Effect of Recombinant Human Lecithin Cholesterol
Acyltransferase Infusion on Lipoprotein Metabolism in Mice□
Xavier Rousset, Boris Vaisman, Bruce Auerbach, Brian R. Krause, Reyn Homan,
John Stonik, Gyorgy Csako, Robert Shamburek, and Alan T. Remaley
Pulmonary and Vascular Medicine Branch, Lipoprotein Metabolism Section, National Heart, Lung, and Blood Institute, National
Institutes of Health, Bethesda, Maryland (X.R., B.V., J.S., R.S., A.T.R.); AlphaCore Pharma, Ann Arbor, Michigan (B.A., B.R.K.,
R.H.); and Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, Maryland (G.C., A.T.R.)
Received April 26, 2010; accepted June 30, 2010
Lecithin cholesterol acyl transferase (LCAT) deficiency is asso-
ciated with low high-density lipoprotein (HDL) and the presence
of an abnormal lipoprotein called lipoprotein X (Lp-X) that con-
tributes to end-stage renal disease. We examined the possibil-
ity of using LCAT an as enzyme replacement therapy agent by
testing the infusion of human recombinant (r)LCAT into several
mouse models of LCAT deficiency. Infusion of plasma from
human LCAT transgenic mice into LCAT-knockout (KO) mice
rapidly increased HDL-cholesterol (C) and lowered cholesterol
in fractions containing very-low-density lipoprotein (VLDL) and
Lp-X. rLCAT was produced in a stably transfected human embry-
onic kidney 293f cell line and purified to homogeneity, with a
specific activity of 1850 nmol/mg/h. Infusion of rLCAT intrave-
nously, subcutaneously, or intramuscularly into human apoA-I
transgenic mice showed a nearly identical effect in increasing
HDL-C approximately 2-fold. When rLCAT was intravenously in-
jected into LCAT-KO mice, it showed a similar effect as plasma
from human LCAT transgenic mice in correcting the abnormal
lipoprotein profile, but it had a considerably shorter half-life of
approximately 1.23 ? 0.63 versus 8.29 ? 1.82 h for the plasma
infusion. rLCAT intravenously injected in LCAT-KO mice
crossed with human apolipoprotein (apo)A-I transgenic mice
had a half-life of 7.39 ? 2.1 h and increased HDL-C more than
8-fold. rLCAT treatment of LCAT-KO mice was found to in-
crease cholesterol efflux to HDL isolated from mice when
added to cells transfected with either ATP-binding cassette
(ABC) transporter A1 or ABCG1. In summary, rLCAT treatment
rapidly restored the normal lipoprotein phenotype in LCAT-KO
mice and increased cholesterol efflux, suggesting the possibil-
ity of using rLCAT as an enzyme replacement therapy agent for
Deficiency of lecithin cholesterol acyltransferase (LCAT;
EC188.8.131.52), first described in 1962 (Glomset, 1962), can
present with two different phenotypes, familial LCAT defi-
ciency (FLD) and fish eye disease (FED) (Santamarina-Fojo
et al., 2001; Rousset et al., 2009). Patients with FLD have
almost an absence of LCAT activity. In contrast, patients
with FED have partial LCAT activity, especially on LDL, and
thus are relatively asymptomatic, with the exception of the
deposition of cholesterol in their corneas. In addition to cor-
neal cholesterol deposits, patients with FLD develop hep-
atosplenomegaly, normochromic normocytic anemia, and
hypertension. Renal disease, however, is the main cause of
morbidity in these patients. It usually presents as protein-
uria that progresses to nephrotic syndrome, glomeruloscle-
rosis, and eventually end-stage renal disease, as early as the
third or fourth decade of life (Santamarina-Fojo et al., 2001).
Low levels of HDL-C (?16 mg/dl for FLD; ?27 mg/dl for FED)
are typically observed in both disorders. In addition, patients
with FLD often have a mild-to-moderate increase in triglyc-
erides and produce an abnormal lipoprotein called Lp-X. This
relatively large lipoprotein is devoid of cholesteryl esters
(CE) and has excess phospholipids, thus it forms vesicular- or
This work was supported in part by the Intramural Research Program of
the National Institutes of Health National Heart, Lung, and Blood Institute
and the National Institutes of Health National Heart, Lung, and Blood Insti-
tute [Grant HL09265601].
Article, publication date, and citation information can be found at
S The online version of this article (available at http://jpet.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: LCAT, lecithin cholesterol acyl transferase; FLD, familial lecithin cholesterol acyl transferase deficiency; FED, fish eye disease;
LDL, low-density lipoprotein; HDL-C, high-density lipoprotein-cholesterol; Lp-X, lipoprotein X; HDL, high-density lipoprotein; RCT, reverse
cholesterol transport; apo, apolipoprotein; rLCAT, recombinant human lecithin/cholesterol acyl transferase; KO, knockout; Tg, transgenic; HEK,
human embryonic kidney; BSA, bovine serum albumin; FPLC, fast protein liquid chromatography; BHK, baby hamster kidney; ABCA1, ATP-
binding cassette; DMEM, Dulbecco’s modified Eagle’s medium; CE, cholesteryl ester(s); CETP, cholesteryl ester transport protein.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
U.S. Government work not protected by U.S. copyright
JPET 335:140–148, 2010
Vol. 335, No. 1
Printed in U.S.A.
multilamellar-like structures. Lp-X is thought to contribute
to the renal disease when it is filtered by the kidney and
accumulates in mesangial cells of the glomerulus (Imbasciati
et al., 1986).
