J. Biomedical Science and Engineering, 2009, 2, 499-505
doi: 10.4236/jbise.2009.25043 Published Online November 2009 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online November 2009 in SciRes. http://www.scirp.org/journal/jbise
Effect of LDL-apheresis on plasma lipids, chitotriosidase and
anti-oxLDL antibodies in heterozygous familial
hypercholesterolemia
Maria Musumeci1, Francesco Pappalardo2,3, GianCarlo Tonolo4, Fernando Torrisi5, Francesca Gullo3,
Salvatore Musumeci6
1Department of Hematology, Oncology and Molecular Medicine, Italian National Institute of Health, Rome, Italy; 2Institute for
Computing Applications “M. Picone”, National Research Council (CNR), Rome, Italy; 3University of Catania, Catania, Italy;
4Diabetology Unit, Azienda Sanitaria Locale 2 Olbia, Italy affiliated to Department of Clinical Sciences, Medical Genetics Unit,
Lund University, Malmö, Sweden; 5Institute of Medical and Environmental Research (IRMA), Acireale (Catania), Italy; 6Department
of Neurosciences and Mother to Child Sciences, University of Sassari, and Institute of Biomolecular Chemistry, National Research
Council (CNR), Li Punti, Sassari, Italy.
Received 16 June 2009; revised 10 July 2009; accepted 17 July 2009.
ABSTRACT
Forty four consecutive subjects aged 29-58 years (21
males and 23 females) with a clinical diagnosis of
heterozygous familial hypercholesterolemia period-
ically treated every 30 days with LDL-apheresis for
statin resistance, were enrolled in this study. A lipid
profile was obtained immediately before starting
LDL-apheresis, a second profile was obtained within
four hours after LDL-apheresis. Chit activity and
anti-oxLDL levels were determined with appropriate
methods in all patients before and after LDL-
apheresis. Total cholesterol, LDL-cholesterol, HDL-
cholesterol and triglycerides decreased significantly
after LDL-apheresis, while the variations of Chit
activity and anti-oxLDL were not significant after
LDL-apheresis. The correlation between Chit and
total cholesterol was negative (r= –0.44 and –0.50 res-
pectively) before and after LDL-apheresis as between
Chit and LDL-cholesterol (r= –0.45 and –0.55
respectively). Anti-oxLDL concentration before and
after LDL-apheresis positively correlated with Chit
activity (r= 0.52 and r = 0.63 respectively), negatively
with total cholesterol (r= –0.33 and r = –0.35 res-
pectively) and with LDL (r = –0.32 and r = –0.21
respectively). We think that removing LDL with
LDL-apheresis the anti-oxLDL/oxLDL ratio could
increase and the excess of anti-oxLDL could induce
macrophage activation through the surface Fc
receptors. Alternatively with high levels of LDL-
cholesterol, the deposition of foam cells represent the
characteristic evolution of atherosclerosis process.
Macrophage activation in the heterozygous familial
hypercholesterolemia could represent an attempt for
re-modeling the vessel wall, reducing the growth of
lipid plaques.
Keywords: LDL-Apheresis; Heterozygous Familial Hyper-
Cholesterolemia; Lipids; Chitotriosidase; Anti-oxLDL Anti-
bodies; Sardinia
1. INTRODUCTION
Familial hypercholesterolemia (FH) is a genetic alte-
ration of lipoprotein metabolism caused by defects in
the low density lipoprotein receptor (LDLR) [1]. High
LDL levels, secondary to the LDLR homozygous
defect, are associated to significant increase of oxidized
LDL (oxLDL), which removed from circulation lead to
massive lipid accumulation, foam cell formation in
endothelial wall, often tendom xanthomas (TX) and
corneal arcus [2]. Also the heterozygous FH (heFH),
shows precocious coronary heart disease before 65 years
old, if they are not treated [3]. Then FH represent a
paradigmatic example of atherosclerosis produced by
oxLDL accumulation and a model to study the role of
macrophage activation in atherosclerosis process [1].
