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Acta Haematologica Polonica 2008, 39, Nr 3, str. 461–467
PRACA ORYGINALNA – Original Article
KAROLINA STACHURA1, AGATA PIETRZYCKA2, ANNA MARCHEWKA3,
ANETA TELEGŁÓW3, MACIEJ DROśDś4, MAREK STĘPNIEWSKI2, HENRYK MARONA1
Red blood cells deformability, aggregation parameters and
plasma total antioxidant activity in blood of patients before
and after hemodialysis
Ocena odkształcalności i agregacji krwinek czerwonych oraz całkowitej zdolności antyoksydacyjnej osocza u pacjentów przed i po
hemodializie
1
Katedra Technologii i Biotechnologii Środków Leczniczych, Uniwersytet Jagielloński Collegium
Medicum, Kraków
Kierownik: Prof. dr hab. K. Kieć- Kononowicz
2
Samodzielna Pracownia Radioligandów, Uniwersytet Jagielloński Collegium Medicum, Kraków
3
Katedra Rehabilitacji Klinicznej, Akademia Wychowania Fizycznego, Kraków
4
Klinika Nefrologii, Uniwersytet Jagielloński Collegium Medicum, Szpital Uniwersytecki, Kraków
SUMMARY
The study presents red blood cells (RBC) aggregability and deformability as well as total antioxidant capacity, expressed as FRAP value in patients with chronic renal failure (CRF). 17
hemodialysis patients (parameters were measured both before and after hemodialysis) and 13
control subjects were studied. Erythrocyte elongation and aggregation were measured using Laser-Assisted Optical Rotational Cell Analyser (LORCA). FRAP was analyzed by Benzi’s
method. The study found that CRF patients had alterations in the kinetics of RBC aggregation,
which was subsequently further decreased by hemodialysis session. RBC aggregates form
rouleau at a shorter time, after their disaggregation, compared to control subjects and their spontaneous ability to aggregate is also significantly higher. The measurement of FRAP value has
shown that total antioxidant capacity is higher in patients before hemodialysis than in control
subjects. Additionally, hemodialysis treatment resulted in a decrease in FRAP values. In conclusion, CRF may be associated with various RBC rheological disturbances and pro/antioxidant imbalance. These results may suggest the necessity of introducing treatment forms to correct erythrocyte rheological properties as well as antioxidant status, which may improve the blood-flow
condition in the microcirculation and prevent posthemodialysis complications.
KEY WORDS: Hemodialysis – Erythrocytes – Deformability – Aggregation – FRAP
STRESZCZENIE
Celem pracy była ocena agregacji i odkształcalności krwinek czerwonych oraz całkowitej zdolności antyoksydacyjnej osocza wyraŜonej jako wartość FRAP u pacjentów z przewlekłą niewydolnością nerek (CRF). Badaniem objęto 17 hemodializowanych (ocena parametrów dotyczyła
462 K. STACHURA et al.
stanu przed oraz po hemodializie), grupa kontrolna liczyła 13 osób. Pomiaru agregacji oraz odkształcalności krwinek czerwonych dokonano przy uŜyciu analizatora LORCA (Laser-Assisted
Optical Rotational Cell Analyser). Oznaczenia wartości FRAP dokonano według Benzie. W wyniku przeprowadzonych badań stwierdzono istotne zmiany w kinetyce agregacji krwinek czerwonych pacjentów z CRF, zmiany te nasilały się w następstwie hemodializy. W grupie hemodializowanych zaobserwowano zwiększoną tendencję do tworzenia stabilnych agregatów krwinkowych. Ponadto wykazano istotny wzrost wartości FRAP we krwi pacjentów przed hemodializą w
stosunku do grupy kontrolnej. W następstwie hemodializy doszło do spadku wartości FRAP.
Uzyskane wyniki wskazują, iŜ CRF moŜe przebiegać z zaburzeniami reologicznymi krwinek
czerwonych oraz zmianami w zakresie równowagi antyoksydacyjno/prooksydacyjnej organizmu.
