The effects of apolipoprotein B depletion on HDL subspecies composition and function[S]

14 Pages·2016·2.38 MB·English

The effects of apolipoprotein B depletion on HDL
subspecies composition and function
W. Sean Davidson, * Anna Heink ,† Hannah Sexmith ,† John T. Melchior , * Scott M. Gordon ,§
Zsuzsanna Kuklenyik, ** Laura Woollett , * John R. Barr , ** Jeffrey I. Jones , **
Christopher A. Toth , †† and Amy S. Shah1 ,†
Center for Lipid and Arteriosclerosis Science,* Department of Pathology and Laboratory Medicine,
University of Cincinnati, Cincinnati, O H 45237; Department of Pediatrics,† Cincinnati Children’s Hospital
Research Foundation, Cincinnati, O H 45229; N ational Heart, Lung, and Blood Institute, § Lipoprotein
Metabolism Section,, Bethesda, M D 20892; Division of Laboratory Sciences,** National Center for
Environmental Health, C enters for Disease Control and Prevention, Atlanta, G A 30341; and B attelle
Memorial Institute, † † Analytical Services, Atlanta, G A 30329
Abstract HDL cholesterol (HDL-C) effl ux function may Epidemiological studies show a strong inverse relation-
be a more robust biomarker of coronary artery disease risk ship between circulating levels of HDL cholesterol (HDL-C)
than HDL-C. To study HDL function, apoB-containing lipo- and coronary artery disease (CAD) in patients with high,
proteins are precipitated from serum. Whether apoB pre-
normal, or low LDL cholesterol (LDL-C) levels (1 , 2) .
cipitation affects HDL subspecies composition and function
However, HDL-C-raising therapies using cholesteryl ester
has not been thoroughly investigated. We studied the effects
transfer protein (CETP) inhibitors have not been fruitful
of four common apoB precipitation methods [polyethylene
glycol (PEG), dextran sulfate/magnesium chloride (MgCl ), with respect to protection from CAD, despite raising HDL-
2
heparin sodium/manganese chloride (MnCl ), and LipoSep C levels signifi cantly. Additionally, Mendelian random-
2
immunoprecipitation (IP)] on HDL subspecies composition, ization studies have revealed that genetic mutations that
apolipoproteins, and function (cholesterol effl ux and re- specifi cally raise HDL-C do not necessarily confer CAD
duction of LDL oxidation). PEG dramatically shifted the benefi ts ( 3–6 ). Thus, HDL-C may not be an appropriate
size distribution of HDL and apolipoproteins (assessed by
biomarker for CAD risk, particularly given new informa-
two independent methods), while leaving substantial amounts
tion about the proteomic and lipidomic complexity un-
of reagent in the sample. PEG also changed the distribution
derlying HDL particles (7 –9 ).
of cholesterol effl ux and LDL oxidation across size frac-
Recently, attention has shifted to HDL function as a
tions, but not overall effl ux across the HDL range. Dextran
sulfate/MgCl , heparin sodium/MnCl , and LipoSep IP did potential biomarker for CAD risk. The function most
2 2
not change the size distribution of HDL subspecies, but commonly associated with HDL is its ability to mobilize
altered the quantity of a subset of apolipoproteins. Thus, or “effl ux” cholesterol from cells in the periphery and
each of the apoB precipitation methods affected HDL com- transport it to the liver for catabolism (1 0 ). It is widely
position and/or size distribution. We conclude that care- believed that HDL-mediated removal of cholesterol from
ful evaluation is needed when selecting apoB depletion
enriched cells in the vessel wall, particularly macrophages,
methods for existing and future bioassays of HDL func-
is a key reason for its association with CAD protection.
tion. —Davidson, W. S., A. Heink, H. Sexmith, J. T. Melchior,
Indeed, many studies have shown that HDL particles iso-
S. M. Gordon, Z. Kuklenyik, L. Woolett, J. R. Barr, J. I. Jones,
C. A. Toth, and A. S. Shah. T he effects of apolipoprotein lated by ultracentrifugation from diseased individuals
B depletion on HDL subspecies composition and function. versus normal controls are defective in effl ux capacity
J. Lipid Res. 2016. 57: 674–686. ( 11–13 ).
However, HDL isolation by ultracentrifugation is a
Supplementary key words polyethylene glycol • dextran sulfate • hep- lengthy procedure that likely disrupts HDL particles ( 14 ),
arin sodium • lipoproteins • high density lipoprotein • low density lipo-
protein • proteomics • cholesterol effl ux
Abbreviations: AF4, asymmetric fl ow fi eld-fl ow fractionation; AUC,
This work was supported by N ational Heart, Lung, and Blood Institute Grants area under the curve; CAD, coronary artery disease; HDL-C, HDL cho-
R01HL67093 (W.S.D.), R 01HL104136 (W.S.D.), and K 23HL118132 lesterol; IP, immunoprecipitation; LDL-C, LDL cholesterol; MgCl ,
(A.S.S.). The authors declare no fi nancial confl icts of interest relevant to this 2
magnesium chloride; MnCl , manganese chloride; MW, molecular
study. The content is solely the responsibility of the authors and does not neces- 2
weight; PEG, polyethylene glycol; STB, standard Tris buffer .
sarily represent the offi cial views of the National Institutes of Health.
