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Malondialdehyde (MDA) and protein carbonyl (PCO) levels as biomarkers of oxidative stress in subjects with familial hypercholesterolemia
Clinical Biochemistry 43 (2010) 1220–1224
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Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m
Malondialdehyde (MDA) and protein carbonyl (PCO) levels as biomarkers of oxidative stress in subjects with familial hypercholesterolemia Ayfer Gözü Pirinccioglu a,⁎, Deniz Gökalp b, Mihdiye Pirinccioglu c, Göksel Kizil c, Murat Kizil c a b c
Department of Pediatrics, Faculty of Medicine, University of Dicle, 21280, Diyarbakir, Turkey Department of Endocrinology, Faculty of Medicine, University of Dicle, 21280, Diyarbakir, Turkey Department of Chemistry, Faculty of Science, University of Dicle, 21280, Diyarbakir, Turkey
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Article history: Received 30 April 2010 Received in revised form 16 July 2010 Accepted 17 July 2010 Available online 4 August 2010 Keywords: Hypercholesterolemia Familial Oxidative stress Lipid peroxidation Malondialdehyde Protein carbonylation Atherosclerosis
a b s t r a c t Objective: Familial hypercholesterolemia (FH) is clinically characterized by elevated total and lowdensity lipoprotein (LDL) cholesterol levels in plasma, which has high risk for developing atherosclerosis. Increased oxidative stress (OS) and FH have been related to atherosclerosis. The study aims to evaluate oxidative stress in patients with hypercholesterolemia by measuring lipid peroxidation and protein carbonyl (PCO) levels in these patients. PCO in these patients may provide a new diagnostic biomarker for oxidative damage in atherosclerosis. Design and method: Total cholesterol (Tc), low-density lipoprotein-cholesterol (LDL-c), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-c), lipoprotein(a) (Lp-a) levels and carotid intima-media thickness were measured to evaluate characteristics of patients (11 homozygous and 25 heterozygous) with FH. 25 age–gender–BMI matched healthy control subjects were included in the study for comparison. Results: MDA and PCO levels were significantly higher in homozygous patients compared with those of heterozygous and controls and it was found that they are positively correlated with LDL-c, Tc, Lp-a and IMT while negatively correlated with HDL-c. The heterozygous group also had significantly higher MDA and PCO levels compared with controls. Conclusion: The data obtained could be important for understanding the alterations presented by FH and could be related to their increased risk of developing atherosclerosis. To our knowledge, measurements of PCO in patients with FH are not recorded before and this may be used as a biomarker for protein oxidation, which may play a role in the increased cardiovascular risk of patients with FH. © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Familial hypercholesterolemia (FH), an autosomal dominant disorder characterized by defects in the low density lipoprotein (LDL) receptor, is associated with a markedly increased risk of developing premature coronary heart disease [1,2]. There are two forms of FH, heterozygous and homozygous. The prevalence of the heterozygous FH is about 1/500 in the general population whereas the homozygous FH is very rare and found about 1 in 1 million people characterized by markedly increased low-density lipoprotein-cholesterol (LDL-c) levels and early onset of atherosclerosis [3,4]. The genetic basis of FH is the lack of functional receptors for LDL on the cell surface in liver and peripheral tissue [3]. As a result, plasma LDL concentrations are elevated and its plasma half-life prolonged, possibly leading to increased susceptibility to free radical attack and oxidation. Endothelial cells, smooth muscle cells, neutrophils and monocytes all have the potential to oxidatively modify LDL, leading to ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (A.G. Pirinccioglu).
the generation of lipid peroxidation products and reactive oxygen species, which is responsible for oxidative stress involved in degenerative disease, including atherosclerosis [5,6]. This can be measured by monitoring the changes in blood malondialdehyde (MDA) and carbonyl content. Determination of carbonyl level is used as an index of the extent of the oxidative damage of protein while malondialdehyde level is a marker of lipid oxidation. Antioxidants work together in human blood cells against toxic reactive oxygen species [7–9]. Reactive oxygen species (ROS) cause lipid peroxidation and oxidation of some specific proteins, thus affecting many intra- and intercellular systems [10]. Some ROSinduced protein modifications can result in unfolding or alteration of protein structure, and some are essentially harmless events. Irreversible protein modifications can lead to inactivation of various proteins and could have lasting detrimental cellular effects [11]. Many different types of protein oxidative modification can be induced by ROS. Carbonylation is an irreversible, non-enzymatic modification of proteins. Carbonyl groups are introduced into proteins by a variety of oxidative pathways. ROS can react directly with the protein or they can react with molecules such as sugars and lipids, generating products (reactive carbonyl species) that then react with protein and
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lead to the formation of protein carbonyl derivatives (aldehydes and ketones) [12,13]. Studies of the formation of protein carbonyls cannot differentiate between those produced through direct protein oxidation and those formed by the addition of previously oxidized molecules, and hence protein carbonyls must be considered as a broad marker of oxidation [12,13]. The present study aims to investigate the status of lipid peroxidation and protein oxidative damage in subjects with FH by measuring serum malondialdehyde and carbonyl levels. Our study is the first to examine protein carbonyl levels in serum of FH patients and to analyse the correlations between protein carbonyl and MDA levels in atherosclerosis in the FH patients. Materials and methods Patients Eleven patients with homozygous FH (6 women and 5 men) with a mean age of 18 ± 6 years (7 children with ages of 8–20 years and 4 adults with ages of 21–28) and a body mass index (BMI) of 21± 