Clinical Hemorheology and Microcirculation 35 (2006) 349–357
IOS Press
349
L-carnitine deficiency and red blood cell
mechanical impairment in β-thalassemia
major
B. Toptas a , A. Baykal a , A. Yesilipek b , M. Isbir c , A. Kupesiz c , O. Yalcin d and
O.K. Baskurt d,∗
a
Department of Biochemistry, Akdeniz University School of Medicine, Antalya, Turkey
Department of Pediatrics, Akdeniz University School of Medicine, Antalya, Turkey
c
Department of Pharmacology, Akdeniz University School of Medicine, Antalya, Turkey
d
Department of Physiology, Akdeniz University School of Medicine, Antalya, Turkey
b
Abstract. L-carnitine is an essential element of intermediary metabolism and also was shown to be effective in maintaining
normal red blood cell (RBC) function. This study aimed at investigating plasma free L-carnitine concentrations and effectiveness of L-carnitine supplementation in protecting deterioration of RBC properties in β-thalassemia major patients. Plasma free
L-carnitine concentrations were determined in the blood samples obtained before their regular transfusion (about one month
after the previous transfusion). Each patient received 100 mg/kg/day oral L-carnitine supplementation. RBC deformability,
lipid peroxidation and intracellular free calcium concentrations were investigated before and after this treatment. Plasma free
L-carnitine levels and RBC deformability before the treatment were found to be lower whereas lipid peroxidation and intracellular calcium concentration in RBC were higher compared to those of the control subjects before the L-carnitine treatment.
After one month supplementation of L-carnitine lipid peroxidation and intracellular calcium concentrations were found to be
decreased and RBC deformability was improved, accompanying the significantly increased plasma L-carnitine concentrations.
These results suggest that L-carnitine can be used as a supplement in β-thalassemic patients, to prevent RBC deterioration.
Keywords: L-carnitine, β-thalassemia, deformability, intracellular calcium, TBARS
1. Introduction
β-thalassemia major is characterized by an imbalance between the synthesis of α and β globin
chains [34]. RBC destruction is increased in β-thalassemic patients due to excess, unmatched α
chains [34], leading to decreased red blood cell (RBC) counts and hemoglobin (Hb) levels. The exact mechanism leading to RBC destruction in thalassemia is still not clear [34]. RBC quality may also
be altered; increased oxidant damage [35], impaired mechanical properties [18,41], and altered membrane composition [26] have been reported. Susceptibility of RBC to oxidative stress is increased in
β-thalassemic patients due to the free α-chains and excess iron [32]. Intracellular calcium levels of RBC
in β-thalassemia major patients were higher than those of normal patients [26,36]. Such alterations in
RBC would increase in vivo destruction and reduce their life span [12], contributing to anemia.
Hematopoetic stem cell transplantation provides curative treatment for β-thalassemic patients [43],
however regular RBC transfusions still remain to be the main therapeutic method. Additionally, therapeutic approaches to reduce oxidative damage and stimulation of fetal hemoglobin production to reduce
*
Corresponding author. E-mail: [email protected].
1386-0291/06/$17.00  2006 – IOS Press and the authors. All rights reserved
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imbalance between α and β chains by increased synthesis of γ chain [30,44] have also been used in
β-thalassemic patients. L-carnitine treatment was also reported to be effective in such patients to prolong
transfusion interval [44]. L-carnitine treatment was effective in decreasing RBC oxidative damage [32]
and to improve cardiac function [19] in β-thalassemic patients.
L-carnitine supplementation has been used to treat RBC abnormalities encountered in other clinical conditions, such as hemodialysis patients [31]. It has been reported that L-carnitine deficiency is
frequently detected in hemodialysis patients [20,33] and disorders that might be related to this deficiency might be corrected by L-carnitine supplementation [20,31,33]. A recent report by Tsagris and
Liapi-Adamidou pointed out that serum L-carnitine concentrations in β-thalassemic patients may also
be lower compared to healthy controls [38]. It has been suggested that L-carnitine deficiency in these patients may play role in muscular dysfunction and the patients may benefit from L-carnitine treatment. It
can be suggested that L-carnitine deficiency in β-thalassemic patients may also play role in deterioration
of RBC properties [18,35,41].