1 To whom correspondence should be addressed.
Manuscript received 16 January 2016 and in revised form 17 February 2016. e-mail: [email protected]
Published, JLR Papers in Press, February 23, 2016 The online version of this article (available at http://www.jlr.org)
DOI 10.1194/jlr.M066613 contains a supplement.
674 Journal of Lipid Research Volume 57, 2016 This article is available online at http://www.jlr.org
This is an Open Access article under the CC BY license.
making this approach problematic for large scale clinical LDL from oxidation. We also tested a proprietary immu-
studies. As a result, clinical studies have begun to evaluate noprecipitation (IP) reagent prepared from delipidated goat
HDL function in situ by precipitating the apoB-containing anti-apoB serum that binds and removes apoB-containing
lipoproteins (LDL and VLDL) out of plasma or serum in lipoproteins. We found that there is no perfect method
order to remove their background activities. with respect to the impact on the HDL-sized species in
In a landmark study, Khera et al. (1 5 ) found that choles- terms of size, protein content, and function.
terol effl ux capacity from cultured macrophages (after
apoB precipitation) was strongly and inversely associated
with both subclinical atherosclerosis (measured by carotid
MATERIALS AND METHODS
artery intima-media thickness) and angiographically con-
fi rmed obstructive CAD. The effl ux capacity outperformed Blood collection
measures of total cholesterol, LDL-C, and HDL-C with
Three healthy nonsmoking normolipidemic females with a
respect to the odds ratios for CAD. Then Rohatgi et al. mean age of 28.7 years were studied. Fasting blood was collected
( 16 ) prospectively confi rmed this association in a large in a red top serum separator (Becton Dickinson Biosciences),
population that was free of disease at baseline. For a review of allowed to clot for 30 min at room temperature, and then spun at
the studies evaluating cholesterol effl ux in human dis- (cid:2) 1,590 g for 15 min to isolate serum. These studies were ap-
ease, see (1 7 ). proved by the Institutional Review Board at Cincinnati Children’s
Hospital Medical Center and informed consent was obtained.
A popular reagent used to precipitate LDL and VLDL
out of serum is polyethylene glycol (PEG) ( 15, 16 ). PEG is
Gel fi ltration chromatography
a neutral polymer that reduces the solubility of LDL and
After collection, 370 (cid:2) l of serum was applied to three Super-
VLDL ( 18 ). Other methods for apoB precipitation include
dex 200 Increase columns arranged in series (10/300; GE Health-
combining polyvalent anions, like heparin and dextran
care), as previously described (2 3 ). Samples were eluted in 10 mM
sulfate, with a divalent cation, such as manganese or mag- Tris and 0.15 M NaCl at pH 8.2, hereafter referred to as stan-
nesium. The polyanions interact with arginine and other dard Tris buffer (STB). The eluate was collected as 47 fractions
positively charged residues on apolipoproteins, while the (fraction collector F9-C; GE Healthcare) of 1.5 ml maintained at
cations help the polyanion-lipoprotein complexes form 4°C. To relate gel fi ltration results to traditional density-centric
via interaction with phospholipids resulting in precipi- defi nitions, we used the presence of apoB, the core constituent
of LDL and VLDL, as a distinguisher (2 3 ). Therefore, the
tation. Dextran sulfate-magnesium chloride (MgCl ) is
2 VLDL/LDL range is defi ned as fractions 15–19 due to the pres-
the most widely used method to quantify HDL-C in most
ence of apoB. We assigned the remaining fractions 20–29 as the
clinical laboratories ( 19 ). In strict terms, none of these
HDL range, because their diameters range from about 15 to
methods have “specifi city” for LDL or VLDL on a molecu- 7 nm in diameter, consistent with measurements for density-
lar basis, such as seen with immunoprecipitations. These isolated HDL and due to the abundance of the major HDL protein,
reagents are titrated into plasma or serum to an extent apoA-I ( 23 ). See supplementary Fig. 1 for these details. Choline-
that their actions have a disproportionate impact on containing phospholipid and total cholesterol were measured in
large apoB-containing lipoproteins versus smaller HDL each fraction using colorimetric kits from Wako (Richmond, VA)
and Pointe Scientifi c (Canton, MI), respectively. Serum was stored
particles.
at 4°C between experiments and was never frozen.