2 kg/m2 and twenty five patients with heterozygous FH (13 women and 12 men) with a mean age of 18 ± 6 years (17 children with ages of 9–20 years and 8 adults ages of 21–29 years) and a body mass index (BMI) (19 ± 2) kg/m2 as well as twenty five age–gender–BMI matched healthy control subjects with a mean age (19 ± 6) years (16 children with ages of 10–20 and 9 adults with ages of 21–30) and a body mass index (BMI) (20 ± 3) kg/m2 were included in the present study. The study was conducted between 2007 and 2009 at the Department of Paediatrics and Department of Endocrinology, University of Dicle. All subjects and their parents gave informed consent prior to study entry and the study was conducted in accordance with the Declaration of Helsinki. The patients were diagnosed as FH based on the following criteria. (i) Cholesterol above 290 mg/dL or low-density lipoprotein-cholesterol (LDL-c) above 190 mg/dL. (ii) The presence of tendon and cutaneous xanthomas at an early age. (iii) An autosomal inheritance mode of hypercholesterolemia in the relatives. (iv) And the presence of primary hypercholesterolemia in the parents of index case. Differential diagnosis of homozygous and heterozygous FH patients were made as follows: patients (children and adults) with severely elevated cholesterol levels were assigned homozygous FH, with total cholesterol (Tc) and LDL-c levels greater than 600 mg/dL and triglyceride levels within the reference range. The assignment of heterozygous FH was made based on the following criteria: an LDL-c level higher than 200 mg/dL in patients younger than 20 years and higher than 290–300 mg/dL in adults. Families were thoroughly examined, particularly for plasma lipids and lipoproteins. Controls consisted of apparently healthy subjects admitted to the outpatient clinics of our institution who had not received any specific diagnosis. Diabetes, hypertension, obesity, and cigarette smoking were exclusion criteria in our study. Patients and controls underwent anthropometric and additionally, in order to exclude Apo B100 mutation, the absence of APOB3500 mutation (R3500Q) was observed using PCR. All measurements were done at the time of the first admission while none of the patients were receiving aphaeresis. Medical therapy (statins, ezetimibe and cholestyramine) was initiated in all patients and 7 of them were also treated with LDL aphaeresis twice a month. Anthropometric evaluation Body weight was measured with light clothing, in kilograms. Height was measured without shoes in centimetres. BMI was expressed as weight (in kilograms) per square of height (in meters). Maximum abdominal girth (in cm) was taken as the waist circumference. Waist circumference was considered as a measure of body fat distribution. All systolic and diastolic blood pressures were measured by the same person using a sphygmomanometer (ERKA® sphygmomanometer,
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İstanbul, Turkey) from the right arm of the subject in a relaxed sitting position. The average of 3 consecutive measurements was used for analysis. Hypertension was defined as a systolic blood pressure higher than 140 mm Hg or a diastolic blood pressure higher than 90 mm Hg for adults. The blood pressure percentile values in patients under 16 were evaluated in terms of their age, gender and height. Measurement of carotid intima-media thickness (IMT) The carotid artery ultrasonographic scans were obtained by A Toshiba SSH-140A ultrasound 7.5 MHz linear probe. The evaluation of the right and left common carotid arteries was 1 cm proximal to the carotid bulb. In each examination, the same operator used different scanning angles to identify the greatest IMT, defined as the distance between the junction of the lumen and intima and that of the media and adventitia. Three measurements of IMT were obtained from the right and left carotid arteries, respectively, and were averaged to determine the mean IMT for both sides combined. The coefficient of variation was 2.6%. Biochemical analysis Blood serum samples were taken from patients and control after overnight fasting. Serum total cholesterol (Tc), high-density lipoprotein-cholesterol (HDL-c) and triglyceride (TG) levels were measured with an autoanalyzer (Abott aeroset autoanalyzer, Toshiba, Japan). LDL-c levels were calculated by using the Friedewald equation [14]. Serum lipoprotein(a) (Lp-a) levels were measured by immunometric assay (Diagnostic Products Corporation 5700, Los Angeles, CA, USA). Presence of Apo B100 mutation was examined by using a Real Time PCR (Roche-Light-cycler) device with Lightcycler-ApoB mutation detection (codon 3500) kit. Protein determination The protein content in serum was measured by method of Lowry et al. with bovine serum albumin as the standard [15]. Lipid peroxidation (MDA) assay Lipid peroxidation in serum was evaluated by the spectrophotometric method based on the reaction between MDA and thiobarbituric acid [16]. Briefly, 0.5 mL each of plasma was mixed with 2.5 mL of 20% (v/v) trichloroacetic acid (TCA) (Sigma, Germany) and centrifuged at 3000 rpm for 10 min. Then, 3 mL of 0.2 g/dL TBA (Sigma, Germany) was added to the supernatant. The mixture was heated in boiling water for 30 min. After cooling on ice, the resulting chromogen was extracted with 4 mL of n-butyl alcohol. The organic phase was separated by centrifugation at 3000 rpm for 10 min and absorbance was recorded at wavelength of 530 nm. MDA solution was made freshly by the hydrolysis of 1,1,3,3-tetramethoxypropane (TMP) (Sigma, Germany) used as the standard. The results are expressed as the nmol MDA/mL serum. Measurement of protein carbonyls Determination of carbonyl content in oxidatively modified proteins was followed by the method of Levine et al. [17].The oxidative damage to proteins was measured by the quantification of carbonyl groups based on their reaction with 2,4-dinitro-phenylhydrazine (DNPH) to form hydrazones. Briefly, 0.1 mL of serum was incubated with 1.0 mL of 20 mM DNPH solution for 60 min. In brief, proteins were precipitated by the addition of 20% (v/v) trichloroacetic acid and re-dissolved in DNPH. Then the proteins were precipitated from the solution with the use of 20% (w/v) trichloroacetate; the protein pellet was washed three times with ethanol and ethyl acetate, and re-suspended in 1 mL of 6 M guanidine. The absorbance was read at 370 nm.