LCAT is secreted into plasma by the liver and associates
mostly with HDL, but also with LDL (Cheung et al., 1986).
It catalyzes the conversion of cholesterol on the surface of
lipoprotein particles to CE. LCAT cleaves fatty acids from
phosphatidylcholine by a phospholipase A2-like activity
and transfers the acyl group to the hydroxyl group on the
A-ring of cholesterol (Rousset et al., 2009). Because CE is
more hydrophobic than cholesterol, it partitions into the
core of lipoproteins, which transforms nascent discoidally
shaped HDL (pre-?-HDL) into spherically shaped HDL
(?-HDL), with a neutral lipid core. When HDL does not
undergo this maturation process, small pre-?-HDL is rap-
idly catabolized, leading overall to low HDL levels. LCAT
is also believed to be a key enzyme in the reverse choles-
terol transport (RCT) pathway, because esterification of
cholesterol on HDL increases the concentration gradient
for the movement of free cholesterol from cells onto HDL
by the various cell transporters that efflux cholesterol
(Zannis et al., 2006). HDL delivers its CE to the liver by
the SR-BI receptor, and cholesterol is then excreted into
the bile as cholesterol or as a bile salt, thus completing the
RCT pathway (Zhang et al., 2005).
There is no specific treatment for FLD; patients are usu-
ally treated symptomatically and are candidates for corneal
and renal transplantation, although the disease can reoccur
in transplanted tissue (Panescu et al., 1997). There have
been several case reports of lipid and lipoprotein abnormal-
ities in patients with FLD being temporarily corrected, after
transfusion of normal plasma containing LCAT (Murayama
et al., 1984). Based on these studies, the half-life of human
LCAT in plasma has been estimated to be 4 to 5 days (Stokke
et al., 1974), which raises the possibility that FLD could be
treated like several lysosomal enzyme storage diseases by
enzyme replacement therapy (Brady, 2006). Because LCAT
acts in the plasma compartment, it does not need to be
targeted to a specific organ or cellular location like the lyso-
some to be effective. Other features that make LCAT an
attractive target for enzyme replacement therapy are that it
is a small single-subunit enzyme of 67 kDa and does not
require any specific cofactors, with the exception of apoA-I,
which is abundant in plasma. LCAT is also relatively stable
and easy to produce in large quantities by recombinant pro-
tein expression systems (Lane et al., 2004). Furthermore, the
concentration of LCAT in normal human plasma is a rela-
tively low, approximately 5 to 6 ?g/ml, and restoration of only
10 to 15% of activity is enough to prevent most of the mani-
festations of FLD, including kidney disease (Rousset et al.,
In this study, we report on the production of human recom-
binant (r)LCAT and investigate its effect on lipid and lipopro-
tein metabolism in several mouse models, including LCAT-
knockout (KO) mice. Intravenous infusion of rLCAT was
found to rapidly raise HDL-C and correct the other lipid
abnormalities in LCAT-KO mice, thus indicating that LCAT
enzyme replacement therapy may be a useful approach for
Materials and Methods
Animals Procedures. LCAT-KO and human LCAT-transgenic
(Tg) mice were described previously (Vaisman et al., 1995; Lambert
et al., 2001). LCAT-KO/apoA-I-Tg mice were produced by crossing
human apoA-I-Tg mice [strain C57BL/6-Tg()1Rub/J, stock 001927;
The Jackson Laboratory, Bar Harbor, ME] with LCAT-KO mice. All
mice were fed ad libitum with a standard chow diet (NIH31 chow
diet; Zeigler Brothers Inc., Gardners, PA). Intravenous treatments
were done by injection into the retro-orbital sinus with a 27.5-gauge
needle. Blood samples were collected from the periorbital sinus of the
contralateral eye, with a heparinized capillary tube (50 or 250 ?l)
and placed into the tubes with K-EDTA as an anticoagulant (final
concentration, 4 mM). Plasma was obtained after centrifugation for
10 min at 3000g at 4°C. LCAT activity in plasma was heat-
inactivated by treatment for 20 min at 56°C, which inhibited more
than 95% of activity. All animal procedures were approved by a
National Institutes of Health Institutional Animal Care Commit-
tee (protocol H-0050R1).
Recombinant Human LCAT Production and Purification.
The plasmid pCMV6-XL4/LCAT, encoding human LCAT cDNA, was
purchased from Origene (Rockville, MD) and ligated into pcDNA3.1/
Hygro (Invitrogen, Carlsbad, CA). Stably transfected HEK293f cells
were selected with 200 ?g/ml hygromycin B and grown in Freestyle
293 serum-free medium (Invitrogen) in 10-liter shake flasks for 4
days. rLCAT was isolated from conditioned cell culture medium by
precipitation with zinc chloride (Zaworski and Gill, 1988), followed
by batch capture with phenyl-Sepharose (GE Healthcare, Little
Chalfont, Buckinghamshire, UK) and batch elution, with 20 mM Tris
and 0.5 M NaCl. Approximately 8 mg of rLCAT could be purified per
liter of conditioned media.