Based on the above mentioned reports, it can be hypothesized that L-carnitine might be effective in
preventing the oxidative damage and related mechanical alterations of RBC in β-thalassemic patients.
This study was designed to investigate plasma L-carnitine levels and the effectiveness of L-carnitine supplementation in preventing the mechanical deteriorations and oxidative damage in RBC of β-thalassemia
major patients.
2. Materials and methods
2.1. Patients and blood sampling
Twenty patients with transfusion dependent beta thalassemia major (3–17 years of age) were followed
up at the Division of Pediatric Hematology, Department of Pediatrics, Akdeniz University School of
Medicine. All thalassemic patients were receiving RBC transfusions, at 3 to 4-week intervals, in order
to maintain their pre-transfusion Hb levels above 85 g/l. They were also being treated with desferroxamine 50 mg/kg/day, s.c., 5 days a week. They received L-carnitine, (Carnitine, Santa Farma, Sigma Tau,
Industrie Farmaceutiche, Pornezia, Italy), 100 mg/kg/day, orally for one month. Plasma free L-carnitine
concentration, RBC lipid peroxidation, intracellular calcium concentration and erythrocyte deformability were investigated in heparinized (10 IU/ml) venous blood samples of these patients before and after
the one month period. Blood samples were collected from all patients before transfusion. RBC count,
hemoglobin and hematocrit values were determined by an electronic hematology analyzer (Cell-Dyn
1600, Abbott Diagnostic Division). The same parameters were also determined in the blood samples
obtained from twenty healthy children in the same age group that served as the control group. All measurements were performed within two hours after the sampling.
2.2. Plasma free L-carnitine measurements
Plasma samples separated by centrifugation at 2000g for 10 min were used for the determination
of free L-carnitine concentration using Cederblad-Lindstedt radioenzymatic method [17]. The incubation of L-carnitine with [1-14 C]acetylcoenzyme A in the presence of carnitine acetyltransferase yields
[1-14 C]acetylcarnitine. An anion exchange resin column was used to retain the excess acetyl coenzyme A. The isotope content of the column effluent was determined by a liquid scintillation counter.
L-carnitine concentrations were calculated using a calibration curve obtained by the measurements in
various dilutions of a standard solution of L-carnitine.
B. Toptas et al. / L-carnitine deficiency and red blood cell mechanical impairment
351
2.3. Measurement of RBC thiobarbituric acid-reactive substances
The extent of lipid peroxidation of RBC was estimated by measuring thiobarbituric acid-reactive substance (TBARS) levels according to the method of Stocks and Dormandy [37]. TBARS levels were estimated by measuring absorbance at 532 nm after reaction with thiobarbituric acid. Trichloroacetic acid
extracts of RBC samples were used to avoid the interference of proteins with TBARS determinations.
Results were expressed as nanomoles per gram Hb.
2.4. Cytosolic calcium measurements
Cytosolic Ca2+ concentration in RBC was estimated using the method described by Cobbold and
Rink [13]. RBC were separated from plasma by centrifugation at 1400g, washed two times in HEPES
buffer (123 mM NaCl, 5 mM KCl, 1 mM MgCl2 , 1.3 mM CaCl2 , 10 mM glucose, 25 mM HEPES) and
then resuspended in HEPES buffer at a hematocrit of 1%. RBC suspension was incubated at 37◦ C, in the
presence of Fura-2AM (Sigma Chemical Company, St. Louis, MO, USA), at a concentration of 1 µM,
for 25 min. The suspension was then centrifuged at 350g for 5 min to eliminate extracellular Fura-2AM
and RBC were resuspended with 2 ml of HEPES buffer. The fluorescence spectrum was measured at an
excitation range of 335–385 nm, with an emission wavelength of 510 nm using a spectrofluorophotometer (Shimadzu RF-5000, Tokyo, Japan). The ratio between the fluorescence intensities of Fura 2-Ca2+
complex and the unchelated Fura 2 (F335/F385) reflects the cytosolic calcium concentrations.