Upon their introduction, each apoB precipitation
method was carefully optimized and verifi ed to quantita- apoB depletion methods
tively deplete LDL-C with minimal impact on HDL-C. For
To remove apoB-containing lipoproteins from fasted serum,
example, in the mid-1980s, methods of apoB depletion on
the following methods were employed: 1 ) PEG [molecular
HDL-C quantifi cation and apoA-I levels were compared weight (MW) 6,000; Sigma-Aldrich, St. Louis, MO] precipita-
( 20, 21) . However, we and others have shown that HDL is tion . PEG (400 (cid:2) l) (20% in water) was added to 1 ml serum,
composed of a heterogeneous population of particles with incubated for 20 min at room temperature, and then centri-
diversity in their proteomic fi ngerprint (7 , 9, 22, 23) and fuged at 10,000 rpm for 30 min. 2 ) Dextran sulfate/MgCl2
physical properties [solubility, size ( 24 ), and ability to in- (Sigma-Aldrich) precipitation. Dextran sulfate (20 (cid:2) l) (5% in
water) and 25 (cid:2) l of 2 M MgCl were added to 1 ml serum, incu-
teract with proteoglycans (2 5 )]. These recent advances 2
bated for 20 min at room temperature, and then centrifuged at
in our understanding of HDL diversity require a reassess-
3,000 g for 10 min (2 6 ). 3 ) Heparin sodium/manganese chlo-
ment of the impact of apoB lipoprotein depletion meth-
ride (MnCl ; Sigma-Aldrich) precipitation. Heparin sodium
2
ods on HDL subspecies composition and function. This (40 (cid:2) l) (1.3 mg/ml in 150 mM saline) and 50 (cid:2) l of 1 M MnCl
2
is important because artifactual alterations in HDL sub- added to 1 ml serum, incubated for 30 min at 4°C, and then
species levels or composition could have profound im- centrifuged at 1,500 g for 30 min ( 27 ). 4 ) LipoSep IP (Sun
pact not only on downstream functional measurements, Diagnostics, New Gloucester, ME). LipoSep IP is a proprietary
like cholesterol effl ux, but also on other HDL functions, IP reagent prepared from delipidated goat anti-apoB antisera.
LipoSep IP reagent (1 ml) was added to an equal volume (1 ml)
including anti-infl ammation and protection of LDL from
of serum, incubated for 10 min at room temperature, and then
oxidation .
centrifuged at 12,000 rpm for 10 min (per the suggested manu-
Here, we assessed the impact of the three most widely
facturer’s recommendations).
used apoB lipoprotein depletion methodologies on HDL
For each method, the pellet (containing apoB lipoproteins)
subspecies profi les, apolipoprotein content, and HDL was discarded and the remaining supernatant (containing HDL)
function in terms of cholesterol effl ux and ability to protect was applied to the Superdex 200 Increase columns as above. To
apoB depletion 675
account for dilution effects of these reagents, prior to use, all mean amount of 3 H-cholesterol present inside the cells at the
apoB depleted samples and control serum were diluted with STB time of treatment addition, 3 H-cholesterol was extracted from
to equal volumes of 1.4 ml (or 2 ml for comparisons using Lipo- radiolabeled cells on a separate T0 plate by washing the wells
Sep IP reagent). with isopropanol, drying down the isopropanol wash, and
then resuspending the dried extract in 150 (cid:2) l toluene. Follow-
Western blot ing incubation, treatment medium was then collected, fi ltered
through a 0.45 (cid:2) m 96-well fi lter plate, and measured for detec-
Confi rmation of complete apoB depletion from serum sam-
tion of 3 H-cholesterol effl uxed to acceptor via scintillation
ples was performed via a Western blot for apoB. Serum, apoB-
counting. Percent cholesterol effl ux is expressed using the
depleted serum from each method, and an ultracentrifugally
following formula: 3 H count in efflux media /(3 H count in
isolated LDL control (75 ng protein) were resolved by SDS-PAGE
effl ux media + mean 3 H count extracted from untreated cells) ×
(4–15%) and transferred to a polyvinylidene difl uoride membrane .
100. All experimental and control samples were assayed in
The membrane was blotted with goat anti-human apoB diluted
triplicate.
1:2,500 (K45253G; Meridian Life Sciences, Memphis, TN) and
Anti-oxidative activity of apoB-depleted serum was assessed
detected using donkey anti-goat IgG-HRP diluted 1:2,500 (SC2056;
toward an exogenous reference ultracentrifugally-isolated LDL
Santa Cruz, Dallas, TX).
sample using the method of Kontush, Chantepie, and Chap-
apoA-I immunoblotting was performed on serum and apoB-
man ( 30 ). LDL was stored at (cid:3) 80°C with 6.25% sucrose (w/v).
depleted serum fractions in the HDL subspecies range (fractions
In a UV transparent 96-well microplate, 20 (cid:2) g LDL-C per well
20–30). Ten microliters of each fraction was resolved by SDS-
was combined with 65 (cid:2) l of serum or apoB-depleted serum
PAGE (4–15%) and transferred to a polyvinylidene difl uoride
fraction and brought up to 200 (cid:2) l with STB. After incubati ng
membrane. The membrane was blotted with rabbit anti-human
the plate for 10 min at 37°C, 100 mM 2,2 ′ -azobis(2-methyl-
apoA-I diluted 1:2,500 (178422; EMD Millipore) and detected us-
propionamidine) dihydrochloride was added to each well at a
ing donkey anti-rabbit IgG-HRP diluted 1:2,500 (NA934V; GE
fi nal concentration of 5 mM to initiate LDL oxidation. Conju-
Healthcare). Densitometry analysis was performed using ImageJ
gated diene absorbance at 234 nm was read every 5 min for 8 h
(National Institutes of Health).