MoŜe to sugerować konieczność wprowadzenia odpowiedniej terapii, której celem będzie poprawa w/w zaburzeń.
SŁOWA KLUCZOWE: Hemodializa – Erytrocyty – Odkształcalność – Agregacja – FRAP
INTRODUCTION
Chronic renal failure (CRF) is connected with various adverse complications and
dialysis-related pathologies. Some of them, including anemia and accelerated atherosclerosis may partly result from Reactive Oxygen Species (ROS) action, as patients
suffering from CRF are constantly exposed to oxidative stress (1, 2). Chronic inflammatory state, high homocysteine concentration and parenteral iron administration are
considered to be the main sources of ROS in patients with renal insufficiency. Moreover, contact of the blood both with bioincompatible membranes of the dialysis system
as well as endotoxin-contaminated dialysate and the loss of some hydrophilic unbound
small-molecular-weight antioxidants during hemodialysis session aggravate the
prooxidant status of uremic patients (3, 4).
As ROS are capable of reacting with most cellular macromolecules, they elicit
various unfavourable changes in red blood cells (RBC), such as augmented membrane
lipid peroxidation and subsequent alteration in membrane deformability. Such changes
may severely influence RBC ability to deform and aggregate and consequently disturb
their passage through microcirculation (5). If damaged during their flow through the
microcirculation, RBC may have decreased survival. Thus, they may contribute to
anemia as well as increased splenic hemolysis in uremic patients (6).
As previous studies in hemodialysis population have led to conflicting results (7),
the purpose of the present study was to determine rheological properties of RBC (deformability and aggregation) as well as total antioxidant capacity, expressed as the
ferric reducing ability of plasma (FRAP) of our hemodialytic population and compare
them with a control group.
MATERIALS AND METHODS
Patients
The study was carried out on 17 hemodialysis patients with end-stage renal disease
(parameters were measured twice for hemodialysed patients: before and after hemodialysis) and 13 control subjects (matched for age and gender). Venous blood samples
Red blood cells deformability
463
were collected in standard sterile polystyrene vacuum tubes containing EDTA as an
anticoagulant. Hemodialysed patients were dialysed in the Department of Nephrology,
University Hospital, Jagiellonian University in Krakow. They were typically dialysed
for four hours, attending three sessions a week, using low-flux polysulfone membranes.
In order to measure RBC deformability 25 µl of blood was diluted 200 times with
0.14 mM polivinylopyrrolidone (PVP, M = 360 000, Sigma) in phosphate-buffered
saline (PBS, viscosity at 37oC up to 31 mPa x s). Aggregation measurement was performed on a sample of 1–2 ml blood. Complete blood was oxygenated for 10–15 minutes before the measurement through the slow rotation in a glass vessel.
Whole blood was centrifugated (2400 × g for 15 min.), then the plasma was carefully separated for FRAP determination.
Instruments and methods
Hemorheological parameters were performed with Laser-Assisted Optical Rotational Cell Analyzer (LORCA, Mechatronics Instruments, The Netherlands) (8).
Changes in RBC deformability were analyzed by the measurement of the elongation of the cells at a range of shear stresses from 0.30 Pa to 59.97 Pa. Finally, the deformation curve was obtained by plotting the calculated values for the elongation index
versus the corresponding shear stress [Pa].
The aggregation measurement is based on the detection of laser back-scattering
from the sheared (disaggregated) to unsheared (aggregated) blood. Back-scattering data
are evaluated by a computer and aggregation index (AI), aggregation half-time (t ½),
the amplitude of aggregation (AMP) and threshold shear stress (Ythr) are calculated on
the basis that there is less light back-scattered from aggregating red cells. Other parameters used in the study are: the intensity of light scattering in the prior disaggregation (Isc disc), time when cells are round and not aggregated (Isc top), and the difference between Isc top and Isc disc (upstroke value).