2.5. RBC deformability measurements
RBC deformability was determined at various fluid shear stresses by laser diffraction analysis using
an ektacytometer (LORCA, RR Mechatronics, Hoorn, The Netherlands) [23]. Briefly, a low hematocrit
suspension of RBC in an isotonic viscous medium is sheared in a Couette system composed of a glass
cup and a precisely fitting bob, with a gap of 0.3 mm between the cylinders. A laser beam is directed
through the sheared sample and the diffraction pattern produced by the deformed cells is analyzed by
a microcomputer. Elongation indexes (EI) are calculated for shear rates between 0.5–50 pascal (Pa);
a higher EI indicates greater cell deformability. Additionally, shear stress for half-maximal deformation
(SS1/2 ) was calculated using this data set for each measurement, by using Lineweaver–Burk analysis as
described elsewhere [3]. Briefly, shear stress–EI curve was linearized by plotting the reciprocal of EI
as the function of the reciprocal of shear stress. The x-intercept of this curve corresponds to the negative reciprocal value of shear stress causing half-maximal deformation (SS1/2 ). A higher SS1/2 indicates
impaired RBC deformability.
2.6. Statistics
Results are expressed as mean ± standard error (SE). Statistical comparison of the EI–shear stress
curve was done by “two-way ANOVA” followed by “Bonferroni post test”. Other parameters were compared by “One-way ANOVA” followed by “Newman–Keuls Post Test”. Statistical significance was accepted at p values less than 0.05.
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3. Results
β-thalassemia major patients were characterized by lower RBC counts, hematocrit and hemoglobin
values compared to the control group. MCV and mean corpuscular hemoglobin (MCH) values were also
significantly lower than control values in these patients. None of the measured hematological parameters
showed a significant alteration after L-carnitine treatment in thalassemic patients (Table 1).
Plasma free L-carnitine concentration was 24.53 ± 4.23 µM/l in thalassemic patients before the treatment, being significantly lower (p < 0.001) than the control value of 43.33 ± 9.55 µM/l (Fig. 1).
L-carnitine concentration increased significantly to 36.69±7.78 µM/l after carnitine treatment (p < 0.01
compared to pre-treatment value).
RBC TBARS concentrations were found to be significantly higher (p < 0.001) in thalassemic patients compared to control (Fig. 2) and significantly diminished after L-carnitine treatment. However,
RBC TBARS concentrations were still significantly higher than the control value after L-carnitine treatment (Fig. 2). The same pattern of alterations was also observed for RBC cytosolic calcium concentration; being significantly higher in thalassemic patients in comparison with the control group (Fig. 3).
A significant decrement in calcium concentration was observed after L-carnitine treatment compared to
pre-treatment value, but calcium concentration was significantly higher than the control value even after
L-carnitine treatment.
RBC EI of thalassemic patients determined at shear stresses above 2.81 Pa before L-carnitine treatment
were found to be significantly impaired (Fig. 4). Deformability of RBC obtained from these patients
significantly enhanced after L-carnitine treatment; post-treatment EI were significantly lower only at
Table 1
Hematological parameters in control and thalassemic patient before and after L-carnitine treatment
Control
Pre-treatment
RBC (1012 /l)
4.58 ± 0.14
3.22 ± 0.14∗
Hematocrit (l/l)
0.39 ± 0.01
0.24 ± 0.01∗
Hemoglobin (g/l)
130.5 ± 3.0
81.3 ± 2.3∗
MCV (fl)
85.13 ± 1.15
76.05 ± 1.66∗
MCH (pg)
28.51 ± 0.92
25.64 ± 0.64∗
MCHC (g/l)
334.4 ± 6.2
336.9 ± 3.2
Values are mean ± SE. ∗ Difference from control; p < 0.001.