at 37°C. The kinetic curves generated for each sample were
analyzed to calculate the maximum rate of the propagation
Immunodot blot
phase ( V ). All data were then normalized to control LDL
max
Immunodot blotting for the detection of apoE in HDL-range (not containing any treatment) by dividing the maximal rate
fractions (fractions 20–26) from serum and apoB-depleted serum of propagation of the sample by the maximal rate of propaga-
was carried out using a Bio-Dot apparatus (Bio-Rad, Hercules, tion of the LDL control condition, and expressing this value as
CA). Thirty-fi ve microliters of each fraction was transferred a percentage. In this assay, protective HDL subspecies de-
onto a nitrocellulose membrane in 300 (cid:2) l Tris-buffered saline creased the rate of LDL oxidation resulting in a decrease in
[20 mM Tris, 150 mM NaCl (pH 7.6)]. The membrane was V ( 30 ).
max
blocked with Odyssey PBS blocking buffer (LiCor Biosciences,
Lincoln, NE) and blotted with mouse anti-human apoE diluted Asymmetric fl ow fi eld-fl ow fractionation
1:1,200 (SC53570; Santa Cruz). Detection was performed using
As an alternative method to study the effects of apoB precipi-
goat anti-mouse IgG-Alexa Fluor 680 conjugate diluted 1:40,000
tation on lipoprotein subspecies, we used asymmetric fl ow fi eld-
(A21058; Life Technologies) in Odyssey blocking buffer contain-
fl ow fractionation (AF4). This separation technique is based on
ing 0.01% SDS. The membrane was imaged using an Odyssey
the fundamental nature of laminar fl ow. A liquid medium passes
infrared imaging system (LiCor Biosciences). Densitometry anal-
through a thin channel, which causes it to adopt a parabolic
yses were accomplished using the Odyssey application software,
velocity profi le across the height of the channel, with stream
version 2.0.41.
velocities faster at the center of the channel and slower near the
walls. When serum is injected into this stream, contained parti-
HDL function
cles are subjected to a second perpendicular fi eld created by
Cholesterol effl ux was assessed as the movement of 3 H-labeled withdrawal of carrier fl uid through a semipermeable membrane
cholesterol from RAW 264.7 macrophages, a murine macro- that drives them toward the accumulation wall, where they expe-
phage cell line (ATCC, Manassas, VA), to the medium contain- rience slower fl ow rates. Once they are concentrated at the ac-
ing fractionated serum samples. Cultured cells were plated in cumulation wall, particle species begin to diffuse up away from
48-well plates. The following day, cells were incubated overnight the accumulation wall via Brownian motion into higher velocity
with labeling medium (high-glucose DMEM with 10% fetal bovine fl ow regimes. Differential retention is caused by the different
serum and 1% penicillin-streptomycin) containing 1 (cid:2) Ci/ml average height achieved by particles of different sizes, with
of 3 H-cholesterol (PerkinElmer, Boston, MA) and 2 (cid:2) g/ml of an smaller particles having a higher average height, which results
ACAT inhibitor (Sandoz 58-035; Sigma-Aldrich). The 8-Br-cAMP in them eluting faster. The AF4 separation requires no fi ltration
(Sigma-Aldrich) at a fi nal concentration of 0.3 mM was added or other sample pretreatment and is achieved by gentle fl uid
to all experimental and control wells to induce efflux via dynamics; both of these elements minimize the risk of intro-
ABCA1, ABCG1, and aqueous diffusion (2 8, 29 ). The next day, ducing artifacts during separation. Serum (50 (cid:2) l) and 50 (cid:2) l
treatments containing 33.3% v/v of each serum fraction or serum after apoB-depletion with PEG, dextran sulfate/MgCl ,
2
apoB-depleted serum fraction were freshly made in basal me- and heparin sodium/MnCl apoB depletion were injected into
2
dium (consisting of DMEM containing 0.2% BSA). It should an AF2000 system . Samples were analyzed in a buffer solution,
be noted that each serum fraction was approximately 50-fold prepared of 10 mM sodium bicarbonate and 150 mM NaCl,
dilute compared with serum. Control treatments containing adjusted to pH 7.4, which was vacuum fi ltered prior to use. The
lipid-free apoA-I and HDL (each at 10 (cid:2) g/ml protein) with and eluent was collected as 40 fractions of approximately 200 (cid:2) l
without 8-Br-cAMP were also made. Labeling medium was re- maintained at 4°C, starting at 5 min in 2.5 min intervals.
moved from the cells, and each well was washed twice with PBS. Choline-containing phospholipid was measured in each fraction
One hundred and fi fty microliters of each treatment were using colorimetric kits from Wako (Richmond, VA). In addition,
added per well and incubated for 6 h. In order to measure the apolipoprotein analysis was performed via trypsin digestion and
676 Journal of Lipid Research Volume 57, 2016
LC-MS/MS. For LC-MS/MS method details, see the supplemen- LDL and VLDL appeared in a single peak between frac-
tary Methods. tions 15 and 19, while the HDL-sized lipoproteins eluted
from large to small in fractions 20–29. The total choles-
Statistics
terol concentration in control serum across lipoprotein
Data are presented as mean ± SD. For any analysis where
subspecies mirrored the phospholipid concentration, ex-
apoB depletion methods were compared with serum, a two-tailed
cept that cholesterol was less prevalent in smaller HDL-
Student’s t -test was used. P < 0.01 was considered signifi cant. A
lower threshold was used to decrease the possibility of false posi- sized particles after fraction 26.
tives given the multiple comparisons across the HDL subspecies When PEG was used to precipitate apoB-containing
distribution. lipoproteins, the concentration of phospholipid ( Fig. 1A )
and cholesterol (F ig. 1B) decreased to baseline in frac-
RESULTS tions 15–19, indicating quantitative removal of these
lipoproteins. This was confi rmed by the complete disap-
Lipoprotein profi les pearance of apoB, shown by the Western blot in F ig. 1C.
