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
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 NaCl) supplemented with 1 g/L sodium EDTA and 1 g/L
NaN3. A 20–100 µL 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.
ria of apo a-i and a-ii
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).
quantification of lipids in column fractions
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; Labsystems), as described previously (74).
agarose gel electrophoresis and immunoblotting
Undiluted plasma samples (1–3 µL) 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 °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 µm pore size nitrocellulose membranes (BA85; Schleicher &
Schuell), 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 NiCl2, and 1 mL/L
H2O2 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 Rf of a bromphenol-blue-stained plasma reference marker) from which the proteins were extracted by brief ultracentrifugation (360 000g for 15 min at 4 °C). Recovery of apo A-I from the agarose gel slices (assessed using 125I-labeled Sm LpA-I) was >95%.
crossed immunoelectrophoresis
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 and density gradient ultracentrifugation
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 °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 °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.
immunoaffinity-prepared hdl
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-HCl, pH 2.5, and
neutralized immediately to pH 7.4 by the addition of Tris base.
statistical analysis
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, log10 transformation was used to
linearize the data before calculating r values. We assessed
differences between grouped variables using the Student
t-test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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-ß2 and
ß3 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.
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method optimization
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 back-pressures, 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 125I-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 µL, 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 µL 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 µL. HP-SEC quantification of Sm LpA-I concentration in very small volumes of biologic fluids (10–25 µL) was accomplished by adjusting the dynamic measurement range of the apo A-I RIA (using more dilute primary antibody and less 125I-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 µL 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 HiLoad 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 µL), 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.
reproducibility and stability studies
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 °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 ± SE, 4.7% ± 0.25% before vs 4.95% ± 0.18% after freezing; P, not significant by paired t-test). Storage of plasma at -70 °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).
corroboration and validation studies
Because Sm LpA-I particles appear to constitute the vast majority
of HDL with pre-ß 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-ß-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-ß 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.
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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).
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It has been shown that Sm LpA-I particles or pre-ß-migrating HDL may
be highly unstable in plasma at 37 °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)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(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 °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 °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 
-mobile species, whereas fractions corresponding to
the third apo A-I peak (Sm LpA-I, fractions 37–41) had pre-ß
electrophoretic mobility.
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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-ß mobile zone of a one-dimensional, preparative agarose gel loaded with 125I-labeled immunoaffinity-purified total HDL. The predominant moiety present in the pre-ß 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-ß2 and pre-ß3 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).
compositional studies
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 HDL2
and HDL3). 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 radioiodinated Sm LpA-I
revealed that only a single band of molecular mass ~28 kDa was
visible (data not shown).
correlates of percentage of apo a-i in Sm LpA-I
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, 0.36–3.83 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).
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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advantages over non-column-chromatographic methods
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)(51)(52). Quantification of apo A-I in the
pre-ß mobile zone in one-dimensional agarose gels by various methods
[e.g., crossed immunoelectrophoresis (CIEP) (56)(57)(58)(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-ß2 and pre-ß3
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).
advantages over other low- and medium-pressure column
chromatographic methods
Several groups have used low-pressure, gravitationally assisted
gel filtration chromatography to study HDL particle size distribution (46)(77)(80)(81)(82)(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.
evidence that Sm LpA-I PARTICLES ARE PHYSIOLOGIC
PARTICLES
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)(22)(23)(24)(25)(26)(27)(28)(29)(30)(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-ß 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 °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 °C or by pretreating the test
samples with (immuno)chemical inhibitors of LCAT, lipase, and/or lipid
transfer protein activity.
prior reports of particles similar to Sm LpA-I USING
OTHER METHODS
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-ß 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 HDL2
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.
composition of Sm LpA-I
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-ß-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-ß1 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-ß1 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-ß 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-ß-migrating HDL in 16 normolipidemic sera because their assay was not sensitive enough to detect pre-ß 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-ß 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).
possible explanations for coelution of third pl peak and apo
a-i (particles larger than Sm LpA-I)
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-ß 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.
correlations of Sm LpA-I PARTICLE CONTENT WITH OTHER
CLINICAL VARIABLES
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)(94)(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-ß1 LpA-I concentrations and
HDL2, HDL3 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.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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