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Table 1 Clinical details and serum profiles in patients (A = homozygous, B = heterozygous) and controls (C).a Parameters
A(n = 11)
B(n = 25)
C(n = 25)
p (ANOVA)
Age (year) Gender (F/M) BMI (kg/m2) Tc (mg/dL) LDL-c (mg/dL) HDL-c (mg/dL) TG (mg/dL) Lp-a (mg/dL) IMT (mm)
18.12 ± 7.24 6/5 20.94 ± 2.27 713 ± 68 650 ± 67 26.78 ± 6.20 184 ± 26 73.31 ± 5.33 1.67 ± 0.48
18.44 ± 7.89 13/12 19.32 ± 2.42 363 ± 76 296 ± 70 31.32 ± 6.12 180 ± 20 51.28 ± 4.64 1.02 ± 0.22
18.75 ± 6.65 12/13 19.87 ± 2.84 229 ± 38 143 ± 33 51.60 ± 6.20 174 ± 19 26.65 ± 2.56 0.54 ± 0.12
NS NS NS b 0.001 b 0.001 b 0.01 NS b 0.01 b 0.001
Abbreviations BMI body mass index. Tc dftotal cholesterol. LDL-c low-density lipoprotein-cholesterol. HDL-c high-density lipoprotein-cholesterol. TG triglycerides. Lp-a lipoprotein(a). IMT intima-media thickness. NS not significant. a Bonferroni multiple comparisons (A–C: P b 0.05; B–C: P b 0.05; A–B: P b 0.05; A–B for HDL-c P N 0.05).
Statistical analyses SPSS 15.0 software package program was employed for the statistical evaluation. The parameter values were all expressed as the mean ± S.D. ANOVA test was used to obtain significant difference among the groups, followed by using post hoc test of Benferroni to obtain pair-wise comparisons. The results were considered significant if the value of p was less than 0.05. Correlation analyses were carried out using Pearson correlation coefficient. Results Mean values of age, gender, BMI, IMT, Tc, LDL-c and Lp-a, LDL-c, HDL-c and TG for patients and controls are recorded in Table 1. Data are expressed as mean ± SD of the mean of 25 controls and 25 heterozygous patients, and 11 homozygous patients. Patients and control groups did not differ in terms of age, gender and BMI. In patients with homozygous FH, TC, LDL-c and Lp-a levels were significantly higher than in patients with heterozygous FH and healthy control subjects. On the other hand HDL-c levels were significantly lower compared to heterozygous and healthy controls. Patients with heterozygous FH had significantly higher Tc, LDL-c and Lp-a levels compared with controls while they had significantly lower HDL-c levels compared with controls. There was not significant correlation between TG levels in patients and controls. Serum levels of MDA and PCO in controls and patients groups are also indicated in Table 2. It can be seen that MDA and PCO levels were significantly higher in patients with homozygous FH compared with those of heterozygous group and controls. The homozygous group also had significantly higher MDA and PCO levels compared with controls (Figs. 1 and 2). Statistical analyses between OS markers and parameters studied are listed in Table 3. They indicated that MDA levels was positively correlated with the serum concentrations of LDL-c (r = 0.84, P b 0.001), Tc (r = 0.84, P b 0.001), TG (r = 0.39, P = 0.02) and Lp-a (r = 0.83, P b 0.01) while negatively correlated with HDL-c (r = −0.43, P = 0.009). A similar trend was found for the correlation of PCO levels
with parameters studied: for LDL-c (r = 0.74, P b 0.001) and for Tc (r = 0.73, P b 0.001) and for Lp-a (r = 0.77, P b 0.001) whereas for HDL-c (r = −0.27, P b 0.001). PCO was not correlated with TG (r = 0.2, P = 0.25). A positive correlation was found between MDA and PCO (r = 0.72, P b 0.001). It was also found that MDA and PCO were positively correlated with IMT (r = 0.82, P b 0.001 and r = 0.63, P b 0.01 respectively). Discussion It is thought that oxidative stress plays a particularly important role in the development of cardiovascular pathology [18–20] and hence their products have a potential use as disease progression markers and consequently are the focus of current biomedical research. It has been demonstrated that increased OS oxidative stress by-products and/or a reduced antioxidant activity are the main cause of atherosclerosis and endothelial dysfunctions [21–23]. Understanding the mechanisms underlying the pathogenesis of cardiovascular diseases (CVD) is extremely important, since they are the leading cause of mortality and morbidity in the world [24]. We investigated the status of lipid peroxidation and protein oxidative damage in FH patients and found that MDA and PCO levels were significantly elevated in these patients compared with controls as indicated in Table 2. Increased blood MDA levels are possible consequent of oxygen free radical-mediated damage of the membrane lipid while the increase in the blood PCO level may be related to the oxidative damage of protein by elevated oxygen free radicals in the FH patients. The oxidation of low density lipoprotein (LDL) is believed to be a critical process in the development of atherosclerosis [25,26]. Carbonylation of proteins is an irreversible oxidative damage, often leading to the loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction [27,28]. To our knowledge, the present study is the first to examine PCO levels in the serum of FH patients. Oxidative stress results from the imbalance between prooxidant and antioxidant mechanisms present on the arterial wall as well as circulating cells. Based on the established relation of increased OS with atherogenesis and the pathogenicity of cardiovascular processes, our results suggest that an increased oxidation status would be able to contribute, at least in part, to the increased risk of cardiovascular disease in FH patients beyond LDL-c plasma values and other traditional risk factors. Therefore, our data support the conclusion obtained by other groups in their studies about FH and oxidative
Table 2 Oxidative stress parameters of patients (A = homozygous, B = heterozygous) and controls (C).a Parameters
A(n = 11)
B(n = 25)
C(n = 25)
p
MDA (nmol MDA equivalent/ 14.66 ± 2.09 10.44 ± 1.22 6.32 ± 1.09 b 0.001 mL serum) PCO (nmol/mg protein) 2.86 ± 0.48 2.12 ± 0.26 1.52 ± 0.28 b 0.001 Abbreviations MDA Malondialdehyde PCO Protein Carbonyl. a Bonferroni multiple comparisons (A–C: P b 0.05; B–C: P b 0.05; A–B: P b 0.05).