Generated oxLDL induces an immune response with
production of anti-oxLDL antibodies and macrophage
cells could remove from circulation immune complexes
anti-oxLDL/oxLDL through the Fc receptor for anti-
bodies [4]. Shoji et al 2000 [5] found a inverse
correlation among anti-oxLDL and oxLDL in healthy
individuals supporting the hypothesis that this mecha-
nism is also operating in condition where oxLDL are

500 M. Musumeci et al. / J. Biomedical Science and Engineering 2 (2009) 499-505
stably low. This mechanism could be not sufficient to
protect from foam cells generation in presence of a
defect in LDLR gene, when the levels of oxLDL are
very high [6].
Chitotriosidase (Chit) is one of the most quantitatively
represented marker of macrophage activation, such as
occurs in Gaucher disease, sarcoidosis, nonalcoholic
liver disease and atherosclerosis [7,8,9]. Plasma Chit
activity has been associated with both the extension and
prognosis of atherosclerotic vascular lesions in humans
[10,11,12] and its phagocyte-specific expression supports
a relevant role in innate immunity [13].
Considering the importance of anti-oxLDL in the
pathogenesis of atherosclerosis lesions [14] and the
involvement of Chit activity in the evolution of athe-
rosclerotic vascular lesions [10,11], we hypothesize a
relation among these two factors. The objective of this
study is to establish the relationship between lipid para-
meters, Chit activity and anti-oxLDL levels in a group of
subjects with clinically and genetically defined heFH
(total cholesterol consistently more than 400 mg/dl)
before and after LDL-apheresis treatment. LDL-aphere-
sis represents an effective therapy in heFH patients, who
had no response to highest doses of statin drugs, and
could restore the physiological mechanism of anti-
oxLDL/oxLDL immunocomplexes clearance altered by
LDLR genetic defect [15].
2. MATERIAL AND METHODS
2.1. Study Subjects
Consecutive 44 subjects from Sardinia (Italy) aged 29–
58 years (21 males and 23 females) with a clinical
diagnosis of heFH were treated periodically with LDL-
apheresis. The diagnosis of heFH was determined
genetically in all patients [2]. They started LDL-aph-
eresis treatment because their previous lipid lowering
therapy (statin and benzafibrate) did not reduce the total
and LDL cholesterol.
A combination of discontinuous blood centrifugation
(MCS 3p Haemonetics Corp., Braintree, MA, USA) and
2 steps membrane differential filtration were performed
at interval of 30 days. Clinical data, history of prior
cardiovascular disease (CVD) at early onset, demogra-
phic and anthropometric measurements, and an accurate
physical examination in search of tendon xanthomas
(TX) were obtained from each subject.
Informed consent was obtained from all subjects and
the ethical committee from each institution approved this
study.
2.2. Lipid Concentrations
To obtain a baseline lipid profile, overnight fasting blood
was drawn immediately before starting LDL-apheresis.
A second profile was obtained within four hours after
LDL-apheresis. Total cholesterol and triglyceride levels
were measured with standardized enzymatic methods.
HDL cholesterol was measured by precipitation methods
and LDL cholesterol was estimated with the Friede-
wald’s formula, since no patient had triglycerides over
300 mg/dl. Lipoprotein (a) was determined in immuno-
nephelometry with specific antibodies (New Scientific
Company S.r.L., Cormano (MI), Italy).
2.3. Chitotriosidase Enzyme Assay
Chitotriosidase enzyme assay was based on the method
described by Hollak et al. 1994 [7], with minor modi-
fications. Briefly, chitotriosidase activity was determined
by incubating 5 µL of plasma with 100 µL of 22 mmol/L
4-methylumbelliferyl-ß-d-N,N′,N′′triacetyl-chitotrioside
fluorogenic substrate (Sigma-Aldrich S.r.L. Milano, Italy,
catalogue M 5639) in McIlvain buffer (100 mmol/L
citric acid and 200 mmol/L sodium phosphate, pH 5.2)
for 15 minutes at 37°C. The reaction was stopped by
using 2 ml of 0.5 mol/L Na2CO3-NaHCO3 buffer, pH
10.7. The substrate hydrolysis by Chit produces the
fluorescent molecule 4-methylumbelliferone, which was
quantified with a Hitachi 2500 fluorometer, excitation at
366 nm and emission at 446 nm, and compared with a
standard 4-methylumbelliferone calibration curve. Chit
activity was expressed as nanomoles of substrate hy-
drolyzed per hour per milliliter of reaction mixture.