Post-treatment
3.13 ± 0.14∗
0.24 ± 0.01∗
78.5 ± 3.2∗
76.38 ± 1.33∗
25.26 ± 0.52∗
330.6 ± 3.6
Fig. 1. Plasma free L-carnitine concentration in control and thalassemic patients before and after L-carnitine treatment. Values
are presented as mean ± SE. ∗ Difference from Control, p < 0.001; † Difference from pre-treatment value, p < 0.01.
B. Toptas et al. / L-carnitine deficiency and red blood cell mechanical impairment
353
Fig. 2. RBC TBARS in control and thalassemic patients before and after L-carnitine treatment. Values are presented as
mean ± SE. ∗ Difference from Control, p < 0.001; † Difference from pre-treatment value, p < 0.001.
Fig. 3. RBC cytosolic calcium concentration in control and thalassemic patients before and after L-carnitine treatment expressed
as the ratio between the fluorescence intensities of Fura 2-Ca2+ complex and the unchelated Fura 2 (F335/F385). Values are
presented as mean ± SE. ∗ Difference from Control, p < 0.01; † Difference from pre-treatment value, p < 0.05.
Fig. 4. RBC elongation indexes (EI) measured at shear stresses between 0.5–50 pascal (Pa) in control and thalassemic patients before and after L-carnitine treatment. Values are presented as mean ± SE. Difference from pre-treatment; ∗ p < 0.05,
∗∗
p < 0.01, ∗∗∗ p < 0.001. Difference from post-treatment: † p < 0.05, †† p < 0.01.
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Fig. 5. Shear stress at half-maximal deformation (SS1/2 ) in control and thalassemic patients before and after L-carnitine treatment. Values are presented as mean ± SE. ∗ Difference from Control, p < 0.001; † Difference from pre-treatment value,
p < 0.05.
shear stresses above 5 Pa, and with smaller p values (Fig. 4). Higher SS1/2 values for thalassemic patients
(Fig. 5) also supported these findings. L-carnitine treatment reduced SS1/2 significantly in thalassemic
RBC; however it remained to be higher than the control value, even after the treatment period.
4. Discussion
An important finding of this study is the decreased plasma free L-carnitine concentration in
β-thalassemia major patients. This study confirms the recent report by Tsagris and Liapi-Adamidou [38]
and further demonstrates that L-carnitine supplementation may partly correct the deterioration in RBC
deformability in these patients as well as preventing oxidative damage in RBC.
The mechanism of L-carnitine deficiency in β-thalassemia major is not known. Tsagris and LiapiAdamidou suggested that this deficiency might be related to reduced hepatic synthesis of carnitine, but
did not provide evidence for this mechanism [38]. The present study also does not provide any evidence
for the mechanism of deficiency. Primary carnitine deficiency is not a common disorder but secondary
deficiency may more frequently occur due to decreased biosynthesis, increased catabolism as in case
of chronic diseases, or non-physiological loses as in case of dialysis [20]. Further studies are needed to
clarify the mechanism of L-carnitine deficiency in β-thalassemia major patients.
L-carnitine is an essential element of energy metabolism, taking part in the mitochondrial fatty acid
oxidation [42]. Therefore it is essential for intermediary metabolism of fatty acids [20]. Additionally,
L-carnitine have demonstrated effects on membrane stability and function that are not related to this
mitochondrial function [24], such as altered sodium-potassium pump activity and activation of enzymes
for lipid incorporation in RBC membranes [16,27]. Arduini et al. reported that L-carnitine increased
RBC membrane stability, probably by interacting membrane skeleton proteins [1]. L-carnitine also stabilized membrane fluidity alterations and morphological changes in RBC under various conditions [25].