Fig. 1. Box plots for malondialdehyde (MDA) levels in familial hypercholesterolemia (FH) patients and controls. Solid horizontal lines = median values; error bars = 95% confidence intervals; Coloured = Interquartile range; *P b 0.05 each patient group vs. controls and between groups.
A.G. Pirinccioglu et al. / Clinical Biochemistry 43 (2010) 1220–1224
Fig. 2. Box plots for PCO levels in familial hypercholesterolemia (FH) patients and controls. Solid horizontal lines = median values; error bars = 95% confidence intervals; Coloured = Interquartile range; *P b 0.05 each patient group vs. controls and between groups.
stress [29–33]. Our findings may also indicate that the mechanism of lipid and protein metabolism is impaired in subjects with FH. The balance between oxidative stress and antioxidant defence mechanism may be impaired by depletion of enzymatic antioxidants and increased blood levels of MDA and PCO in subjects with FH. The mechanism of the difference in the levels of LDL-c and HDL-c in homozygous and heterozygous patients is not well-established. So the difference in MDA and PCO levels in two groups may be rationalized based on the correlation of MDA with LDL-c, that is, the higher LDL-c the higher MDA. Higher levels in Lp-a compared to controls may be ascribed to the fact that Lp-a is the contributory factor for the development of premature coronary heart disease. Individuals with high cholesterol plasma levels may develop very early vascular damage even without other additional cardiovascular risk factors. In particular, carriers of genetic forms of hypercholesterolemia, who are exposed to high concentration of plasma cholesterol since childhood, may develop premature cardiovascular event [3]. Children with FH has a rapid progression to clinical CVD as they become young adults compared to normocholesterolemic children. Even in the pediatric period, they exhibit evidence of subclinical atherosclerosis with increased carotid IMT [34,35] and endothelial dysfunction compared with similar-aged controls [36–38]. We have established that IMT values were positively correlated with LDL-c and Tc while negatively correlated with HDL-c. They were also positively correlated with both MDA and PCO. Large numbers of our homozygous patients (7 out of 11) were under 20 years and five of them had IMT over 1.0 mm. This implies that atherosclerosis may develop in pediatric period. So earlier diagnosis of the existance of OS in these patients by simple biochemical markers is possible and this
Table 3 Pearson correlations of MDA and PCO with parameters studied. Pairs
r
p
MDA-LDL-c MDA-Tc MDA-Lp-a MDA-HDL-c MDA-IMT PCO-LDL-c PCO-Tc PCO-Lp-a PCO-HDL-c PCO-IMT MDA-PCO
0.84 0.84 0.83 − 0.43 0.82 0.74 0.73 0.77 − 0.55 0.63 0.72
b 0.001 b 0.001 b 0.001 0.009 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001
r is correlation coefficient.
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may be an interect indication of coronary disease, which is an important cause of morbidity and mortality during childhood in this group of patients. The pathophysiologic role of Lp-a in atherogenesis is not completely understood. Similar to LDL, Lp-a undergoes oxidative modification in the vascular subendothelium. In vitro studies revealed that oxidized Lp(a) impairs endothelium-dependent vasodilatation and induces apoptotic endothelial cell death [39]. We have found that MDA and PCO are positively correlated with Lp-a. To sum up, our study showed that MDA and PCO levels were significantly increased in serum of ADH patients compared with the levels in control subjects. These indirectly suggest an increased production of oxygen free radicals in FH. Highly reactive oxygen metabolites, especially hydroxyl radicals, act on unsaturated fatty acids of phospholipid components of membranes to produce MDA, a lipid peroxidation product. Also, the increase in the blood carbonyl level is related to the oxidative damage of the protein by increased oxygen free radicals in the FH subjects. There is a need for more detailed studies on the relevance of protein oxidation, which is stated to play a significant role in the aetiology and pathogenesis of various diseases, including atherosclerosis. Identification of PCO may provide new diagnostic biomarkers for oxidative damage which occurs in atherosclerosis; clarify the correlation of atherosclerosis with several diseases and conditions such as diabetes-mellitus, cardiovascular diseases, menopause and smoking, in which free radical production and oxidative stress increase, and yield basic information for antioxidant treatment approaches in the treatment of atherosclerosis in FH patients. In conclusion, the data obtained could be important for understanding the alterations presented by FH and could be related to their increased atherosclerosis development risk. The increased PCO levels in the serum of FH patients suggest that protein oxidation may play a role in the increased cardiovascular risk of FH patients. However, further prospective and intervention studies are necessary to evaluate the impact of OS in the pathogenesis of cardiovascular disease in FH population. Conflicts of interest No further conflicts to disclose. Acknowledgment We thank Professor M. Yusuf Çelik for his valuable contribution to the statistical analysis. References [1] Goldstein JL, Brown MS. The LDL receptor focus and relation to genetics of familial hypercholesterolaemia. Annu Rev 1979;13:259–89. [2] Scientific Steering Committee on behalf of the Simon Broome Register Group. Risk of fatal coronary heart disease in familial hypercholesterolemia. BMJ 1991;303: 893–6. [3] Goldstein JL, Hobbs H, Brown MS. Familial hypercholesterolemia. In: Scriver C, Beaude A, Sly W, Vall D, editors. The methabolic and molecular basis of inherited disease. New York, USA: McGraw-Hill; 2001. p. 2863–913. [4] Rader DJ, Cohen J, Hobbs HH. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. J Clin Invest 2003;111:1795–803. [5] Hartvigsen K, Chou MY, Hansen LF, Shaw PX, Tsimikas S, Binder CJ, Witztum JL. The role of innate immunity in atherogenesis. J Lipid Res 2009:S388–93. [6] Duarte MMMF, Rocha JBT, Moresco RN, Duarte T, Da Cruz IBM, Loro VL, Schetinger MRC. Association between ischemia-modified albumin, lipids and inflammation biomarkers in patients with hypercholesterolemia. Clin Biochem 2009;42:666–71. [7] Vassalle C, Pratali L, Boni C. An oxidative stress score as a combined measure of the pro-oxidant and anti-oxidant counterparts in patients with coronary artery disease. Clin Biochem 2008;41:1162–7. [8] Cederberg J, Basu S, Eriksson UJ. Increased rate of lipid peroxidation and protein carbonylation in experimental diabetic pregnancy. Diabetologia 2001;44:766–74. [9] Kirkova M, Ivancheva E, Russannova E. Lipid peroxidation and antioxidant enzyme activity in aspirin-treated rats. Gen Pharmacol 1995;26:613–7.
