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Annals of Clinical & Laboratory Science 32:399-403 (2002)
© 2002 Association of Clinical Scientists

Immobilization-Induced Changes in Erythrocyte Membrane Fluidity in Rabbits: a Spin-Label Electron Spin Resonance Study

Ming Ju Liu1, Jing Xian Li2, Ling Qin1, Kwong Man Lee3 and Kai Ming Chan1
1 Department of Orthopaedics & Traumatology, 2 Department of Sports Science and Physical Education, 3 Lee Hysan Clinical Research Laboratory, The Chinese University of Hong Kong, Hong Kong, People’s Republic of China

Address correspondence to Ming Ju Liu, Ph.D., 5/F Clinical Science Building, Department of Orthopaedics and Traumatology, Chinese University of Hong Kong, Prince of Wales Hospital, Shantin, New Territories, Hong Kong, People’s Republic of China; tel 852 26 32 3311; fax 852 26 46 3020; e-mail liumingju{at}ort.cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been shown that many diseases are linked to abnormalities of the erythrocyte membrane. This study observed the changes in erythrocyte membrane fluidity during an immobilization period of 21 days. The right hindlimbs of male adult New Zealand white rabbits were immobilized for 21 days. Blood samples were collected and heparinized prior to immobilization and on days 1, 3, 7, 14, and 21 of immobilization. The membrane fluidity of erythrocytes was measured by spin-label electron spin resonance (ESR). The membrane fluidity was expressed by the value of order parameter (S). The results showed a significant increase of S on days 7 and 14 of immobilization (p <0.01). The highest value of S was found on day 7 of immobilization; thereafter, S gradually declined. Compared with the value measured before immobilization, there was no significant difference in the value of S on day 21. The results show that immobilization significantly affected erythrocyte membrane fluidity, indicating a systemic and temporal response of the immobilized body. This study is the first to document the time-course of changes in erythrocyte membrane fluidity in rabbits with single hindlimb immobilization during 21 days. These results may be helpful in studying the pathophysiology of immobilization.

(received 29 June 2002; accepted 8 July 2002)

Keywords: erythrocyte membrane fluidity, electron spin resonance, immobilization, oxidative stress


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immobilization is one of the most common clinical procedures in orthopaedics. There are obvious benefits of stabilizing an injured limb during the healing of a fracture or severe soft tissue injury. However, immobilization results in dysfunction of the limb and obvious atrophy of the affected muscle [1–3]. Recent studies that reported serum biochemical alterations, evidence of oxidative stress, and hematological changes in immobilized animals indicated that the systemic changes go far beyond the local response [4,5]. Studies on long-term bed rest revealed negative effects on most organs and systems, with the most significant changes in the locomotor and circulatory systems [6].

In muscle atrophy caused by immobilization in rats, Kondo et al [7,8] found significant increases in the ratio of oxidized glutathione (GSSG) to total glutathione and in the activities of CuZn-superoxide dismutase (CuZn-SOD), and xanthine oxidase (XOD). These findings suggested that the higher level of oxidative stress in immobilized tissue was related to muscle atrophy. Recently, significantly increased levels of thiobarbituric acid reactive substances (TBARS) in plasma and high antioxidant activities of catalase and superoxide dismutase in erythrocytes have been reported in rats that were immobilized for 6 hr [5]. The study showed a relationship between immobilization and elevated indices of oxidative stress in blood.

Erythrocytes are rich in antioxidants and enzymes, but the abundant polyunsaturated fatty acids in the erythrocyte membrane result in membrane being susceptible to oxidation by chain reaction. Lipid composition and lipid-protein interactions are known to be the major contributing factors to changes in membrane fluidity, which is an important factor in modulating cell functions, such as rheological behavior and membrane microviscosity [9].

Many studies have shown that disorders of various systems are linked to abnormalities of erythrocyte membrane fluidity [10–13]. However, to our knowledge, there has been no study of the effects of single hindlimb immobilization on erythrocyte membrane fluidity. Such a change in erythrocyte membrane fluidity might provide relevant information about systemic responses during immobilization of limbs.

The objective of this study was to investigate the changes in erythrocyte membrane fluidity at successive intervals of immobilization, using the spin-label electron spin resonance (ESR) method. This is a sensitive method of evaluating the physicochemical properties of cell membranes and their changes in disorders that are attended by disturbances of membrane fluidity [10,13–15].