LCAT Activity Assay. LCAT activity was measured, using a
proteoliposome as a substrate (Albers et al., 1986), with the following
modifications (Vaisman and Remaley, 2010). Proteoliposomes were
made by mixing of [14C]cholesterol (1.25 ?Ci/ml; PerkinElmer Life
and Analytical Sciences, Boston, MA) and cholesterol (final concen-
tration, 72 ?M; Sigma-Aldrich, St. Louis, MO) and phosphatidylcho-
line (1.2 mM, ?-L-lecithin; Calbiochem-EMD, La Jolla, CA) in chlo-
roform and drying down under nitrogen. Lipids were solubilized in
an assay buffer (110 mM Tris-HCl, 140 mM NaCl, and 1 mM EDTA,
pH 7.4) containing an amphipathic peptide ETC-642 (PVLDL-
FRELLNELLEALKQKLK; Busseuil et al., 2008) and sodium cholic
acid (final concentration, 62 mM; Sigma-Aldrich). After dialysis,
proteoliposomes were stabilized by adding of equal volume of 2%
BSA (w/v) with ?-mercaptoethanol (10 mM) in the assay buffer and
then incubated 20 min at 37°C. Proteoliposomes were then diluted
1.2-fold with assay buffer containing ?-mercaptoethanol (35 mM)
and stored in liquid nitrogen. To measure LCAT activity, 1 to 5 ?l of
sample was incubated with 60 ?l of proteoliposomes for 30 min at
37°C. Reaction was stopped by adding of 1 ml of 100% cold (?20°C)
ethanol, and samples were kept 20 min on dry ice and centrifuged for
10 min at 10,000g at 4°C. Supernatant was removed, evaporated in
a Speed-Vac concentrator (Thermo Fisher Scientific, Waltham, MA),
and resuspended in 30 ?l of chloroform containing cholesterol (0.1
mg/ml) and cholesteryl oleate (0.1 mg/ml). After separation by thin
layer chromatography (PE SIL G plates; Whatman, Clifton, NJ) with
a mixture of diethyl ether (60 ml), petroleum ether (293 ml), and
acetic acid (3 ml), radioactivity in the cholesterol and cholesteryl
ester spots were measured by liquid scintillation counting. One unit
of LCAT activity was defined as 1 nmol/ml/h of cholesteryl ester
produced at 37°C.
Lipid and Lipoprotein Analysis. Total cholesterol (Wako
Chemicals, Richmond, VA), free cholesterol (Wako Chemicals), and
triglycerides (Roche Diagnostics, Indianapolis, IN) were determined
enzymatically, using a Victor3 plate reader (PerkinElmer Life and
Analytical Sciences). Lipoproteins were fractionated by FPLC (Akta
FPLC; GE Healthcare) on two Superose 6 columns in series. HDL
(density ? 1.21–1.063 g/ml) was isolated by density gradient ultra-
Effect of Recombinant LCAT on Lipoproteins
centrifugation (Schumaker et al., 1986). HDL-C was determined
enzymatically from serum after precipitation of apoB-containing li-
poproteins, with dextran sulfate/magnesium (Raichem, San Diego,
CA). A semiautomated electrophoretic system (Hydrasys; Sebia,
Norcross, GA), with agarose gels (Hydragel Lipoprotein[e] 15/30),
was used for separation and detection of serum lipoproteins, after
staining with Sudan black. Detection of apolipoproteins in gels was
done by immunofixation, using a modified Sebia immunofixation electro-
phoresis method. Purified goat anti-human apoA-I (Meridian Life Science,
Inc., Saco, ME) and anti-human apoB-48/100 (Meridian Life Science, Inc.)
acid violet stain. Human serum from a 47-year-old FLD male, with the
following lipid and lipoprotein test values, was obtained under an institu-
tional review board-approved protocol/ total cholesterol, 175 mg/dl;
triglycerides, 499 mg/dl; HDL-C, ?5 mg/dl; apoA-I, 28 mg/dl; and
apoB, ?30 mg/dl.
Cholesterol Efflux Assay. Baby hamster kidney (BHK) cells
stably transfected with human ABCA1 (Oram et al., 2001), human
ABCG1 (Vaughan and Oram, 2005), or human SR-BI, using the
mifepristone inducible expression system (Invitrogen), were labeled
with [3H]cholesterol (1 ?Ci/ml) for 18 h in DMEM with 10% fetal
bovine serum. The cells were then washed and induced with mife-
pristone (10 nM) in DMEM plus 1 mg/ml BSA for 24 h. Efflux media
were added for the time interval indicated, and released [3H]choles-
terol was monitored by liquid scintillation counting. Residual counts
in the cells were measured after solubilization in hexane/isopropanol
(1:2). Efflux results are expressed as percentage of total radioactive
counts effluxed into the media over time.
Statistical Analysis. Unless otherwise indicated, all results are
presented as the mean ? 1 S.D. of at least three replicates. Differ-
ences between groups were analyzed by unpaired t test and differ-
ences with P ? 0.05 were considered to be significant.