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Clinical Biochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c l i n b i o c h e m
Malondialdehyde (MDA) and protein carbonyl (PCO) levels as biomarkers of oxidative stress in subjects with familial hypercholesterolemia Ayfer Gözü Pirinccioglu a,⁎, Deniz Gökalp b, Mihdiye Pirinccioglu c, Göksel Kizil c, Murat Kizil c a b c
Department of Pediatrics, Faculty of Medicine, University of Dicle, 21280, Diyarbakir, Turkey Department of Endocrinology, Faculty of Medicine, University of Dicle, 21280, Diyarbakir, Turkey Department of Chemistry, Faculty of Science, University of Dicle, 21280, Diyarbakir, Turkey
a r t i c l e
i n f o
Article history: Received 30 April 2010 Received in revised form 16 July 2010 Accepted 17 July 2010 Available online 4 August 2010 Keywords: Hypercholesterolemia Familial Oxidative stress Lipid peroxidation Malondialdehyde Protein carbonylation Atherosclerosis
a b s t r a c t Objective: Familial hypercholesterolemia (FH) is clinically characterized by elevated total and lowdensity lipoprotein (LDL) cholesterol levels in plasma, which has high risk for developing atherosclerosis. Increased oxidative stress (OS) and FH have been related to atherosclerosis. The study aims to evaluate oxidative stress in patients with hypercholesterolemia by measuring lipid peroxidation and protein carbonyl (PCO) levels in these patients. PCO in these patients may provide a new diagnostic biomarker for oxidative damage in atherosclerosis. Design and method: Total cholesterol (Tc), low-density lipoprotein-cholesterol (LDL-c), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-c), lipoprotein(a) (Lp-a) levels and carotid intima-media thickness were measured to evaluate characteristics of patients (11 homozygous and 25 heterozygous) with FH. 25 age–gender–BMI matched healthy control subjects were included in the study for comparison. Results: MDA and PCO levels were significantly higher in homozygous patients compared with those of heterozygous and controls and it was found that they are positively correlated with LDL-c, Tc, Lp-a and IMT while negatively correlated with HDL-c. The heterozygous group also had significantly higher MDA and PCO levels compared with controls. Conclusion: The data obtained could be important for understanding the alterations presented by FH and could be related to their increased risk of developing atherosclerosis. To our knowledge, measurements of PCO in patients with FH are not recorded before and this may be used as a biomarker for protein oxidation, which may play a role in the increased cardiovascular risk of patients with FH. © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Familial hypercholesterolemia (FH), an autosomal dominant disorder characterized by defects in the low density lipoprotein (LDL) receptor, is associated with a markedly increased risk of developing premature coronary heart disease [1,2]. There are two forms of FH, heterozygous and homozygous. The prevalence of the heterozygous FH is about 1/500 in the general population whereas the homozygous FH is very rare and found about 1 in 1 million people characterized by markedly increased low-density lipoprotein-cholesterol (LDL-c) levels and early onset of atherosclerosis [3,4]. The genetic basis of FH is the lack of functional receptors for LDL on the cell surface in liver and peripheral tissue [3]. As a result, plasma LDL concentrations are elevated and its plasma half-life prolonged, possibly leading to increased susceptibility to free radical attack and oxidation. Endothelial cells, smooth muscle cells, neutrophils and monocytes all have the potential to oxidatively modify LDL, leading to ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (A.G. Pirinccioglu).