Total antioxidant capacity, expressed as FRAP value, was analyzed by Benzi’s
method (9). FRAP values were presented as µmol per g of protein. Protein concentration of plasma samples was measured by the method of Bradford (10).
Statistics
The statistical package “Statistics for Windows” (Statsoft, Poland, version 6.0) was
used. p values ≤ 0.05 were accepted as statistically significant.
RESULTS
On the basis of EI values study a slight decrease in RBC deformability was found
in the blood taken from patients before hemodialysis for shear stress values 0.30, 0.58
and 1.13 Pa as compared to the control group (Table 1).
464 K. STACHURA et al.
Table 1. Descriptive statistics of rheological parameters in study groups: control [C], before dialysis [B]
and after dialysis [A]
Tabela 1. Parametry reologiczne krwinek czerwonych w grupach badanych: kontrola [C] przed dializą
[B], po dializie [A]
Median
C
EI 0.30 [Pa]
EI 0.58 [Pa]
EI 1.13 [Pa]
Isc disc [au]
Isc top [au]
Upstroke [au]
AMP [au]
Ythr [1/s]
5th
Perc.
C
95th
Perc.
C
–1.00
1.10
2.80
33.40
40.73
4.49
22.40
125.00
3.70
4.50
11.60
53.48
59.19
8.49
31.86
400.00
0.40
2.40
8.50
45.78
51.31
5.99
27.72
350.00
Median
B
–0.80
1.00
7.40
37.58
41.81
4.03
22.84
600.00
5th
Perc.
B
95th
Perc.
B
–13.00
–0.20
5.60
20.95
26.11
1.91
12.38
125.00
2.30
2.80
8.70
50.88
56.07
9.17
31.56
800.00
Median
A
–0.70
1.40
7.40
37.96
44.15
4.19
23.25
600.00
5th
Perc.
A
95th
Perc.
A
Significant
differences
at p ≤ 0.05
–3.00
0.00
6.60
14.47
16.29
1.82
12.04
200.00
3.30
2.90
9.40
51.40
58.56
10.31
34.13
800.00
(1)
(1), (2)
(1)
(1), (2)
(1), (2)
(1)
(1), (3)
(1), (2)
Notes: (1) – differences between median in group C v. B; (2) – differences between median in group C v.
A; (3) – differences between median in group B v. A.
Uwagi: (1) – róŜnice pomiędzy medianami w grupach C v. B; (2) – róŜnice pomiędzy medianami w grupach C v. A; (3) – róŜnice pomiędzy medianami w grupach B v. A.
When analyzing aggregation parameters, a significant increase was found in aggregation index (AI) between the blood taken from posthemodialysed patients and control
blood samples (Fig. 1A). Aggregation half time (t1/2) was higher in control group than
in hemodialysed patients (both before and after hemodialysis) (Fig. 1B).
[A]
[B]
Fig. 1. Comparison of aggregation index (AI) [A] and aggregation half time (t1/2) [B] in control group and
patients before and after hemodialysis
Ryc. 1. Porównanie wartości indeksu agregacyjnego (AI) [A] oraz czasu potrzebnego do osiągnięcia
połowy agregacji (t1/2) [B] w grupie kontrolnej oraz u pacjentów przed i po hemodializie
Red blood cells deformability
465
A significant rise was found in the Ythr value in patients both before and after
hemodialysis when compared with control samples. At the same time a decline was
found in the values of AMP, Isc disc and Isc top. For the upstroke values the decrease
was significant only between the blood taken from control group and prehemodialysed
patients (Table 1).
FRAP measurement has shown that total antioxidant capacity is significantly
higher in patients before hemodialysis than in control group. Additionally, hemodialysis treatment resulted in a decrease in FRAP values, the differences between prehemodialysed and posthemodialysed blood were also significant (Fig. 2).