Effect of Infusion of Plasma from LCAT-Tg Mice into
LCAT-KO Mice. To test the feasibility of using rLCAT for
enzyme replacement therapy, we first examined the effect
of infusion of plasma from human LCAT-Tg mice into
LCAT-KO mice. LCAT-Tg plasma contains 6600 units/ml
LCAT activity, which is 220-fold higher than normal mouse
plasma (Vaisman et al., 1995). Fresh plasma or plasma heat
treated to inactivate LCAT was infused intravenously into
LCAT-KO mice, and then plasma lipids were measured (Fig. 1).
One hour after plasma infusion, total cholesterol nearly dou-
bled for both the fresh and heat-inactivated plasma (Fig. 1A).
After 24 h, total cholesterol in the heat-inactivated control
group returned to baseline, whereas the mice treated with
fresh plasma still had a 2-fold increase in total cholesterol.
By 48 h, total cholesterol for both groups returned to near
baseline. As expected, the level of CE at baseline (Fig. 1B)
was very low, but it markedly increased after plasma infu-
sion. Although this occurred in both treatment groups, there
was a much larger increase of CE in mice treated with fresh
plasma, particularly at 24 h, when CE was still more than
10-fold increased above baseline. Because LCAT-Tg mice
have a substantial elevation of not only human LCAT but
also more than a 2-fold increase in HDL-C and a 5-fold
increase in HDL-CE (Vaisman et al., 1995), the exogenous
lipids from the plasma infusion is the most likely source for
the observed increase in total cholesterol and CE observed at
1 h, especially for the heat-inactivated plasma-treated group.
In contrast, the difference in total cholesterol and CE ob-
served between the fresh plasma and heat-inactivated
plasma at later times when most of the exogenous lipids were
catabolized and removed from the plasma compartment is
probably due to the effect of the infused LCAT activity from
the fresh plasma on endogenous lipoproteins.
The level of LCAT activity was monitored after injection of
fresh plasma into LCAT-KO mice (Fig. 1C). Immediately
after infusion of LCAT-Tg plasma, a greater than 10-fold
Fig. 1. Effect of plasma infusion from hLCAT-Tg mice on lipids in
LCAT-KO mice. Heat-inactivated plasma (control; open bars) or fresh
(LCAT; solid bars) plasma (220 ?l) from hLCAT-Tg mice, containing 3300
units of LCAT, was injected intravenously into LCAT-KO mice (n ? 5).
Plasma from the treated mice was collected at the indicated times and
measured for total cholesterol (A) or CE (B). Data represent mean ? S.D.
(n ? 5). ??, p ? 0.001; ???, p ? 0.0001, LCAT versus control group. LCAT
activity after plasma infusion in LCAT-KO mice was measured (C) at
the indicated times after the plasma infusion. Results are expressed
as the percentage of first time point (5 min) after treatment. Results
are the mean ? 1 S.D. of five replicates.
Rousset et al.
increase above baseline in LCAT activity was observed.
LCAT activity then followed a monoexponential decay, with
an estimated half-life of 8.29 ? 1.82 h, which is close to the
reported half-life of mouse HDL (Tape and Kisilevsky, 1990).
Although the major change in CE occurred at 24 h (Fig. 1B),
less than 20% of the original dose of infused LCAT was still
present in the circulation after 24 h, which suggests that
there is a delay in the catabolism of CE formed earlier by
LCAT. No significant increase in LCAT activity was observed
after infusion of heat-inactivated plasma (data not shown).
Changes in lipoprotein distribution and lipid composition
of mice after plasma infusion was examined at the 24-h time
point by FPLC analysis (Fig. 2), when it appeared that the
infused LCAT had its greatest effect on plasma lipids (Fig. 1).
For the heat-inactivated plasma group (Fig. 2B), the FPLC
lipoprotein profile was similar to that of untreated LCAT-KO
mice (Fig. 2A). The major cholesterol peak was found in
fractions corresponding to where VLDL and Lp-X elute (elu-
tion volume, 12–19 ml), and only a relatively small fraction of
cholesterol was esterified. A smaller cholesterol peak corre-
sponding to LDL also was observed, similar to untreated
LCAT-KO mice. In contrast, the major cholesterol peak after
treatment of LCAT-KO mice with fresh plasma was in the
HDL region (Fig. 2C). Cholesterol in VLDL fractions from
mice treated with fresh plasma was decreased by approxi-
mately 40% compared with baseline (Fig. 2A). Unlike the
heat-inactivated plasma treatment group, a large fraction,
approximately 60% of cholesterol, was esterified in mice
treated with fresh LCAT-Tg plasma.
Production and Characterization of rLCAT. Human
rLCAT produced in HEK cells was purified from conditioned
serum-free medium as described under Materials and Meth-
ods. When analyzed on a 10% SDS-polyacrylamide gel elec-
trophoresis gel and stained with Coomassie Blue-R250, a
single band of approximately 70 kDa was observed, consis-
tent with the reported size of the full-length glycosylated
protein (Fig. 3; Supplemental Fig. 1). The band was found to
stain with an anti-human LCAT antibody (Supplemental
Fig. 1). The purified protein had a specific activity of 1850
nmol/mg/h, which is similar to previous reports of purified
LCAT, using a proteoliposome substrate (Lane et al., 2004).
Purified LCAT stored in an aqueous solution (PBS, pH 7.4),
with 10% glycerol, was found to retain greater than 90%
activity after storage at ?70°C for at least 3 months.