the generation of lipid peroxidation products and reactive oxygen species, which is responsible for oxidative stress involved in degenerative disease, including atherosclerosis [5,6]. This can be measured by monitoring the changes in blood malondialdehyde (MDA) and carbonyl content. Determination of carbonyl level is used as an index of the extent of the oxidative damage of protein while malondialdehyde level is a marker of lipid oxidation. Antioxidants work together in human blood cells against toxic reactive oxygen species [7–9]. Reactive oxygen species (ROS) cause lipid peroxidation and oxidation of some specific proteins, thus affecting many intra- and intercellular systems [10]. Some ROSinduced protein modifications can result in unfolding or alteration of protein structure, and some are essentially harmless events. Irreversible protein modifications can lead to inactivation of various proteins and could have lasting detrimental cellular effects [11]. Many different types of protein oxidative modification can be induced by ROS. Carbonylation is an irreversible, non-enzymatic modification of proteins. Carbonyl groups are introduced into proteins by a variety of oxidative pathways. ROS can react directly with the protein or they can react with molecules such as sugars and lipids, generating products (reactive carbonyl species) that then react with protein and
0009-9120/$ – see front matter © 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2010.07.022
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lead to the formation of protein carbonyl derivatives (aldehydes and ketones) [12,13]. Studies of the formation of protein carbonyls cannot differentiate between those produced through direct protein oxidation and those formed by the addition of previously oxidized molecules, and hence protein carbonyls must be considered as a broad marker of oxidation [12,13]. The present study aims to investigate the status of lipid peroxidation and protein oxidative damage in subjects with FH by measuring serum malondialdehyde and carbonyl levels. Our study is the first to examine protein carbonyl levels in serum of FH patients and to analyse the correlations between protein carbonyl and MDA levels in atherosclerosis in the FH patients. Materials and methods Patients Eleven patients with homozygous FH (6 women and 5 men) with a mean age of 18 ± 6 years (7 children with ages of 8–20 years and 4 adults with ages of 21–28) and a body mass index (BMI) of 21± 2 kg/m2 and twenty five patients with heterozygous FH (13 women and 12 men) with a mean age of 18 ± 6 years (17 children with ages of 9–20 years and 8 adults ages of 21–29 years) and a body mass index (BMI) (19 ± 2) kg/m2 as well as twenty five age–gender–BMI matched healthy control subjects with a mean age (19 ± 6) years (16 children with ages of 10–20 and 9 adults with ages of 21–30) and a body mass index (BMI) (20 ± 3) kg/m2 were included in the present study. The study was conducted between 2007 and 2009 at the Department of Paediatrics and Department of Endocrinology, University of Dicle. All subjects and their parents gave informed consent prior to study entry and the study was conducted in accordance with the Declaration of Helsinki. The patients were diagnosed as FH based on the following criteria. (i) Cholesterol above 290 mg/dL or low-density lipoprotein-cholesterol (LDL-c) above 190 mg/dL. (ii) The presence of tendon and cutaneous xanthomas at an early age. (iii) An autosomal inheritance mode of hypercholesterolemia in the relatives. (iv) And the presence of primary hypercholesterolemia in the parents of index case. Differential diagnosis of homozygous and heterozygous FH patients were made as follows: patients (children and adults) with severely elevated cholesterol levels were assigned homozygous FH, with total cholesterol (Tc) and LDL-c levels greater than 600 mg/dL and triglyceride levels within the reference range. The assignment of heterozygous FH was made based on the following criteria: an LDL-c level higher than 200 mg/dL in patients younger than 20 years and higher than 290–300 mg/dL in adults. Families were thoroughly examined, particularly for plasma lipids and lipoproteins. Controls consisted of apparently healthy subjects admitted to the outpatient clinics of our institution who had not received any specific diagnosis. Diabetes, hypertension, obesity, and cigarette smoking were exclusion criteria in our study. Patients and controls underwent anthropometric and additionally, in order to exclude Apo B100 mutation, the absence of APOB3500 mutation (R3500Q) was observed using PCR. All measurements were done at the time of the first admission while none of the patients were receiving aphaeresis. Medical therapy (statins, ezetimibe and cholestyramine) was initiated in all patients and 7 of them were also treated with LDL aphaeresis twice a month. Anthropometric evaluation Body weight was measured with light clothing, in kilograms. Height was measured without shoes in centimetres. BMI was expressed as weight (in kilograms) per square of height (in meters). Maximum abdominal girth (in cm) was taken as the waist circumference. Waist circumference was considered as a measure of body fat distribution. All systolic and diastolic blood pressures were measured by the same person using a sphygmomanometer (ERKA® sphygmomanometer,
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İstanbul, Turkey) from the right arm of the subject in a relaxed sitting position. The average of 3 consecutive measurements was used for analysis. Hypertension was defined as a systolic blood pressure higher than 140 mm Hg or a diastolic blood pressure higher than 90 mm Hg for adults. The blood pressure percentile values in patients under 16 were evaluated in terms of their age, gender and height. Measurement of carotid intima-media thickness (IMT) The carotid artery ultrasonographic scans were obtained by A Toshiba SSH-140A ultrasound 7.5 MHz linear probe. The evaluation of the right and left common carotid arteries was 1 cm proximal to the carotid bulb. In each examination, the same operator used different scanning angles to identify the greatest IMT, defined as the distance between the junction of the lumen and intima and that of the media and adventitia. Three measurements of IMT were obtained from the right and left carotid arteries, respectively, and were averaged to determine the mean IMT for both sides combined. The coefficient of variation was 2.6%. Biochemical analysis Blood serum samples were taken from patients and control after overnight fasting. Serum total cholesterol (Tc), high-density lipoprotein-cholesterol (HDL-c) and triglyceride (TG) levels were measured with an autoanalyzer (Abott aeroset autoanalyzer, Toshiba, Japan). LDL-c levels were calculated by using the Friedewald equation [14]. Serum lipoprotein(a) (Lp-a) levels were measured by immunometric assay (Diagnostic Products Corporation 5700, Los Angeles, CA, USA). Presence of Apo B100 mutation was examined by using a Real Time PCR (Roche-Light-cycler) device with Lightcycler-ApoB mutation detection (codon 3500) kit. Protein determination The protein content in serum was measured by method of Lowry et al. with bovine serum albumin as the standard [15]. Lipid peroxidation (MDA) assay Lipid peroxidation in serum was evaluated by the spectrophotometric method based on the reaction between MDA and thiobarbituric acid [16]. Briefly, 0.5 mL each of plasma was mixed with 2.5 mL of 20% (v/v) trichloroacetic acid (TCA) (Sigma, Germany) and centrifuged at 3000 rpm for 10 min. Then, 3 mL of 0.2 g/dL TBA (Sigma, Germany) was added to the supernatant. The mixture was heated in boiling water for 30 min. After cooling on ice, the resulting chromogen was extracted with 4 mL of n-butyl alcohol. The organic phase was separated by centrifugation at 3000 rpm for 10 min and absorbance was recorded at wavelength of 530 nm. MDA solution was made freshly by the hydrolysis of 1,1,3,3-tetramethoxypropane (TMP) (Sigma, Germany) used as the standard. The results are expressed as the nmol MDA/mL serum. Measurement of protein carbonyls Determination of carbonyl content in oxidatively modified proteins was followed by the method of Levine et al. [17].The oxidative damage to proteins was measured by the quantification of carbonyl groups based on their reaction with 2,4-dinitro-phenylhydrazine (DNPH) to form hydrazones. Briefly, 0.1 mL of serum was incubated with 1.0 mL of 20 mM DNPH solution for 60 min. In brief, proteins were precipitated by the addition of 20% (v/v) trichloroacetic acid and re-dissolved in DNPH. Then the proteins were precipitated from the solution with the use of 20% (w/v) trichloroacetate; the protein pellet was washed three times with ethanol and ethyl acetate, and re-suspended in 1 mL of 6 M guanidine. The absorbance was read at 370 nm.
