The relation between serum MDA and cystatin C levels in chronic spinal cord injury patients

Clinical Biochemistry 38 (2005) 1034 – 1037

The relation between serum MDA and cystatin C levels in chronic spinal cord injury patients ¨ ner-I˙yidog˘an a, Figen Gu¨rdo¨l a, Taner Koc¸ak b, Deniz Esin c Hikmet Koc¸ak a,*, YNldNz O a

Department of Biochemistry, Istanbul Faculty of Medicine, Istanbul University, C¸apa 34093, Istanbul, Turkey b Department of Urology, Istanbul Faculty of Medicine, Istanbul University, C¸apa 34093, Istanbul, Turkey c Taksim Training and Research Hospital, 2nd Division of Internal Medicine, Taksim 34433, Istanbul, Turkey Received 20 April 2005; received in revised form 29 July 2005; accepted 16 August 2005 Available online 9 September 2005

Abstract Objectives: The assessment of renal function is particularly important in patients with spinal cord injury (SCI). Creatinine (Cr) is known to be unsuitable as a marker of renal function in SCI because of muscle wasting. Although cystatin C (cys-C) is more reliable than Cr, its expression may be affected by oxidative stress accompanying SCI. The aim of the study was to estimate the cys-C levels in SCI patients with normal functioning kidneys. The prooxidant/antioxidant state in plasma of the patients and controls was measured, and any correlations between these parameters and cys-C were determined. Design and methods: Blood samples from 41 chronic SCI patients and 13 controls were obtained. Serum Cr was assayed by the Cobas Integra 400 autoanalyzer and cys-C by particle-enhanced immunoturbidimetry. Heparinized plasma was used for biochemical determinations of vitamin E, total antioxidant status (TAS) and malondialdehyde (MDA). Results: Cr, TAS and MDA levels were significantly lower in SCI patients compared to the controls ( P = 0.007, P = 0.019, P = 0.000, respectively), whereas no difference was seen in cys-C and vit E concentrations. Body mass indices (BMI) of SCI patients were less than those of the controls ( P = 0.03). No correlation existed between cys-C and MDA in SCI patients and controls. Cys-C levels were independent from the body mass indices of subjects. Conclusion: In our study, although BMI and MDA were both affected in SCI patients, cys-C levels were unchanged. Therefore, the measurement of cys-C appeared to be of value for the follow-up of renal function in SCI. The low MDA levels observed in these patients suggest that various adaptation mechanisms may be relevant for subjects undergoing prolonged stress situations. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Cystatin C; Malondialdehyde; Spinal cord injury

Introduction Cystatin C (cys-C), a non-glycosylated low molecular weight protein (MW 13,359 Da), is produced by all nucleated cells at a constant rate [1] and is freely filtered in the gromeruli and reabsorbed and catabolized in the proximal tubuli [2]. It is one of the most important extracellular inhibitors of cysteine proteases and plays a pivotal role in tissue remodeling together with the other protease inhibitors by regulating protease activities. Severely reduced cys-C levels in atherosclerotic and aneurysmal lesions in vascular smooth muscle cells * Corresponding author. Fax: +90 212 5494656. E-mail address: [email protected] (H. Koc¸ak).

suggest an increased activity of proteases due to lack of inhibition which might be causative for the disease [3]. In several studies, alterations in cys-C expression have been shown in response to oxidative stress and inflammation. In vitro, alveolar macrophages from smokers or monocytes stimulated by interferon-gamma (IFN-g) secreted less cys-C than unstimulated cells, raising the possibility of reduced cys-C levels at sites of inflammation [4,5]. In a recent study, cys-C was thought to have important roles in neuronal death, owing to its high rate of expression caused by reactive oxygen species (ROS) [6]. The involvement of oxidative stress in neuronal death has been shown in various conditions such as cerebrovascular injury, aging and Alzheimer’s disease [7 – 9]. Furthermore, the high ROS levels induced gene expression for some