In Vitro Effect of rLCAT on Lipoproteins. The in vitro
effect of rLCAT on lipoproteins was examined after incuba-
tion of rLCAT with fasting human serum from an FLD sub-
ject and control subject (Fig. 3). rLCAT was incubated with
serum for 15 h at 37°C and analyzed by agarose gel electro-
phoresis. As indicated, purified human apoA-I also was
added to some samples. The addition of rLCAT to the control
serum either in the presence or absence of exogenous apoA-I
was found to shift LDL from its ?-position and HDL from its
?-position to faster migrating forms (Fig. 3A). Serum from a
patient with FLD had no detectable HDL (?-lipoprotein), but
it had a slow migrating band cathodal to the ?-region and
some residual lipoprotein trapped in the origin. After incu-
bation of FLD serum with rLCAT, faint bands were detected
in the ?- and pre-?-region where HDL from normal serum
also was found to migrate after rLCAT treatment. In addi-
tion, the major band cathodal to the ?-region disappeared
and the predominant band appeared with the same pre-?-
position as the major band from normal serum treated with
rLCAT. When the gels were immunofixed for apoA-I (Fig.
3B), the majority of apoA-I was found in the ?-region for the
control serum, and it shifted to a slightly faster migrating
position after LCAT treatment. In FLD plasma, most of the
apoA-I was present in the pre-?-region just in front of the
major band observed with Sudan black staining (Fig. 3A).
After treatment with LCAT, apoA-I shifted to the ?- and
pre-?-region similar in position observed for control serum
after rLCAT treatment. When control serum was stained for
apoB (Fig. 3C), the major Sudan black band in the ? region
was found to contain apoB and migrated slightly faster after
treatment with LCAT. The slow-migrating band observed
with Sudan black in FLD plasma also stained with apoB and
shifted to the same position as the control serum after rLCAT
The effect of rLCAT incubation with serum from patients
with FLD on lipid levels is presented in Fig. 3D. Total cho-
lesterol, as expected, showed no change, but CE increased
from approximately 20% of total cholesterol to approximately
75%, which is the typical percentage of CE in human serum
(Rousset et al., 2009). HDL-C more than doubled, and there
was a corresponding proportional decrease in cholesterol on
Fig. 2. Lipoprotein profile of LCAT-KO mice infused hLCAT-Tg mice
plasma. LCAT-KO mice were infused with plasma from LCAT-Tg mice,
as described in Fig. 1. FPLC fractions from pooled plasma from untreated
LCAT-KO mice (n ? 5; A), heat-inactivated plasma-treated mice (n ? 5;
B), and fresh plasma-treated mice (n ? 5; C) were analyzed for total
cholesterol (solid line) and CE (dashed line).
Effect of Recombinant LCAT on Lipoproteins
non-HDL lipoproteins; thus, the in vitro incubation of rLCAT
with serum from FLD subjects resulted in the net transfer of
cholesterol to HDL from other lipoproteins.
Investigation of Different Routes of rLCAT Admin-
istration in Mice. The effect of intravenous rLCAT admin-
istration on plasma lipids was tested in human apoA-I-Tg
mice and compared with intramuscular and subcutaneous
delivery of rLCAT in human apoA-I-Tg mice. We first se-
lected mice overexpressing human apoA-I, because it has
been shown previously that human LCAT has a preference
for human apoA-I as an activator over mouse apoA-I (Fran-
cone et al., 1995). Compared with the saline-treated control
group, rLCAT delivered intravenously, intramuscularly or
subcutaneously showed a nearly identical effect on raising
plasma CE over time (Fig. 4A). Likewise, all three routes of
treatment showed a similar effect in raising HDL-C (Fig. 4B).
HDL-C increased almost 2-fold by 24 h after rLCAT treat-
ment, by all three routes, and thereafter began to decline to
baseline. Even after 72 h, however, HDL-C was still elevated
by approximately 25% in the rLCAT-treated mice.
In Vivo Effect of rLCAT Infusion in LCAT-KO Mice.
rLCAT was infused intravenously into LCAT-KO mice, and
plasma was analyzed for lipids after separation by FPLC
(Fig. 5). Four hours after rLCAT injection, cholesterol on
VLDL was reduced by more than 80% (Fig. 5A). This was
associated with the appearance of a prominent HDL-C peak,
which contained the majority of total cholesterol. At 24 h,
cholesterol in the HDL fractions began to decrease, the size of
the HDL also became smaller, whereas cholesterol in the
LDL fractions increased and cholesterol on VLDL reap-
peared. At baseline, CE was only observed in the VLDL peak,
but 4 h after infusion of rLCAT, almost all CE was observed
in HDL (Fig. 5B). By 24 h, CE was still present on HDL but
was reduced and on a smaller sized particle. CE also reap-
peared on VLDL at 24 h, and more CE was found on LDL
compared with baseline.
When the activity of rLCAT was monitored after infusion
into LCAT-KO mice (Fig. 5C), it was found that rLCAT had
a much shorter half-life of approximately 1.23 ? 0.63 h
compared with that of hLCAT from plasma (Fig. 1C). By 24 h,
Fig. 3. In vitro effect of rLCAT on human FLD plasma.