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Table 1 Clinical details and serum profiles in patients (A = homozygous, B = heterozygous) and controls (C).a Parameters
A(n = 11)
B(n = 25)
C(n = 25)
p (ANOVA)
Age (year) Gender (F/M) BMI (kg/m2) Tc (mg/dL) LDL-c (mg/dL) HDL-c (mg/dL) TG (mg/dL) Lp-a (mg/dL) IMT (mm)
18.12 ± 7.24 6/5 20.94 ± 2.27 713 ± 68 650 ± 67 26.78 ± 6.20 184 ± 26 73.31 ± 5.33 1.67 ± 0.48
18.44 ± 7.89 13/12 19.32 ± 2.42 363 ± 76 296 ± 70 31.32 ± 6.12 180 ± 20 51.28 ± 4.64 1.02 ± 0.22
18.75 ± 6.65 12/13 19.87 ± 2.84 229 ± 38 143 ± 33 51.60 ± 6.20 174 ± 19 26.65 ± 2.56 0.54 ± 0.12
NS NS NS b 0.001 b 0.001 b 0.01 NS b 0.01 b 0.001
Abbreviations BMI body mass index. Tc dftotal cholesterol. LDL-c low-density lipoprotein-cholesterol. HDL-c high-density lipoprotein-cholesterol. TG triglycerides. Lp-a lipoprotein(a). IMT intima-media thickness. NS not significant. a Bonferroni multiple comparisons (A–C: P b 0.05; B–C: P b 0.05; A–B: P b 0.05; A–B for HDL-c P N 0.05).
Statistical analyses SPSS 15.0 software package program was employed for the statistical evaluation. The parameter values were all expressed as the mean ± S.D. ANOVA test was used to obtain significant difference among the groups, followed by using post hoc test of Benferroni to obtain pair-wise comparisons. The results were considered significant if the value of p was less than 0.05. Correlation analyses were carried out using Pearson correlation coefficient. Results Mean values of age, gender, BMI, IMT, Tc, LDL-c and Lp-a, LDL-c, HDL-c and TG for patients and controls are recorded in Table 1. Data are expressed as mean ± SD of the mean of 25 controls and 25 heterozygous patients, and 11 homozygous patients. Patients and control groups did not differ in terms of age, gender and BMI. In patients with homozygous FH, TC, LDL-c and Lp-a levels were significantly higher than in patients with heterozygous FH and healthy control subjects. On the other hand HDL-c levels were significantly lower compared to heterozygous and healthy controls. Patients with heterozygous FH had significantly higher Tc, LDL-c and Lp-a levels compared with controls while they had significantly lower HDL-c levels compared with controls. There was not significant correlation between TG levels in patients and controls. Serum levels of MDA and PCO in controls and patients groups are also indicated in Table 2. It can be seen that MDA and PCO levels were significantly higher in patients with homozygous FH compared with those of heterozygous group and controls. The homozygous group also had significantly higher MDA and PCO levels compared with controls (Figs. 1 and 2). Statistical analyses between OS markers and parameters studied are listed in Table 3. They indicated that MDA levels was positively correlated with the serum concentrations of LDL-c (r = 0.84, P b 0.001), Tc (r = 0.84, P b 0.001), TG (r = 0.39, P = 0.02) and Lp-a (r = 0.83, P b 0.01) while negatively correlated with HDL-c (r = −0.43, P = 0.009). A similar trend was found for the correlation of PCO levels
with parameters studied: for LDL-c (r = 0.74, P b 0.001) and for Tc (r = 0.73, P b 0.001) and for Lp-a (r = 0.77, P b 0.001) whereas for HDL-c (r = −0.27, P b 0.001). PCO was not correlated with TG (r = 0.2, P = 0.25). A positive correlation was found between MDA and PCO (r = 0.72, P b 0.001). It was also found that MDA and PCO were positively correlated with IMT (r = 0.82, P b 0.001 and r = 0.63, P b 0.01 respectively). Discussion It is thought that oxidative stress plays a particularly important role in the development of cardiovascular pathology [18–20] and hence their products have a potential use as disease progression markers and consequently are the focus of current biomedical research. It has been demonstrated that increased OS oxidative stress by-products and/or a reduced antioxidant activity are the main cause of atherosclerosis and endothelial dysfunctions [21–23]. Understanding the mechanisms underlying the pathogenesis of cardiovascular diseases (CVD) is extremely important, since they are the leading cause of mortality and morbidity in the world [24]. We investigated the status of lipid peroxidation and protein oxidative damage in FH patients and found that MDA and PCO levels were significantly elevated in these patients compared with controls as indicated in Table 2. Increased blood MDA levels are possible consequent of oxygen free radical-mediated damage of the membrane lipid while the increase in the blood PCO level may be related to the oxidative damage of protein by elevated oxygen free radicals in the FH patients. The oxidation of low density lipoprotein (LDL) is believed to be a critical process in the development of atherosclerosis [25,26]. Carbonylation of proteins is an irreversible oxidative damage, often leading to the loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction [27,28]. To our knowledge, the present study is the first to examine PCO levels in the serum of FH patients. Oxidative stress results from the imbalance between prooxidant and antioxidant mechanisms present on the arterial wall as well as circulating cells. Based on the established relation of increased OS with atherogenesis and the pathogenicity of cardiovascular processes, our results suggest that an increased oxidation status would be able to contribute, at least in part, to the increased risk of cardiovascular disease in FH patients beyond LDL-c plasma values and other traditional risk factors. Therefore, our data support the conclusion obtained by other groups in their studies about FH and oxidative
Table 2 Oxidative stress parameters of patients (A = homozygous, B = heterozygous) and controls (C).a Parameters
A(n = 11)
B(n = 25)
C(n = 25)
p
MDA (nmol MDA equivalent/ 14.66 ± 2.09 10.44 ± 1.22 6.32 ± 1.09 b 0.001 mL serum) PCO (nmol/mg protein) 2.86 ± 0.48 2.12 ± 0.26 1.52 ± 0.28 b 0.001 Abbreviations MDA Malondialdehyde PCO Protein Carbonyl. a Bonferroni multiple comparisons (A–C: P b 0.05; B–C: P b 0.05; A–B: P b 0.05).