These membrane stabilizing actions of L-carnitine may also contribute to the observed protective action
in β-thalassemic RBC. RBC mechanical properties are closely related to membrane properties and function [11,28,29] and these regulatory actions of L-carnitine may play role in maintaining normal RBC
deformability.
The protective effect of L-carnitine on RBC mechanical properties may also be related to its antioxidant action. Palmieri et al. reported that L-propionylcarnitine prevented oxidative stress in β-thalassemic
B. Toptas et al. / L-carnitine deficiency and red blood cell mechanical impairment
355
RBC in vitro, dose dependently [32], although the mode of action is not clear. L-carnitine has been reported to behave as a free radical scavenger, reducing lipid peroxidation in vivo or in vitro [2,7,40].
The results of this study also suggest such a relationship; L-carnitine supplementation decreased RBC
TBARS concentrations in β-thalassemia major patients, while preventing the impairment in RBC deformability in part.
Oxidative damage in RBC is one of the well established mechanisms of mechanical impairment [5].
RBC deformability is determined by cellular geometry, cytoplasmic viscosity of RBC (determined by
cytosolic hemoglobin concentration) and viscoelastic properties the RBC membrane [11]. Membrane
viscoelasticity is in turn determined by RBC membrane skeleton, which is mainly a spectrin network
attached to the integral proteins [9]. The contribution of lipid bilayer to the overall viscoelasticity is
negligible [9], however oxidative reactions that start in the lipid components (i.e., lipid peroxidation)
lead to the formation of cross-linkages within the membrane skeleton and/or hemoglobin and membrane
skeletal proteins [39], increasing membrane viscosity. Additionally, oxidative damage may affect the
transport processes through the RBC membrane, affecting the cell geometry and cytosolic viscosity,
accompanied by the alterations in the cytosolic concentration of cations. Calcium homeostasis is an
important determinant of normal RBC deformability; increased cytosolic calcium concentration leads
to impaired RBC deformability [21]. As it was shown in previous studies certain drugs and chemicals
such as cyclosporin A, nifedipine and cadmium may affect RBC deformability via intracellular calcium
alterations [4,6]. Therefore, both increased lipid peroxidation and cytosolic calcium concentration may
explain the impaired RBC deformability in β-thalassemia major patients.
Free radical scavenging action of L-carnitine may also be related to the mechanism of L-carnitine
deficiency in β-thalassemia major patients. It has been reported that plasma carnitine cannot be freely
exchanged with RBC [8,15] however, RBC carnitine was closely related to plasma carnitine levels [15].
It can be suggested that increased oxidant stress in thalassemic RBC leads to increased L-carnitine
turnover, resulting in decreased RBC and plasma L-carnitine concentrations.
It should be noted that the general hematological picture was not altered in β-thalassemia major patients after the L-carnitine treatment of one month duration; RBC counts, hemoglobin and hematocrit
values, MCV and MCH remained at pre-treatment levels, being significantly lower than the control group
(Table 1). However, RBC quality was improved after L-carnitine treatment. RBC lipid peroxidation and
cytosolic calcium concentrations were found to be decreased significantly, and RBC deformability was
improved. RBC deformability is an important determinant of blood rheology, either in bulk flow conditions or in microcirculation [10]. Normal RBC deformability is essential for proper tissue perfusion
and oxygenation [11], as well as the normal survival of RBC in the circulation [12]. Therefore, maintaining normal RBC mechanical properties should be an important objective in therapeutic approach to
hematological disorders including β-thalassemia major.
The significant role of L-carnitine in maintaining normal RBC physiology under various conditions
has been demonstrated previously [7,14,22,31,32]. This study provided additional evidence for the importance of “normal” L-carnitine level for maintaining normal RBC properties. The results of this study
suggest that L-carnitine supplementation can be used to improve RBC quality in β-thalassemia major
patients. Further studies are warranted for clarification of the mode of action of L-carnitine in RBC
pathophysiology.
Acknowledgement
This study was supported by Akdeniz University Research Projects Unit (Project No: 20.01.0103.21).
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