Serum (450 ?l) from a normal subject (lanes 1–4) or an
FLD subject (lanes 5–8) was incubated with rLCAT (185
units) (lanes 3, 4, 7, and 8) or the same volume of saline
(lanes 1, 2, 5, and 6) at 37°C for 15 h. Then, 30 ?g of
purified human apoA-I was added to lanes 2, 4, 6, and 8.
After rLCAT treatment, samples were analyzed by agarose
gel electrophoresis and stained with Sudan black (A), im-
munofixed for apoA-I (B), or immunofixed for apoB (C).
D, serum from an FLD subject (60 ?l) was incubated with
rLCAT (25 units) (LCAT; solid bars) or saline (control; open
bars) at 37°C for 6 h. Samples were analyzed for the indi-
cated lipids and lipoproteins. Results represent the mean ?
1 S.D. of triplicates. ???, p ? 0.0001, experimental versus
Rousset et al.
there was nearly no residual LCAT activity detected, even
though there were still changes in the lipoprotein distribu-
tion at this time (Fig. 5, A and B).
In Vivo Effect of rLCAT Infusion in LCAT-KO/apoA-
I-Tg Mice. The effect of intravenous infusion of rLCAT into
LCAT-KO/apoA-I-Tg mice is presented in Fig. 6. In contrast
to LCAT-KO mice (Fig. 2), at baseline LCAT-KO/apoA-I-Tg
mice had a much smaller cholesterol peak on VLDL and had
low but detectable level of cholesterol on a relatively large-
sized HDL subfraction. It is interesting to note that they also
had a prominent peak in fractions (elution volume, 32–35 ml)
corresponding to a small-sized pre-?-HDL. Four hours after
rLCAT injection, both the large- and small-sized HDL peaks
disappeared, and an intermediate-sized HDL peak was
formed. These changes largely persisted for 24 h, but the
level of HDL-C continued to increase and by 24 h was ap-
proximately 8-fold higher compared with baseline (Fig. 6B).
rLCAT had a half-life of 7.39 ? 2.1 h (Fig. 6C), which was
similar to what was observed for hLCAT from LCAT-Tg
plasma (Fig. 1C).
Effect of rLCAT on Cholesterol Efflux. The function-
ality of HDL formed in plasma after rLCAT treatment was
tested by assessing its ability to stimulate cholesterol efflux
(Fig. 7). HDL was isolated by density gradient ultracentrif-
ugation (density ? 1.21–1.063 g/ml) from the plasma of
LCAT-KO mice 4 h after infusion of either saline or rLCAT.
The same volume of HDL from the isolated density fraction
was then used from each group of mice to stimulate choles-
terol efflux from BHK cells stably transfected with either
ABCG1, SR-BI, or ABCA1. Increased cholesterol efflux was
observed from the ABCA1- and ABCG1-transfected cell lines
after rLCAT treatment, but no significant difference was
observed in cholesterol efflux for the saline- versus rLCAT-
treated group of mice from nontransfected BHK cells or
The results of this study support the feasibility of using
rLCAT as an enzyme replacement therapy agent for FLD.
Infusion of plasma from LCAT-Tg mice into LCAT-KO mice
yielded results similar to previous reports of the rapid cor-
rection of the abnormal lipoproteins in FLD subjects after
infusion with normal plasma (Norum and Gjone, 1968; Mu-
rayama et al., 1984). The half-life of rLCAT in mice, however,
was significantly shorter than the 4- to 5-day half-life de-
scribed in humans (Stokke et al., 1974). In LCAT-KO mice,
which have very low HDL, the half-life of LCAT activity was
Fig. 4. Effect of route of administration of rLCAT on mouse lipids. rLCAT
(4200 units) was injected intravenously into the tail vein (squares), in-
tramuscularly into hind limb (triangles) or subcutaneously (diamonds)
into the back of hapoA-I-Tg mice, and then plasma was removed at the
indicated times and analyzed for total cholesterol (A) and HDL-C (B). The
control group was treated subcutaneously with saline (circles). Data
represent mean ? S.D. (n ? 10). ?, p ? 0.005; ??, p ? 0.001; and ???, p ?
0.0001, experimental versus control group.
Fig. 5. In vivo effect of rLCAT on lipoprotein profile of LCAT-KO mice.
rLCAT (2900 units) was injected intravenously into LCAT-KO mice (n ?
5). Control group of LCAT-KO mice (n ? 5) was injected with PBS. Pooled
plasma at baseline (dotted line), 4 h (large dashed line) and 24 h (solid
line) after rLCAT treatment was fractionated by FPLC and analyzed for
cholesterol (A) and CE (B). C, LCAT activity was measured after intra-
venous rLCAT injection for the indicated times. Results are expressed as
percentage of first time point (5 min) after treatment. Results are the
mean ? 1 S.D. of five replicates.