Plasma Chit activity was measured by duplication and
three QC samples from healthy adults were added in
every set of determinations. The coefficient of variation
was less than 5% in all cases.
2.4. Oxidized Low-Density Lipoprotein
Antibodies
An enzyme-linked immunosorbent assay (ELISA) for
the detection and quantification of IgG antibodies to
oxidized low-density lipoprotein (oxLDL) in human
plasma was used (ImmuLisa™, IMMCO Diagnostics,
Buffalo, NY, USA). The intensity of the color changes,
proportionally to the antibodies concentration, was read
as absorbance at 405 nm. Three QC samples from
healthy adults were also added in each plate containing a
calibration curve. The absorbance values on native LDL
were subtracted from the absorbance obtained on oxLDL
for control, calibrators and specimens. The concentration
of anti-oxLDL was determined from the calibration
curve and the results are expressed in Enzyme Units per
milliliter (EU/ml).
2.5. Apolipoprotein E Genotyping
DNA from patients isolated by peripheral blood cells
was used in a polymerase chain reaction (PCR) and
Apolipoprotein E genotypes were determined by HhaI
digestion as the methods described by Hixon and Vernier
[16], modified by Tsukamoto et al [17].
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2.6. LDLR Genetic Analyses
For LDLR gene analysis the entire gene was sequenced.
Mutations in the LDLR gene causing FH were classified
when possible, as receptor-defective or receptor-negative
on the basis of the residual LDLR activity. Mutation
leading to a frame-shift and/or a truncated receptor were
considered as receptor-negative [2,6].
2.7. Chitotriosidase Polymorphism
DNA from patients were used as template in subsequent
PCR reactions. The duplication mutation analysis was
performed using specific primers [Chs9 (AGCTATCT
GAAGCAGAAG) and Chas8 (GGAGAAGCCGGCA-
AAGTC)] and two fragments of 75 and 99 base pairs
were amplified from the wild and mutant CHIT gene,
respectively. Electrophoresis in Metaphore gel (4%),
allowed the detection of both fragments.
3. STATISTICAL METHODS
The data are expressed as mean values with standard
deviation (SD) for variables with normal distribution and
as medians and range for variables with a skewed distri-
bution. Statistical differences were computed by using
the Student’s t test or the Mann-Whitney U-test, respec-
tively. Correlation and statistical analyses were perform-
ed with SSPS software (version 13.0), with significance
set at P<0.05.
4. RESULTS
In Table 1, we show the clinical and biochemical
characteristics of the 44 heFH subjects, including also
the LDLR and CHIT genotype polymorphisms. No
signi- ficant difference, among men and women,
regarding age, body mass index, blood pressure was
found and neither presence of hypertension nor diabetes.
Differences were found in smoking habit, premature
CVD, and age of first CVD event, being more smoker
among the men group, who had more premature CVD,
with minor age at the first event. Mean values of total
cholesterol, LDL- cholesterol, triglycerides in the last
few months were elevated in all subjects with no
significant difference between men and women (see
Table 1). Only men had significantly lower HDL
cholesterol levels compared to women. No Chit deficient
patients was found at the genotype analysis, 27 (61.4 %)
were wild/wild and 17 (38.6%) wild/mut. Chit activity
was found elevated before LDL-apheresis, without
difference between males (11.94+8.68 nmol/ml/h) and
females (12.26+7.70 nmol/ml/h), considering that
7.3±1.9 nmol/ml/h activity was found in healthy control
of the same age in our laboratory. Also the anti-oxLDL
were found elevated before LDL-apheresis in males
(38.20+17.64 UI/ml) and in females (55.80+38.97),
considering that < 20 UI/ml was found in healthy control
Ta b le 1 . Clinical and laboratory characteristics of 44 heteroz-
ygous FH patients (21 men and 23 women).