The phospholipid and total cholesterol concentrations in Interestingly, the HDL-sized lipoproteins underwent
control serum across lipoprotein subspecies separated by gel a marked rightward shift in their phospholipid and
fi ltration chromatography are shown in black in F ig. 1A, B . cholesterol profi le, implying a change to overall smaller
Fig. 2. Phospholipid and cholesterol distribution across lipopro-
Fig. 1. Phospholipid and cholesterol distribution across lipoprotein tein subspecies separated by gel fi ltration chromatography before
subspecies separated by gel fi ltration chromatography before and and after apoB depletion of serum by dextran sulfate/MgCl . Data
2
after apoB depletion of serum by PEG. Data are mean and SD (n = 3). are mean and SD (n = 3). Serum control versus apoB depletion us-
Serum control versus apoB depletion using PEG. All samples were ing dextran sulfate/MgCl . All samples were diluted with STB to
2
diluted with STB to equal volume prior to gel fi ltration chromatogra- equal volume prior to gel fi ltration chromatography. Phospholipid
phy. Phospholipid distribution (A), cholesterol distribution (B), and distribution (A), cholesterol distribution (B), and AUC of LDL
area under the curve (AUC) of LDL and HDL fractions in serum and HDL fractions in serum versus apoB-depleted serum (C). The
versus apoB-depleted serum (C). The inset in (C) shows Western blot inset in (C) is a Western blot detection of apoB before and after
detection of apoB after serum apoB depletion with PEG, demon- serum apoB depletion with dextran sulfate/MgCl . Serum control
2
strating full precipitation of apoB-containing particles. Serum control (black diamond); dextran sulfate/MgCl apoB-depleted serum
2
(black diamond); PEG apoB-depleted serum (open circle ) . (gray square).
apoB depletion 677
size ( Fig. 1A, B) . However, the AUC of phospholipid for apoB ( Fig. 4A–C ), with the peak of phospholipid and cho-
the HDL-sized particles did not change significantly lesterol shifted by one fraction.
( Fig. 1C ).
Protein distributions
Dextran sulfate/MgCl treatment compared with serum
2
also showed a quantitative depletion of VLDL/LDL phos- apoA-I. apoA-I distribution was evaluated by Western
pholipid, cholesterol, and apoB ( Fig. 2A–C ). In this case, blotting across the HDL size range after the various pre-
the peak in the HDL size range did not shift toward smaller cipitation strategies. F igure 5A shows that the apoA-I dis-
particles as it did for PEG. However, there was a trend to- tribution in the HDL size range was shifted to the right
ward a lower AUC for phospholipid in the HDL size range after the PEG treatment, mirroring the phospholipid and
after apoB depletion, P = 0.050. cholesterol changes. The apoA-I distribution tracked with
Heparin sodium/MnCl exhibited the least impact on the phospholipid for both dextran sulfate/MgCl and with
2 2
lipid content of particles in the HDL size range ( F ig. 3A–C) . heparin sodium/MnCl and did not change the overall
2
Phospholipid and cholesterol in the HDL size range were pattern or levels of apoA-I ( Fig. 5B, C) . LipoSep IP re-
superimposed on that of the control serum, even after re- sulted in lower levels of apoA-I across the HDL size range.
moval of the VLDL/LDL. LipoSep IP also quantitatively
depleted VLDL/LDL of phospholipid, cholesterol, and apoE. Given that large HDL particles are known to be
enriched in apoE, we assessed the impact of the various
apoB depletion methods on apoE in the HDL size range.
Fig. 3. Phospholipid and cholesterol distribution across lipopro-
tein subspecies separated by gel fi ltration chromatography before Fig. 4. Phospholipid and cholesterol distribution across lipopro-
and after apoB depletion of serum by heparin sodium/MnCl . tein subspecies separated by gel fi ltration chromatography before
2
Data are mean and SD (n = 3). Serum control versus apoB deple- and after apoB depletion of serum by LipoSep IP. Data are mean
tion using heparin sodium/MnCl . All samples were diluted with and SD (n = 3). Serum control versus apoB depletion using Lipo-
2
STB to equal volume prior to gel fi ltration chromatography. Phos- Sep IP. All samples were diluted with STB to equal volume prior to
pholipid distribution (A), cholesterol distribution (B), and AUC of gel fi ltration chromatography. Phospholipid distribution (A), cho-
LDL and HDL fractions in serum versus apoB-depleted serum (C). lesterol distribution (B), and AUC of LDL and HDL fractions in
The inset in (C) is Western blot detection of apoB before and after serum versus apoB-depleted serum (C). The inset in (C) is Western
serum apoB depletion with heparin sodium/MnCl . Serum control blot detection of apoB before and after serum apoB depletion with
2
(black diamond); heparin sodium/MnCl apoB-depleted serum LipoSep IP. Serum control (black diamond); LipoSep IP apoB-
2
(open triangle). depleted serum (open diamond).