Effect of Recombinant LCAT on Lipoproteins
only 1.23 ? 0.63 h (Fig. 5C). In contrast, hLCAT from
LCAT-Tg plasma injected into LCAT-KO mice (Fig. 1) or
rLCAT infused into LCAT-KO/apoA-I-Tg mice (Fig. 6) had a
considerably longer half-life of 7 to 8 h. This is similar to the
reported half-life of apoA-I in mice (Tape and Kisilevsky,
1990), which suggests that the half-life of rLCAT is shorter in
mice than humans, because of the more rapid turnover of
HDL in mice. Furthermore, the presence of endogenous HDL
in mice infused with rLCAT, such as LCAT-KO/apoA-I-Tg
mice (Fig. 6), may allow the association of LCAT onto HDL,
which may stabilize it against rapid clearance. This suggests
that for FLD subjects with low levels of HDL, a larger initial
loading dose of rLCAT may be needed, but once some HDL is
formed, less rLCAT may be needed to maintain a normal
lipoprotein profile. That rLCAT produced in cells had a sim-
ilar half-life in the presence of HDL (Fig. 6) as hLCAT endo-
genously produced in transgenic mice (Fig. 1) suggests that
the two forms of LCAT have undergone a similar post-trans-
lational processing, such as glycosylation, which is well
known to affect the half-life of proteins (Brady, 2006).
Based on the promising plasma infusion studies, we devel-
oped a system to produce rLCAT. Similar to a previous report
synthesized relatively large amounts of rLCAT. More work is
but preliminary results suggest that it should be possible to
produce sufficient quantities of pure rLCAT to treat patients.
Assuming a half-life of 4 to 5 days and that only 10 to 15% of
LCAT activity is necessary to prevent renal disease (San-
tamarina-Fojo et al., 2001), we estimate that one treatment
per week containing approximately 10 to 15 mg of pure LCAT
should be sufficient to treat an average-sized adult with FLD.
Based on Fig. 4, which shows a similar effect with three
different routes of delivery, it may be possible to deliver
rLCAT either subcutaneously or by intramuscular injection,
which could potentially make possible the self-administra-
tion of rLCAT by patients with FLD.
Incubation of rLCAT with FLD plasma transformed the
abnormal lipoproteins and changed their electrophoretic mi-
gration position to the same region as lipoproteins from nor-
mal plasma treated with rLCAT (Fig. 3). In FLD subjects,
apoA-I was primarily in the pre-?-region, which did not stain
with Sudan black, suggesting that it is relatively devoid of
CE or triglycerides. This band probably represents pre-?-
HDL, which is phospholipids-rich but neutral lipid-poor par-
ticle and is known to accumulate in FLD (Rousset et al.,
2009). The esterification of cholesterol on pre-?-HDL by
rLCAT resulted in the formation of normal-sized HDL. The
change in the migration position also may be due to the
production of lysophosphatidylcholine by rLCAT, which
would increase on the electronegativity of HDL, therefore
increasing its anodal migration. The main lipoprotein ob-
served in FLD plasma was an apoB-containing particle that
migrated at a position where both abnormal VLDL and Lp-X
Fig. 6. In vivo effect of rLCAT on lipoprotein profile of LCAT-KO ?
hapoA-I-Tg mice. rLCAT was injected (2900 units) intravenously into
LCAT-KO/hapoA-I-Tg mice (n ? 5). Pooled plasma at baseline (dotted
line), 4 h (large dashed line), and 24 h (solid line) after rLCAT treatment
was fractionated by FPLC and analyzed for total cholesterol (A).
B, HDL-C was measured in plasma of control group (LCAT-KO mice, n ?
5; injected with saline; open bars) and in LCAT group (injected with
rLCAT; solid bars). C, LCAT activity was measured after intravenous
rLCAT injection for the indicated times. Results are expressed as per-
centage of first time point (5 min) after treatment. Results are the
mean ? 1 SD of five replicates. ???, p ? 0.0001, experimental versus
Fig. 7. Effect of rLCAT on cholesterol efflux. rLCAT (2900 units; solid
squares) or saline (open squares) was injected intravenously into
LCAT-KO mice (n ? 4), and plasma was collected 4 h after treatment.
HDL was isolated by density gradient ultracentrifugation from 1.1 ml of
plasma and contained 44 and 23 mg/dl total cholesterol from the rLCAT
and saline-treated group, respectively. Volume of HDL corresponding to
10 ?l of plasma was added to 1 ml of serum-free DMEM containing 1
mg/ml BSA and used to efflux cholesterol from the indicated transfected
BHK cell lines or the control BHK cell line for 6 h. Results represent the
mean percentage of total radioactive counts efflux ? 1 S.D. of triplicates.
?, p ? 0.005 and ???, p ? 0.0001, LCAT versus control group (injected
Rousset et al.
migrates (O K and Frohlich, 1995). After treatment with
rLCAT, it shifted to a more anodal position to where LDL
from normal plasma migrated after treatment with rLCAT.
Treatment of LCAT-KO mice with rLCAT largely restored
their lipoprotein profile to a more normal pattern (Fig. 5). In
as short as 4 h, cholesterol in VLDL-sized particles was
markedly reduced, and a large increase in cholesterol on
HDL was observed and most of it was esterified. Based on the
in vitro incubation of rLCAT with FLD plasma (Fig. 3), this
may have occurred due to the conversion of pre-?-HDL in
mice to larger ?-HDL by the esterification of cholesterol.