Men
N=21
Women
N=23 p
Age, years 35.2 (29-54) 36.9 (32-58)N.S.
Body mass index, kg/m2 28.0+3.68 28.5+4.84 N.S.
Smoking status:
Never, n (%)
Current, n (%)
Former, n (%)
7 (33.3)
3 (14.3)
11 (52.4)
15 (65.2)
5 (21.7)
3 (13.0)
0.0001
Smoking, packs x years 26.1+10.0 26.5+7.42 N.S.
Systolic blood pressure, mmHg130.3+17.1 129.4+19.1 N.S.
Diastolic blood pressure, mmHg77.4+10.3 76.8+11.3 N.S.
Hypertension, n (%) 4 (19.0) 5 (21.7) N.S.
Diabetes, n (%) 1 (4.8) 1 (4.3) N.S.
Premature CVD, n (%) 8 (38.1) 6 (26.1) 0.016
Age of first CVD event, years45.4+6.16 49.5+7.53 0.0578
Family history premature CVD,
n. (%) 10 (47.6) 11 (47.8) N.S.
Tendon xanthomas, n. (%) 8 (38.1) 11 (47.8) N.S.
Total cholesterol, mmol/L 8.99+4.03 8.29+1.53 N.S.
Triglycerides, mmol/L 1.46+1.24 1.04+0.53 N.S.
LDL cholesterol, mmol/L 7.25+1.20 6.82+1.33 N.S.
HDL cholesterol, mmol/L 1.33+0.28 1.48+0.35 0.0228
Chitotriosidase (nmol/ml/hr) 11.94+8.68 12.26+7.70 N.S.
Anti-oxLDL (UI/ml) 38.20+17.64 55.80+38.97 0.0646
ApoE genotype
E3/E3, n (%)
E3/E4, n (%)
E3/E2, n (%)
E4/E4, n (%)
E2/E2, n (%)
16 (76.2)
5 (23.8-0)
0 (0)
0 (0)
0 (0)
16 (69.6)
5 (21.7)
2 (8.7)
0 (0)
0 (0)
N.S.
LDLR gene mutation
Defective, n (%)
Negative, n (%)
Undefined, n (%)
9 (45.0)
7 (35.0)
4 (20.0)
8 (33.7)
9 (39.1)
6 (26.1)
N.S.
Chit gene mutation
Mut/Mut, n (%)
Wild/Mut, n (%)
Wild/Wild,n (%)
0 (0)
13 (61.9)
8 (38.1)
0 (0)
14 (60.8)
9 (39.2)
N.S.
of the same age in our laboratory. The difference
between males and females was not significant (P =
0.0646). In Table 2 are reported the levels of total
cholesterol, LDL-cholesterol, HDL-cholesterol, trigly-
cerides, Chit and anti-oxLDL before and after LDL-
apheresis, considering separately males and females. All
lipid parameters significantly decreased after LDL-
apheresis (P<0.0001). The reduction of Chit activity
after LDL-apheresis was not significant as well as the

502 M. Musumeci et al. / J. Biomedical Science and Engineering 2 (2009) 499-505
Table 2. Lipid parameters before and after plasmapheresis in 44 heterozygous FH patients (21 men and 23 women).
Before Plasmapheresis After Plasmapheresis Student T test
Total cholesterol, mmol/L (men)
‘’ ‘’ ‘’ ‘’ (women) 8.99+4.03 8.29+1.53
3.23+0.85
2.98+0.70
0.0001
0.0001
Triglycerides, mmol/L (men)
‘’ ‘’ ‘’ ‘’ (women) 1.46+1.24
1.04+0.53
0.62+0.26
0.44+0.21
0.0001
0.0001
LDL cholesterol, mmol/L (men)
‘’ ‘’ ‘’ ‘’ (women) 7.25+1.20
6.82+1.33
2.10+0.74
1.97+0.63
0.0001
0.0001
HDL cholesterol, mmol/L (men)
‘’ ‘’ ‘’ ‘’ (women) 1.33+0.28
1.48+0.35
0.93+0.21
1.03+0.22
0.0001
0.0001
Chitotriosidase (nmol/ml/hr) (men)
‘’ ‘’ ‘’ ‘’ (women) 11.94+8.68
12.26+7.70
9.04+4.36
9.16+4.92
N.S.