0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.08.005

H. Koc¸ak et al. / Clinical Biochemistry 38 (2005) 1034 – 1037

antioxidant enzymes, and motoneuronal death has been found to be positively correlated along spinal cord development [10]. The preservation of renal function is the primary urological goal in managing patients with spinal cord injury (SCI) and neurogenic bladder dysfunction. The most widely accepted surveillance protocol of renal function in this population is the serial measurement of the serum creatinine (Cr) level. Although this value usually closely reflects creatinine clearance and is sufficient to monitor renal function in normal patients [11], it is often significantly decreased as a result of the diffuse muscle atrophy in patients with SCI [12]. As a result, the measured serum Cr level may be normal in these patients despite clinically significant reductions in GFR [13]. Being independent of gender, age and muscle mass [14], cys-C has been accepted as an accurate index for GFR and renal impairment [15 –17]. However, the presence of oxidative stress conditions in SCI patients may cause the increased levels of cys-C, thereby leading to inaccurate estimations of GFR. The primary aim of this study was to estimate whether serum cys-C levels are affected in SCI patients. We also wanted to investigate the prooxidant activity and antioxidant capacity in the plasma of patients and controls by estimating MDA, vitamin E and total antioxidant status (TAS) and to see if these parameters correlated with the cys-C levels. Methods The study population Forty-one SCI patients with normal renal functions were included in the study. The patients who have developed neurogenic bladder (n = 33) were routinely admitted to the Department of Urology in order to handle their bladder dysfunction. In these subjects, renal functions were evaluated periodically by renal scintigraphy and IVP (intravenous pyelography), while the remainder with a normal bladder were controlled with daily urine output and urinary analyses and with the addition of scintigraphy if necessary. When evaluating the IVPs of the SCI patients after the injection of the contrast material, the graphics obtained during the early phase of excretion showed homogeneous opacification of the renal parenchyma which can only be seen in normal functioning kidneys due to the collection of the excreted contrast material in the renal tubules. The contrast material started to fill the renal pelvis within 2 or 3 min being the other sign of a normally functioning kidney. In scintigraphical assessment of renal function using 99mTc-DTPA as the radionuclide agent, subjects were observed for at least 20 min, and all exhibited both adequate concentration in a normal period of time and an adequate excretion starting at the normal time. The control group consisted of thirteen healthy laboratory staff with a median age of 36.5 years (range 31– 55). None of the subjects took any drug or vitamin supplements. The SCI patients were aged between 18 and 60 years (median 34), and twelve of them either smoked and/or had alcoholic beverages in negligible amounts. The study protocol was approved by the

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Ethical Committee of the Istanbul Faculty of Medicine, and informed consent was obtained from all patients in accordance with the ethical standards set down in the 1964 Declaration of Helsinki. The causes of the SCI in our patients were: motor vehicle accident (18); fall from height (13); injuries due to earthquake (3); gun shot injury (5); and occupational injury (2). All SCI patients have been suffering for at least 1.5 years (1.5 –12 years since injury). Sample collection and processing In each patient, blood samples from the axillary vein were collected and used for biochemical measurements. The determinations of Cr and cys-C were carried out in sera collected in VacutainerR tubes. An aliquot of serum was stored at 35-C for the determination of cys-C. Cr was immediately studied by the Cobas Integra 400 autoanalyzer from Roche, with the reference intervals being 53.41 – 106.82 Amol/L. Serum cys-C was determined using a particle-enhanced immunoturbidimetric assay in a Hitachi 717 autoanalyzer (Hoffmann, La Roche) using reagents from Dako Diagnostics (Copenhagen, Denmark). The reference interval for the serum levels of cys-C in our laboratory was 0.63– 1.33 mg/L. Heparinized plasma was used for determinations of vitamin E, total antioxidant status (TAS) and malondialdehyde (MDA) and stored at 80-C until analyzed. Vitamin E was determined by the spectrophotometric method of Desai et al. [18], with the reference intervals being 12– 42 Amol/L. TAS was assayed by the enzymatic kit (Randox Laboratories, Antrim, UK) with reference values ranging from 1.28 to 1.83 mmol/L. Malondialdehyde was determined by the 2-thiobarbituric acid reactive substances (TBARS) method [19], in which mean value was reported as 1.3 T 0.23 Amol/L for healthy adults [20]. 1,1,3,3Tetraethoxypropane (Sigma, UK) was used as a standard. Statistical methods Data were presented as mean T SD, and comparisons were made by the independent sample t test. The correlations were calculated according to Pearson’s test. Statistical analyses were performed with the SPSS 10.0 for Windows statistical software (SPSS, Chicago, IL, USA). Results The concentrations of serum Cr, cys-C and plasma vitamin E, TAS and MDA levels are shown in Table 1. Cr, plasma TAS and MDA concentrations were significantly lower in SCI patients compared to the control group ( P = 0.007, P = 0.019 and P = 0.000; respectively), whereas there was no significant difference with regard to serum cys-C and plasma vit E concentrations between these groups. Body mass indices (BMI) of SCI patients were found to be significantly less than those of the controls ( P = 0.03). None of the oxidative stress parameters correlated with the plasma cys-C concentrations in either SCI patients or controls

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H. Koc¸ak et al. / Clinical Biochemistry 38 (2005) 1034 – 1037

Table 1 Comparison of biochemical parameters and BMI between SCI and control groups (mean T SD) Parameters

Control (n = 13)

Cr (Amol/L) Cys C (mg/L) MDA (Amol/L) TAS (mmol/L) Vit E (Amol/L) BMI (kg/m2)

67.81 0.73 1.58 1.15 33.87 26.01

SCI vs. a P< b P< c P< d P<

T T T T T T

8.3 0.20 0.50 0.13 5.56 2.9

SCI (n = 41) 53.76 0.68 0.79 0.96 32.02 23.16

T T T T T T

13.49a 0.23 0.35b 0.20c 9.05 3.5d

control group. 0.01. 0.001. 0.02. 0.05.