ApoA-I and other exchangeable apolipoproteins also have
been observed on Lp-X (Santamarina-Fojo et al., 2001) as
well as VLDL (Hamilton et al., 1991), so the esterification of
cholesterol on these particles also could have resulted in the
displacement of exchangeable apolipoproteins from these
particles and the de novo formation of HDL. A similar process
has been described to occur, during the postprandial lipolysis
of VLDL and chylomicrons, which can generate de novo HDL
(Sloop et al., 1983). That the ex vivo treatment of plasma
with rLCAT caused the net transfer of cholesterol from non-
HDL lipoproteins to HDL (Fig. 3C) is also consistent with
It is interesting to note that overexpression of hapoA-I in
LCAT-KO mice reduced the level of cholesterol in the VLDL-
sized fractions (Fig. 6). A peak corresponding to small, phos-
pholipid-rich HDL also was observed (data not shown), which
probably represents pre-?-HDL. Increased production of
hapoA-I in the absence of sufficient LCAT would be expected
to result in the production of more pre-?-HDL. Shortly after
rLCAT treatment, almost of all the small-sized HDL was
converted to an intermediate-sized HDL. In addition, a much
greater increase in HDL-C was observed in these mice com-
pared with LCAT-KO mice (Fig. 6). This probably occurs
because there was more substrate, i.e., small-sized HDL for
LCAT, and possibly because human apoA-I is a better acti-
vator for LCAT than mouse apoA-I (Francone et al., 1995).
As would be expected, if HDL formed by LCAT was func-
tional, more cholesterol efflux was observed from ABCA1-
and ABCG1-transfected cells to HDL isolated from mice
treated with rLCAT (Fig. 7). Cholesterol efflux from ABCG1
is known to occur to lipid-rich forms of HDL, which were
generated by the rLCAT treatment (Figs. 2 and 5). In con-
trast, ABCA1 effluxes cholesterol to lipid-poor HDL like pre-
?-HDL (Zannis et al., 2006). The observed increase in choles-
terol efflux from ABCA1-transfected cells was not anticipated.
Several recent studies, however, have shown that lipid-rich
HDL also can serve as an acceptor for cholesterol from ABCA1,
possibly when apoA-I dissociates from HDL (Favari et al.,
2009). As discussed, the displacement of exchangeable apoli-
poproteins from VLDL or Lp-X particles after treatment with
rLCAT also could generate nascent-like HDL particles that
could stimulate cholesterol efflux by ABCA1.
The increase in cholesterol efflux from cells after rLCAT
treatment suggests that rLCAT also may be used for the
treatment of atherosclerosis. LCAT has long been proposed to
increase RCT, by trapping cholesterol effluxed from cells
until it can be removed as CE by the liver (Glomset, 1968).
Because cholesterol efflux is considered to be the first step in
RCT, the cholesterol efflux data are consistent with a recent
study showing that the flux of radiolabeled cholesterol from
peritoneal macrophages to the stool was reduced by 45% in
mice lacking LCAT (Tanigawa et al., 2009). LCAT expressed
with adeno-associated virus did not however increase the
amount of radiotracer cholesterol in the stool of apoA-I trans-
genic mice. It should be noted however that studies in mice
that do not express CETP can lead to contradictory results
compared with the effect of various genes on human lipopro-
tein metabolism (Briand et al., 2010). LCAT transgenic mice
are not protected against atherosclerosis (Be ´rard et al.,
1997), unless CETP also is expressed (Fo ¨ger et al., 1999). In
hamsters, which have CETP, overexpression of LCAT not
only increased HDL but also increased biliary sterol excre-
tion (Zhang et al., 2004). In rabbits, which express CETP,
LCAT overexpression decreased the cholesterol content of
the aorta (Van Craeyveld et al., 2009), and LCAT transgenic
rabbits were protected from diet-induced atherosclerosis
(Hoeg et al., 1996). A recent preliminary report showed that
subcutaneous injection of rLCAT in rabbits stimulated
whole-body RCT and reduced atherosclerosis (Zhou et al.,
2009). Although the question whether FLD subjects have
increased risk for atherosclerosis have yielded contradictory
results (Hovingh et al., 2005; Calabresi et al., 2009), a recent
study, using a more sensitive method (i.e., carotid 3.0 Tesla
MRI; R. Duivenvoorden, unpublished data).
rLCAT treatment may be particularly useful when used in
conjunction with HDL replacement therapy (Remaley et al.,
2008). Under these conditions, LCAT levels may become rate-
limiting, as evidenced by the increase in plasma pre-?-HDL
(LCAT substrate) after administration of certain apoA-I mi-
metic peptides (Navab et al., 2004). If so, a combination of
apoA-I mimetics along with rLCAT may show not only syn-
ergy in increasing HDL-C but also enhance the ability of HDL
to remove cholesterol from atherosclerotic arteries. This is con-
sistent with the result observed with the apoA-I Tg mice; de-
spite having normal LCAT levels and increased apoA-I, they
showed a much greater increase in HDL-C after rLCAT treat-
ment than did just LCAT-KO mice (Figs. 5 and 6).
In summary, rLCAT treatment in LCAT-KO mice was
observed to rapidly increase HDL-C and to decrease abnor-
mal lipoproteins. These results demonstrate the potential of
using rLCAT as an enzyme replacement therapy agent for
patients with FLD.
We thank Rene Costello for excellent technical assistance.
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Address correspondence to: Dr. Xavier Rousset, National Heart, Lung, and
Blood Institute, National Institutes of Health, Bldg. 10, Room 8N226, 10
Center Dr., Bethesda, MD 20892. E-mail: email@example.com
Rousset et al.