N.S.
Anti ox-LDL (UI/ml) (men)
‘’ ‘’ ‘’ ‘’ (women) 30.20+17.64
55.80+38.97
26.45+15.48
48.00+30.01
N.S.
N.S.
(a) (b)
(c) (d)
Figure 1.
reduction of anti-oxLDL concentration. Before and after
LDL-apheresis the correlation between Chit and total
cholesterol was negative (r=–0.44 and –0.50 respectively)
and the same between Chit and LDL-cholesterol (r =
–0.45 and –0.55 respectively) Figures 1a, b, c, d). The
correlation between HDL-cholesterol and Chit before
and after LDL-apheresis was not significant. The anti-
oxLDL concentration positively correlated with Chit
activity (r = 0.52) and negatively with LDL-cholesterol
(r = –0.32) and total cholesterol (r = –0.33) before
LDL-apheresis (Figures 2a, c, e). After LDL-apheresis
the correlation with Chit was maintained high (r = 0.63)
and the correlation with LDL-cholesterol and total
cholesterol remained significant (r = –0.21 and r = –0.35
respectively) (see Figures 2b, d, f).
However negative correlations were not maintained
for high levels of total cholesterol and LDL-cholesterol,
before LDL-apheresis. This was supported by the order-
2 polynomial trend behavior showed in Figures 1a and c,
Figures 2c and e. On the contrary, these trends were
maintained, when the level of total cholesterol and
LDL-cholesterol decreased after LDL-apheresis.
5. DISCUSSION
Plasma filtration represent an effective therapy in heFH
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M. Musumeci et al. / J. Biomedical Science and Engineering 2 (2009) 499-505 503
patients, who had no response to highest doses of statin
and could restore through a macrophage activation the
physiological mechanism of remotion of anti-oxLDL/
oxLDL immunocomplexes altered by LDLR genetic
defect [15].
In fact in this study we demonstrated a negative
correlation among Chit activity, total and LDL-choles-
terol before and after LDL-apheresis, which seems to be
dependent by active macrophage removal of anti-
oxLDL/oxLDL immunocomplexes.
By removing LDL with LDL-apheresis, the anti-
oxLDL/oxLDL ratio could increase and the excess of
anti-oxLDL could induce macrophage activation through
the surface Fc receptors. Alternatively with high levels
of LDL-cholesterol, the deposition of foam cells repre-
sent the natural evolution of atherosclerosis process (as
highlighted by the trend inversion above mentioned).
Hulthe et al 1998 [18] found, in a post hoc analysis,
lower antibody titers in patients with a history of
myocardial infarction, suggesting that antibodies against
(a) (b)
(c) (d)
(e) (f)
Figure 2.
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504 M. Musumeci et al. / J. Biomedical Science and Engineering 2 (2009) 499-505
oxLDL could have a protective effect. This observation
is in agreement with Tinahones et al 2002 [19], who
reported in literature a negative correlation among
anti-oxLDL and total cholesterol in a population of 400
subjects from Malaga (Spain), suggesting again the
active role of macrophage cells in the remotion of
oxLDL. The same authors [20] in a larger population
(1354 subjects) found a very significant increase (P <
0.0001) of anti-oxLDL: younger persons (16-35 years)
had higher levels of anti-oxLDL (MDA-LDL) anti-
bodies than persons older than 35 years (P = 0.05) and
immune complexes were significantly higher (P = 0.05)
in persons aged 5–15 years than in persons older than 40
years. On the contrary Artieda et al 2003 [10] reported
an increased plasma Chit activity, independent from the
distinct allelic or genotype distribution, in Spanish
patients with atherothrombotic stroke and correlated
plasma Chit activity to the severity of the atherosclerotic
lesion [11]. Moreover, in Spanish hypercholesterolemic
patients Canudas et al [21] found that no correlation
exist between plasma Chit activity and the variation of
plasma lipid levels before and after treatment with
atorvastatin or bezafibrate. In our study plasma Chit
activity, total and LDL-cholesterol levels correlated
negatively before and after LDL-apheresis. These con-
trasting results could be due to different mechanism
depending from the high level of oxLDL in plasma,
which could inhibit the re-modeling of vessel wall in
defect of anti-oxLDL.