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immobilization animal model.  Male adult New Zealand white rabbits (age 3 mo) with mean body weight of 2.5 kg (range, 2.35 – 2.65 kg) were used in this study. The animals were housed individually at 23°C and had food and water ad libitum. The light was on from 8:00 to 20:00. All experiments complied with regulations of the Animal Ethics Committee of our university.

The right hindlimb of each rabbit was immobilized using two splints, a non-adhesive bandage, and an adhesive elastic bandage, keeping the knee joint flexed at an angle of 90° and the ankle joint in fully extended position [16]. The immobilization devices were inspected daily and adjusted as required.

Sample preparation.  Blood samples were collected from a central ear artery and heparinized. The blood samples were obtained before immobilization and on days 1, 3, 7, 14, and 21 of immobilization. The plasma and buffy coat were carefully removed after the blood was centrifuged at 2000 x g for 3 min at 4°C. Washed erythrocytes were suspended in isotonic buffer (140 mM, NaCl, 20 mM Tris-HCl, pH 7.4) at a hematocrit of 50% [15]. The erythrocyte suspension was incubated in NaCl-Tris buffer for 30 min at 37°C before labeling.

Spin-label of erythrocytes.  Twenty µl of fatty acid spin label agent (5-nitroxide stearic acid [5-NS] Sigma, St. Louis, MO), diluted in NaCl-Tris buffer before the measurement (5 x10-5 M), was added to 100 µl of the erythrocyte suspension and shaken gently. After incubation for 1 hr at 37°C, the mixture was centrifuged at 3000 x g for 3 min. The deposit was washed 3 times with NaCl-Tris buffer to remove free spin label. ESR measurements were performed immediately [13,15].

ESR measurements.  Membrane fluidity of the labeled erythrocytes was measured using a Bruker ESP-300 ESR instrument. Measurement conditions were as follows: microwave power, 20 mw; modulation amplitude, 2.0 G; central magnetic field, 3490 G; sweep width, 200 G; band, X. The temperature was maintained at 25°C.

Fig. 1Go illustrates a typical ESR spectrum of erythrocytes labeled with 5-NS. Outer and inner hyperfine splitting (2T// and 2T{bot} in gauss, respectively) were evaluated in each ESR spectrum, and the order parameter (S) calculated from 2T// and 2T{bot} values according to the following formula: S = [(T// - T{bot})/(TZZ - TXX)] x (an/a'n), where TZZ, TXX = hyperfine constants, an/a'n = isotropic coupling constant [13]. The higher the value of S, the lesser freedom of motion of the spin-labels in the biomembrane bilayers, indicating lower membrane fluidity.



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  		Fig 1. Typical ESR spectrum of rabbit erythrocyte membrane labeled with 5-NS. The order parameter (S) was calculated by the equation described in the text. T// and T{bot} are parallel and perpendicular parts of hyperfine splitting (hfs), which correspond to one-half of the separation of the outer peaks and the inner peaks in the spectrum respectively.

 
Statistics.  All data are reported as the mean ± SD. Statistical analyses were performed by one-way repeated measures ANOVA. The Bonferroni procedure was used for pair-wise comparisons. Statistical significance was set at p <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As shown in Fig. 2Go, there was a significant increase in the value of order parameter (S) on days 7 and 14 of immobilization (p <0.01), indicating that erythrocyte membrane fluidity was decreased during immobilization. The highest value of S was found on day 7 of immobilization. Thereafter, the S value gradually decreased. Compared with the value before immobilization, there was no significant difference in S on day 21. The mean values of S obtained before immobilization and on days 1, 3, 7, 14, and 21 of immobilization were: 0.6719 ± 0.0093, 0.6748 ± 0.0123, 0.7067 ± 0.0169, 0.7331 ± 0.0077, 0.7145 ± 0.0042, and 0.6869 ± 0.0161, respectively.