678 Journal of Lipid Research Volume 57, 2016
Fig. 5. apoA-I across HDL subspecies before and after apoB depletion. apoA-I levels measured across gel
fi ltration fractions from serum (black bars) compared with apoA-I in fractions after apoB depletion with
PEG (white bars) (A), dextran sulfate/MgCl (dark gray bars) (B), heparin sodium/MnCl (light gray bars)
2 2
(C), and LipoSep IP (diagonal patterned bars) (D). apoA-I was detected via Western blotting. Bands were
quantifi ed by densitometry.
The results of a single apoE dot blot are shown in Fig. 6 . ( Fig. 7A) ; however, the total effl ux summed across all
The LipoSep IP experiment was not quantifi ed because of HDL-sized fractions was not different between the control
the presence of cross-reacting goat apoE present in the serum and PEG ( P = 0.773).
reagent. Unlike for apoA-I, each of the three precipitation After dextran sulfate/MgCl , the effl ux to each of the
2
methods depleted apoE levels in the larger HDL size HDL-sized fractions was essentially unchanged from con-
range. PEG had the least effect on summed total apoE lev- trol serum in both magnitude and overall pattern. The to-
els across the HDL range, but consistent with our previous tal effl ux summed across HDL size fractions was also not
analyses, there was a clear rightward shift with smaller statistically different ( P = 0.767, see Fig. 7B ). Similar results
HDL-sized particles exhibiting increased levels of apoE were seen after heparin sodium/MnCl apoB depletion
2
versus control. Dextran sulfate/MgCl and heparin so- ( Fig. 7C) . Again the summed total effl ux across fractions
2
dium/MnCl methods signifi cantly reduced apoE levels in was not different for heparin sodium/MnCl apoB deple-
2 2
the fractions containing larger particles with differences tion compared with control serum, P = 0.878. LipoSep IP
of (cid:3) 33 and (cid:3) 50%, respectively, in fraction 20. Fractions reagent showed slightly lower effl ux in fraction 21 relative
containing smaller particles were less affected by dextran to control serum. However, the summed effl ux across the
sulfate/MgCl and heparin sodium/MnCl depletion, and HDL size range was not different from the control, P =
2 2
neither precipitation method caused a noticeable shift in 0.582 (F ig. 7D) .
overall pattern similar to PEG apoB depletion.
HDL anti-oxidation. Figure 8A shows a typical functional
HDL function readout resulting from HDL-sized particles in serum.
Cholesterol effl ux. Figure 7A shows the typical pattern of There were two peaks of anti-oxidation activity (noted by a
cholesterol effl ux from cultured macrophages to each of the decrease in the propagation rate). One was centered on
HDL-sized lipoprotein fractions from control serum. Most fraction 23, where the largest amount of HDL phospho-
of the cholesterol effl ux was promoted by fractions con- lipid and apoA-I was located. A second peak appeared at
taining medium-sized HDL particles (around fractions fraction 28 in small and poorly lipidated species. PEG apoB-
23–24) with less in the fractions containing small HDL depleted serum ( Fig. 8A) showed a loss of the peak in the
particles. When serum was depleted of VLDL/LDL with large HDL size range (fraction 22, P < 0.01 compared with
PEG, there was again a rightward shift of the effl ux pattern control serum). Additionally, the antioxidant activity per-
in the HDL size range, which matched tightly with the shift sisted much later in the gradient in the presence of PEG
in phospholipid, cholesterol, and apoA-I observed above compared with the control (fraction 29, P < 0.01 compared
apoB depletion 679
We next quantifi ed the amount of PEG that remained
in serum after the VLDL/LDL precipitation. Typically,
apoB depletion is performed at 20% (v/v) PEG to plasma
volume. To see how much of this comes out of solution
with the precipitated LDL and VLDL, we obtained a fl uo-
rescein-labeled PEG (MW 6,000; Nanocs, New York, NY)
and tracked the reagent in plasma. F igure 9 compares
the total PEG that remained in plasma after a typical
VLDL/LDL precipitation reaction with the same amount
of labeled PEG in buffer. After separation on the gel
filtration system, most of the PEG ran near the total
volume of the columns (i.e., the very small range) in both
plasma and buffer. Interestingly, the process of VLDL/
LDL precipitation only consumed about 20% of the PEG
used in the protocol, leaving about 80% (or about 53 mg/ml)
behind.