In fact the activation of macrophages within the
atherosclerotic lesion could represent an attempt for
re-modeling the vessel wall, which may control the
growth of lipid plaques. In condition where the total
cholesterol and LDL-cholesterol levels are maintained
elevated, the negative trend which seems to demonstrate
an active mechanism of immunocomplexes removal
disappears, supporting the foam cells formation and
subsequent deposition. Some years ago we demonstrated
“in vitro” that the activation of macrophages was
induced by formation of immunocomplexes anti-oxLDL/
oxLDL through the Fc fragment receptor on the
macrophage cell surface [22]. Recently this hypothesis
was resumed by Fostergard et al 2007 [23] who
demonstrated the importance of antibodies response,
comparing two populations with different cardiovascular
disease prevalence from New Guinea and from Sweeden.
The discriminant factor among this two groups was a
significant level of anti-phosphorylcholine antibodies of
IgM subclass, which are active in the remotion of
oxLDL. Also in atherosclerosis mice model, the in-
duced increase of anti-oxLDL appears to be protective,
in fact several investigators have demonstrated with
oxLDL immunization [24,25] or with S. Pneumoniae
immunization [26], that in such model the atherosc-
lerosis development attenuates. This was also confirmed
by an “in vivo” study in Watanabe heritable hyper-
lipidemic rabbits, lacking the LDL receptor and
mimicking the human familial hypercho-lesterolemia. In
this mice model the continuous auto-immunization with
malondialdehyde-modified LDL, miming oxLDL, results
in a very high concentration of antibodies against oxLDL,
leading to significantly reduced progression of athero-
sclerosis [27]. This experiment opens the way to a model
for the oxLDL vaccination, with the aim to activate the
macrophage system to clean the atheromatosis lesions.
Moreover it is evident that the mechanism of active
cleaning of atheromatosis lesions is working when the
level of total cholesterol and LDL-cholesterol is kept
sufficiently low [5].
This evidence was also supported by our computa-
tional model that was able to reproduce experimental
data and is actually used for making predictions and
evaluate biological hypothesis [28].
In fact, LDL-apheresis is, up to date, the more active
system for lowering LDL level in FH, and we found a
negative correlation between anti-oxLDL, total chole-
sterol and LDL-cholesterol before and after LDL-
apheresis, even plasma Chit activity and total and
LDL-cholesterol correlated negatively before and after
treatment. This stable response is explained by active
remotion of immunocomplexes in these patients in the
interval between LDL-apheresis. The increase of Chit
activity was not found after statin treatment, probably
because the modification of total cholesterol and LDL
was not rapid enough to induce an activation of macro-
phage cells [21]. On the contrary Orem et al 2002 [29]
reported after lipid lowering therapy with atorvastatin
(10 mg/day) a significant decrement of anti-oxLDL and
an increase of anti oxidant capacity of plasma LDL,
which suggested again an active removal of immune
complexes. They suggest also that the measurement of
antibodies against oxLDL during lipid-lowering therapy
may be used as an important marker for monitoring
in-vivo LDL oxidation and atherosclerosis processes.
In individuals where the LDL were reduced by LDL-
apheresis or by statine treatment, an active mechanism
may be operative clearing the surface of vessel as was
reported in our beta-thalassemia patients [30] and in
diabetes patients in statine treatment [22] protecting
from oxLDL vascular alteration.
6. ACKNOWLEDGEMENTS
Authors are grateful to Mister Rapicavoli Giuseppe, laboratory tech-
nician, for his precious assistance in the preparation of samples and in
the determination of chitotriosidase activity.
7. AUTHORS CONTRIBUTION
Authors have equally contributed to the manuscript.
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