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  		Fig 2. Changes in the order parameter (S) of the erythrocyte membrane before and during 21 days of single hindlimb immobilization. A higher S value indicates lower membrane fluidity. This value peaked on day 7 after immobilization (values are mean ± SD; n = 4). Significant differences are indicated as ** p < 0.01, versus the value before immobilization.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that hindlimb immobilization resulted in a decrease in erythrocyte membrane fluidity, indicating a systemic and temporal response to local immobilization. The results showed that the decrease in erythrocyte membrane fluidity was evident on the first day of immobilization, reached the lowest level on day 7 of immobilization, and then gradually underwent recovery. This is the first study to demonstrate the time-course of changes in erythrocyte membrane fluidity in rabbits during 21 days of single hindlimb immobilization.

Most previous studies focused on the morphological, biochemical, and metabolic changes in atrophic muscle. Recently, Kondo et al [7,8] found that the level of GSSG/T-GSH, and activities of CuZn-SOD and XOD increased significantly in the immobilized muscle, and proposed a mechanism of oxidative stress in atrophic muscle induced by immobilization. In studying patients during long-term bed-rest [6] and in animal with whole body immobilization for 6 hr [5], the authors found increased lipid peroxidation products in plasma and erythrocytes, which documented reactive oxidative stress in blood as a result of immobilization stress.

Cohen et al [4] observed alterations of serum total protein, albumin, and urea during single hindlimb immobilization in rabbits. The results showed that immobilization induced systemic changes beyond the local response. A study of the time-course of muscular atrophy during immobilization of hindlimbs in rats, conducted by Booth [17], showed that the time taken to decrease to one-half of the the new apparent steady state level was about 4–6 days for muscle mass and selected proteins. Studies of strength [2] and weight loss [1] indicated that major adaptations to immobilization took place during the first week. The time-course of changes in erythrocyte membrane fluidity in the present study are consistent with these reports.

The erythrocyte membrane is rich in polyunsaturated fatty acids; it also has an anion channel through which superoxide anions can enter. Erythrocytes also act as "sinks" for hydrogen peroxide and superoxide produced in plasma [18–20]. Although erythrocytes are resistant to oxidative damage owing to their efficient protective mechanisms, excessive reactive oxygen species can peroxidize the membrane lipid and disrupt the membrane structure, impairing its functions of defense and transport.

5-NS is a stearic acid analogue that contains a nitroxide radical ring at the 5th carbon position from the carboxyl group of the acyl-chain. Therefore, the order parameter (S) for 5-NS gives information regarding the hydrophilic region of the membrane. Lipid composition and lipid-protein interactions are known to be major contributing factors to changes in membrane fluidity [9,21]. Membrane fluidity is influenced by the composition of phospholipids, the content of cholesterol, and products of lipid peroxidation [11,21]. The present study suggests that hindlimb immobilization can affect the lipid composition, leading to an alteration of membrane fluidity, which may be related to the membrane-bound enzyme function.

Studies of various forms of muscular dystrophy revealed membrane defects manifested in erythrocytes as well as in muscle fibers [10,22]. Alterations in the biophysical and biochemical states of erythrocyte membranes in each disorder are specific to that disease state [22]. Using the spin-label ESR method, Butterfield et al [10] detected alterations of erythrocyte membrane fluidity in myotonic muscular dystrophy (MMD), Duchenne muscular dystrophy (DMD), and congenital myotonia (CM). The results showed that MMD erythrocyte membranes are more fluid near the membrane surface than those of normal controls. The data in CM indicated similarly high erythrocyte membrane fluidity, whereas erythrocytes from DMD had normal membrane fluidity. These results suggest a correlation of increased membrane fluidity with the presence of myotonia. In the present study, erythrocyte membrane fluidity was decreased after immobilization, which suggests that there may be related pathophysiological changes in red blood cells and in the atrophic muscles.

In summary, this study showed decreased erythrocyte membrane fluidity in hindlimb immobilized animals, suggesting that the abnormal membrane fluidity may be related to the pathophysiological changes in muscle that result from immobilization. The lowest value of erythrocyte membrane fluidity occurred on day 7 of immobilization, indicating that the most serious damage of tissues occurred during this period. The diminution of erythrocyte membrane fluidity persisted for at least one week. The possible relationship of the altered erythrocyte membrane fluidity and the atrophy of immobilized muscle fibers requires further study.


   Acknowledgments
 
Acknowledgments

This study was supported by RGC Earmarked Grant (CUHK 4344/99M), funded by the Hong Kong Government.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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