We hypothesized that PEG might interact with the gel
fi ltration column medium to cause an artifactual retention
of the HDL particles on the column, thus driving the right-
ward shift in the profi le without a direct effect on HDL
particle size per se. We attempted to size the HDL particles
in the presence of PEG by several methods that were inde-
pendent of gel fi ltration, such as dynamic light scattering
and NMR particle sizing. Unfortunately, in each case, the
presence of PEG interfered with the analysis, resulting in
uninterpretable results (data not shown). We also attempted
to remove PEG via dialysis using a membrane with a mo-
Fig. 6. apoE across HDL subspecies before and after apoB deple- lecular weight cutoff of 10,000 (Thermo Fisher Scientifi c).
tion. A: apoE in serum (black bars) versus apoE after apoB deple- For this experiment, we used fl uorescein-labeled PEG.
tion with PEG (white bars), dextran sulfate/MgCl2 (dark gray After six 4-liter exchanges with STB, 84.7% of the PEG
bars), and heparin sodium/MnCl (light gray bars) as determined
2 remained in the sample, demonstrating the impracticality
by a single apoE immunodot blotting. Dots were quantifi ed by den-
of PEG removal by this method .
sitometry. B: AUC of apoE for the HDL-sized fractions.
However, we were able to analyze these apoB depletion
treatments using AF4. This method was chosen because it
with serum) consistent with a general shift in the elution does not require a separation medium like gel fi ltration.
pattern. Dextran sulfate/MgCl and heparin sodium/MnCl The phospholipid profi le of untreated serum is shown in
2 2
( Fig. 8B, C ) had little effect versus the control on either Fig. 10 , where the fi rst peak (fractions 3–15) represents
activity peak. We were unable to obtain interpretable data HDL-sized particles and the second peak (fractions 25–30)
with the LipoSep IP reagent in this assay, possibly due to represents VLDL/LDL-sized particles (again, using the
the abundant goat proteins in the preparation. presence of apoB as a distinguisher). Upon precipitation
with PEG, dextran sulfate/MgCl , and heparin sodium/
2
Effects of PEG on HDL particles MnCl , the VLDL/VLDL peak disappeared consistent with
2
Due to the striking differences that we noted with PEG apoB depletion. However, after PEG precipitation, the
using the gel fi ltration approach, we further explored the phospholipid associated with HDL-sized lipoproteins un-
impact of PEG on HDL particle properties. We tested a derwent a clear leftward shift compared with unprecipi-
higher molecular weight PEG (MW 8,000), a shorter incu- tated serum. Dextran sulfate/MgCl and heparin sodium/
2
bation of serum for 5 min instead of 20 min with PEG, and MnCl did not shift the peak, though small shoulders ap-
2
a different buffer system with this reagent (glycine buffer peared in the main HDL peak that could be consistent
instead of water). However, each method recapitulated with some particle rearrangement.
the rightward shift seen in the data above (not shown). We Quantitative MS was performed on the fractions after
also sought to determine whether the rightward shift of AF4 separation using isotope-labeled peptide standards for
HDL subspecies upon PEG treatment was related to the several of HDL’s most abundant apolipoproteins. F igure 11
process of VLDL/LDL depletion. For this, we took HDL shows that, in unprecipitated serum, the various apolipopro-
that was isolated by ultracentrifugation (lacking any VLDL/ teins distribute in distinct profi les across the HDL size range.
LDL) at roughly the same concentrations found in serum. Upon VLDL/LDL precipitation by any method, apoB was
PEG addition promoted the same rightward shift in the quantitatively removed. apoA-I and apoA-II were largely
gel fi ltration elution of HDL measured by both phospho- retained after apoB precipitation, but the apoA-I and apoA-II
lipid and cholesterol as it did in situ in serum (shown in peaks showed a clear leftward shift with PEG that paralleled
supplementary Fig. 2). the lipid profi le seen by gel fi ltration. Dextran sulfate/MgCl
2
680 Journal of Lipid Research Volume 57, 2016
Fig. 7. Cholesterol effl ux across lipoprotein subspecies before and after apoB depletion of serum. Data are
mean and SD. Each fraction was measured in triplicate. Serum control effl ux (black bars) versus apoB deple-
tion effl ux with PEG (white bars) (A), dextran sulfate/MgCl (dark gray bars) (B), heparin sodium/MnCl
2 2
(light gray bars) (C), and LipoSep IP (diagonal line patterned bars) (D). The inset shows the sum of all effl ux
in the HDL size range. Equal volumes were used across fractions. * P < 0.01 compared with control serum.
and heparin sodium/MnCl depletion had minimal effects and heparin sodium/MnCl depleted levels of apoC-III
2 2
on both apoA-I and apoA-II levels and size. However, we in the HDL size range, but dextran sulfate/MgCl mini-
2
noted distinct changes in other apolipoproteins. Both PEG mally affected apoC-III. All three precipitation methods
apoB depletion 681
Fig. 8. Ability of HDL to inhibit LDL oxidation across lipoprotein subspecies after apoB depletion of se-
rum. Data are mean and SD. Each fraction was measured in triplicate. Serum control propagation rate
(black bars) versus apoB depletion propagation rate with PEG (white bars) (A), dextran sulfate/MgCl (dark
2
gray bars) (B), and heparin sodium/ MnCl (light gray bars) (C). Equal volumes were used across fractions.
