Effect of LDL-apheresis on plasma lipids, chitotriosidase and anti-oxLDL antibodies in heterozygous familial hypercholes-terolemia

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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.

Email: [email protected]

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)

16 (69.6)

5 (21.7)

2 (8.7)

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

Triglycerides, mmol/L (men)

‘’ ‘’ ‘’ ‘’ (women) 1.46+1.24

1.04+0.53

0.62+0.26

0.44+0.21

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

HDL cholesterol, mmol/L (men)

‘’ ‘’ ‘’ ‘’ (women) 1.33+0.28

1.48+0.35

0.93+0.21

1.03+0.22

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.

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.

(a) (b)

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)

(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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