(data not shown). In order to see whether the BMI difference had any effect on plasma MDA, the SCI patients were divided into two subgroups according to their body mass indices (Table 2). Mean MDA values were similar in the subgroups either with BMI < 24 or BMI > 26. No correlation existed between serum cys-C and plasma MDA concentrations in each subgroup with different BMI values. Discussion Patients with SCI and neurogenic bladder dysfunction are at increased risk of developing renal insufficiency [13]. Among various methods, none is ideal for measuring GFR in these subjects and the best method to be used has not been determined yet. It was reported that serum Cr and the Cockcroft – Gault formula should not be used for the former is insensitive and the latter is inaccurate in identifying early deterioration of renal function [13]. In our study, since the SCI patients had significantly low levels of serum Cr, this parameter appeared to be unsuitable as a marker of renal function in the advanced period of SCI due to muscle wasting, as suggested previously [12]. Cys-C, another marker for GFR, has also previously been investigated in patients with SCI, and data from these studies have revealed that it is more accurate than Cr to estimate renal function as it is independent of the degree of muscle atrophy [14,21]. In recent reports, however, certain pathological conditions such as neuronal degeneration and oxidative stress have been shown to influence the expression of cys-C [6,22]. Evidence from in vivo and in vitro studies indicates that both of these factors are related to the spinal cord injury as either a cause or a consequence. Wozniak et al. observed increased free radical generation and decreased antioxidant capacity in the plasma of patients with cervical spinal cord injury [23]. In vitro experiments revealed that high ROS levels participate in the cell death during spinal cord development. Furthermore, gene expression for some antioxidant enzymes and motoneuronal death were found to be positively correlated along spinal cord development [10]. Cellular injury originated from ROS has been implicated in many muscle disorders [24]. An increment in activities of the antioxidant enzymes in muscle cells during the oxidative

challenges has implied that muscle cells regulate antioxidant defenses in response to oxidative stress [25]. It was shown that varying degrees of muscle atrophy exist in spinal cord injury. Therefore, it was thought that in SCI patients the low muscle mass may indirectly contribute to the oxidative-stress-induced cell damage and alteration in cys-C expression. In our study, SCI patients were free of any change in cys-C levels despite their low BMI and MDA. Therefore, the stability of cys-C seems to be relevant in patients with SCI, thus making its usage reliable for early detection of renal impairment. Both their Cr and BMI values have revealed that these subjects had a considerable degree of muscle atrophy. The degree of Cr decrement was more pronounced in men, indicating that low Cr levels are due to muscle wastage, since muscle mass contribution to body mass in men is relatively higher than women. Interestingly, our spinal cord patients had significantly lower MDA levels than those obtained from the healthy controls. Abovementioned studies emphasizing the involvement of oxidative stress in spinal cord injury have been carried out during the early phase of SCI. In contrast, our study group consisted of patients with SCI lasting from 1.5 to 12 years. We hypothesized that low levels of MDA in these patients might be related to the caloric restriction owing to diminished physical activity. Caloric restriction is commonly suggested to patients with restricted physical activity for minimizing the negative conditions due to a long-lasting sedentary life. In a recent study, the effect of weight reduction on free radical generation was investigated, and plasma MDA levels were found significantly reduced by weight loss and correlated with the BMI [24]. The relatively low body mass indices observed in our subjects seemed to support this report. However, when the patients were grouped according to their BMI (<24 vs. >26); MDA levels in each group neither differed nor correlated with the BMI of subjects. Caloric restriction has been shown to be stressful to animals due to metabolic substrate deprivation [26,27] and is used as a tool to examine the biological effects of stress in experimental studies. When animals were subjected to a long-term caloric restriction plus repeated immobilization stress, no additional effects on the characteristic biological responses were observed. Thus, it was thought that animals adapt more rapidly to simultaneous administration of two stressors as opposed to a single stress regimen [28]. The patients with spinal cord injury may particularly be considered as a population who are under prolonged effects of multiple stressors. The low MDA levels observed in these patients suggest that various adaptation Table 2 Comparison of MDA concentrations between SCI patients with lower and higher BMI (mean T SD) na

MDA (Amol/L) Range BMI (kg/m2) a b

BMI < 24 kg/m2

BMI > 26 kg/m2

24

11 b

0.79 T 0.35 0.29 – 1.45 20.88 T 2.04

0.87 T 0.44 0.27 – 1.44 27.83 T 1.16

Subjects with BMI values between 24 and 26 kg/m2 are not included. P > 0.05 (n.s.).

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