2
* P < 0.01 compared with control serum.
markedly reduced apoE levels, consistent with the gel fi l- many of the initial characterizations of these methodolo-
tration method. Each of the precipitation methods also gies done in the 1980s, including the ability to fully re-
affected apoA-IV, apoC-I, and apoC-II in the HDL size range. move VLDL and LDL from human serum/plasma and
We also attempted to use native gel electrophoresis to preserve apoA-I and apoA-II. However, when we probed
determine whether HDL size is affected by the presence deeper into the size distribution of the HDL subspecies,
of PEG. We found that PEG did not affect the apparent we noted some troubling effects. Each apoB depletion
diameter of total HDL (isolated by ultracentrifugation) method was found to alter HDL in different ways, whether
when separated by native PAGE (not shown). However,
we also noted that the PEG (as tracked by the fl uorescent
analog) rapidly separated from the HDL during the gel
run, even during the stacking portion of the gel. This in-
dicates that the size-altering effects of PEG are apparent
only when present with the HDL particles during the sepa-
ration (as in the gel fi ltration and A4F techniques), but
once separated from the particles, PEG does not cause
these effects.
DISCUSSION
With the realization that HDL function may be more
important than HDL-C with regard to CAD risk, many
Fig. 9. Fluorescein-PEG (MW 6,000) detection in serum frac-
laboratories have initiated large scale efforts to quantify
tions separated by gel fi ltration chromatography compared with
cholesterol effl ux in patient serum samples. To reduce fl uorescein-PEG in water. The elution patterns of PEG within an
variability, the apoB-containing lipoproteins are commonly apoB-depleted serum sample (black circles) and in a control buffer
removed by precipitation to focus primarily on HDL. With (open diamonds) on the triple Superdex Increase column setup
were determined by PEG apoB-depleting serum with 20% PEG
recent advances in HDL separation techniques, we sought
6000 containing 1% fl uorescein-conjugated PEG (MW 6,000;
to assess the impact of the most commonly used apoB
Nanocs, New York, NY). Fluorescence was measured in each frac-
precipitation methodologies on the distribution of HDL tion using a BioTek Synergy HT plate reader at excitation and
subspecies and their functions. Our results confirmed emission wavelengths of 495 and 520 nm, respectively.
682 Journal of Lipid Research Volume 57, 2016
Fig. 10. Phospholipid distribution across lipoprotein subspecies as separated by AF4 before and after PEG
apoB depletion of serum. Lipoproteins were separated by AF4. Serum control (black circles) versus apoB
depletion with PEG (open circles) (A), dextran sulfate/MgCl (gray squares) (B), and heparin sodium/
2
MnCl (open triangles) (C). D: AUC of phospholipid for HDL fractions in serum (black) versus apoB-
2
depleted serum for PEG (white), dextran sulfate/MgCl (dark gray), heparin sodium/MnCl (light gray ) .
2 2
Equal volumes were used.
it was by altering HDL protein content, or changing ap- found that PEG separates from HDL when examined by
parent particle size, or a combination of both. Arguably native gel electrophoresis. Furthermore, we were unable
the most commonly used method, PEG, had profound ef- to fi nd evidence of strong binding of fl uorescent PEG
fects on apparent HDL particle size, while leaving signifi - to HDL, despite the fact that we found PEG diffi cult to re-
cant amounts of the reagent behind in the sample. First, move by dialysis. Thus, PEG must either: 1 ) affect the re-
we will discuss the impact of VLDL/LDL precipitation on solving power of the gel fi ltration column media; or 2 )
HDL subpopulations and then comment on the overall modify the hydrodynamic properties of the HDL particles
implications on HDL functional analyses. themselves.
The profound rightward shift in the HDL elution profi le The fact that the AF4 technique, which does not rely on
seen with gel fi ltration after PEG depletion initially sug- a separation medium, showed a similar size reduction of
gested that the reagent causes a rearrangement of HDL HDL by PEG, strongly supports the latter explanation.
particles to a smaller size. PEG is a polymer that promotes The exact effects of PEG on the effective hydrodynamic
protein precipitation by steric exclusion. Essentially, proteins diameter of HDL particles cannot be determined from
are excluded from regions of the solvent occupied by PEG, these studies, but given the known effects of PEG as an
such that the effective protein concentration increases. Once “inert solvent sponge,” we suggest that the residual PEG
concentration exceeds the lipoprotein solubility, precipi- remaining in solution reduces the hydration shell around
tation occurs. In such a case, it is not diffi cult to envision a HDL particles. This, in turn, alters the size profi le of HDL
scenario where certain HDL particles might also affected. proteins. The reduced hydrodynamic size results in faster
PEG has been shown previously to affect the apparent diffusion during AF4 and increased partition into the sta-
molecular weight of proteins analyzed by gel fi ltration. tionary phase of the gel fi ltration medium. Another way
Yan et al. (3 1 ) noted that PEG concentrations as low as 5% in which PEG could alter HDL particles is by causing the
can cause a shift of protein elution curves to higher elution dissociation of certain apolipoproteins, perhaps due to
volume on Sephadex G-75 columns. The magnitude of the changes in particle hydration. For example, although
effect increased with the size of the protein, with larger apoC-III underwent a minor shift to smaller size in F ig. 11,
proteins having a more pronounced effect than smaller it is clear that PEG treatment also resulted in decreased
ones. Studies by Atha and Ingham ( 32 ) indicated that this levels of this protein in the HDL size range. This loss of
does not occur because PEG binds to proteins. Indeed, we apolipoprotein content could also account for some of the
apoB depletion 683