Fig. 2. Comparison of FRAP values in control group and patients before and after hemodialysis
Ryc. 2. Porównanie wartości FRAP w grupie kontrolnej oraz u pacjentów przed i po hemodializie
DISCUSSION
In the present study we observed a slight decrease in RBC deformability in the
blood taken from patients before hemodialysis for the lowest shear stress values (i.e.
0.30, 0.58 and 1.13 Pa) as compared to the control group. According to the results obtained, erythrocyte deformability did not seem to be much altered both before and
after hemodialysis treatment, which was also stated formerly by other researchers (11).
However, similar studies demonstrated a significant increase in RBC deformability in
patients before hemodialysis for shear stress value 30.00 Pa as compared to control
subjects (12). Additionally, in the same study they did not observe any changes in this
parameter in RBC coming from posthemodialysed group. Other authors applying filtra-
466 K. STACHURA et al.
tion techniques to investigate RBC deformability found reduced erythrocyte deformability with respect to controls (13).
Some authors revealed erythrocyte hyperaggregation before hemodialysis, which
was subsequently increased during hemodialysis in comparison to the control group
(14). These results are in good agreement with our data. Our study found that CRF
patients have alterations in the kinetics of RBC aggregation expressed by shortened
aggregation half-time, which was subsequently further decreased by hemodialysis session. This indicates that RBC aggregates form rouleau at a shorter time, after their disaggregation, compared to control subjects and their spontaneous ability to aggregate is
also significantly higher. As upstroke value is regarded as the measure of elasticity of
RBC, we noted that RBC taken from prehemodialysed patients are more rigid than
those in the control group. Thus, CRF affects the elasticity of erythrocytes making
them more rigid. A significant rise in the threshold shear stress value between the control group and patients both before and after hemodialysis signifies an increased tendency towards the formation of aggregates and of their stability. As aggregation half
time was higher in control subjects than in hemodialysed patients we can state the faster rate of the aggregation process in CRF patients.
Oxidative stress has been reported in patients with CRF treated with hemodialysis
(15). Earlier studies had indicated defective antioxidant enzyme activities in End Stage
Renal Disease (ESRD) patients undergoing hemodialysis and in predialysis uremic
patients (3, 16). We found an increase in FRAP values in those prehemodialysed versus controls. These results indicate that regardless of the factors involved, the antioxidant ability of plasma is improved in this group of patients in response to numerous
sources of ROS they are subjected to. According to further observations, hemodialysis
resulted in a decline in FRAP values. Such a decline may be associated with the loss of
some small-molecular-weight antioxidants such as uric acid and bilirubin during
hemodialysis session, both of which considerably contribute to FRAP value.
The analyses carried out immediately after hemodialysis showed in general that the
rheological and antioxidant profiles were deteriorated during the treatment. The rheological and antioxidant alterations observed in patients with CRF may be associated with
the development of various cardiovascular disorders, which include among others:
atherosclerosis, impaired erythrocyte deformability, increased hemolysis, platelet dysfunction and arthropathy. Today there is no doubt that the correction of the
pro/antioxidant imbalance and improvement in RBC rheology in patients with chronic
renal failure is an important approach for the reduction of the risk of these patients to
develop cardiovascular complications.
Acknowledgements
We wish to thank Professor Zbigniew Dąbrowski from Institute of Zoology, Jagiellonian University, Laboratory of Experimental Hematology for his helpful comments
and advice.
Red blood cells deformability
467
Technical assistance from Wanda Jabłońska from Radioligand Laboratory, Jagiellonian University Medical College, Faculty of Pharmacy for the execution of the
measurements is greatly appreciated.
This work was supported by the Jagiellonian University Programs No.
501/P/203/F and No. 501/P/191/F.
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Received: 25.02.2008 r. and accepted: 25.04.2008 r.
Adres Autora:
Dr hab. Henryk Marona
Katedra Technologii i Biotechnologii Środków Leczniczych UJCM
ul. Medyczna 9, 30-688 Kraków