Fig. 1. Box plots for malondialdehyde (MDA) levels in familial hypercholesterolemia (FH) patients and controls. Solid horizontal lines = median values; error bars = 95% confidence intervals; Coloured = Interquartile range; *P b 0.05 each patient group vs. controls and between groups.
A.G. Pirinccioglu et al. / Clinical Biochemistry 43 (2010) 1220–1224
Fig. 2. Box plots for PCO levels in familial hypercholesterolemia (FH) patients and controls. Solid horizontal lines = median values; error bars = 95% confidence intervals; Coloured = Interquartile range; *P b 0.05 each patient group vs. controls and between groups.
stress [29–33]. Our findings may also indicate that the mechanism of lipid and protein metabolism is impaired in subjects with FH. The balance between oxidative stress and antioxidant defence mechanism may be impaired by depletion of enzymatic antioxidants and increased blood levels of MDA and PCO in subjects with FH. The mechanism of the difference in the levels of LDL-c and HDL-c in homozygous and heterozygous patients is not well-established. So the difference in MDA and PCO levels in two groups may be rationalized based on the correlation of MDA with LDL-c, that is, the higher LDL-c the higher MDA. Higher levels in Lp-a compared to controls may be ascribed to the fact that Lp-a is the contributory factor for the development of premature coronary heart disease. Individuals with high cholesterol plasma levels may develop very early vascular damage even without other additional cardiovascular risk factors. In particular, carriers of genetic forms of hypercholesterolemia, who are exposed to high concentration of plasma cholesterol since childhood, may develop premature cardiovascular event [3]. Children with FH has a rapid progression to clinical CVD as they become young adults compared to normocholesterolemic children. Even in the pediatric period, they exhibit evidence of subclinical atherosclerosis with increased carotid IMT [34,35] and endothelial dysfunction compared with similar-aged controls [36–38]. We have established that IMT values were positively correlated with LDL-c and Tc while negatively correlated with HDL-c. They were also positively correlated with both MDA and PCO. Large numbers of our homozygous patients (7 out of 11) were under 20 years and five of them had IMT over 1.0 mm. This implies that atherosclerosis may develop in pediatric period. So earlier diagnosis of the existance of OS in these patients by simple biochemical markers is possible and this
Table 3 Pearson correlations of MDA and PCO with parameters studied. Pairs
r
p
MDA-LDL-c MDA-Tc MDA-Lp-a MDA-HDL-c MDA-IMT PCO-LDL-c PCO-Tc PCO-Lp-a PCO-HDL-c PCO-IMT MDA-PCO
0.84 0.84 0.83 − 0.43 0.82 0.74 0.73 0.77 − 0.55 0.63 0.72
b 0.001 b 0.001 b 0.001 0.009 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001 b 0.001
r is correlation coefficient.
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may be an interect indication of coronary disease, which is an important cause of morbidity and mortality during childhood in this group of patients. The pathophysiologic role of Lp-a in atherogenesis is not completely understood. Similar to LDL, Lp-a undergoes oxidative modification in the vascular subendothelium. In vitro studies revealed that oxidized Lp(a) impairs endothelium-dependent vasodilatation and induces apoptotic endothelial cell death [39]. We have found that MDA and PCO are positively correlated with Lp-a. To sum up, our study showed that MDA and PCO levels were significantly increased in serum of ADH patients compared with the levels in control subjects. These indirectly suggest an increased production of oxygen free radicals in FH. Highly reactive oxygen metabolites, especially hydroxyl radicals, act on unsaturated fatty acids of phospholipid components of membranes to produce MDA, a lipid peroxidation product. Also, the increase in the blood carbonyl level is related to the oxidative damage of the protein by increased oxygen free radicals in the FH subjects. There is a need for more detailed studies on the relevance of protein oxidation, which is stated to play a significant role in the aetiology and pathogenesis of various diseases, including atherosclerosis. Identification of PCO may provide new diagnostic biomarkers for oxidative damage which occurs in atherosclerosis; clarify the correlation of atherosclerosis with several diseases and conditions such as diabetes-mellitus, cardiovascular diseases, menopause and smoking, in which free radical production and oxidative stress increase, and yield basic information for antioxidant treatment approaches in the treatment of atherosclerosis in FH patients. In conclusion, the data obtained could be important for understanding the alterations presented by FH and could be related to their increased atherosclerosis development risk. The increased PCO levels in the serum of FH patients suggest that protein oxidation may play a role in the increased cardiovascular risk of FH patients. However, further prospective and intervention studies are necessary to evaluate the impact of OS in the pathogenesis of cardiovascular disease in FH population. Conflicts of interest No further conflicts to disclose. Acknowledgment We thank Professor M. Yusuf Çelik for his valuable contribution to the statistical analysis. References [1] Goldstein JL, Brown MS. The LDL receptor focus and relation to genetics of familial hypercholesterolaemia. Annu Rev 1979;13:259–89. [2] Scientific Steering Committee on behalf of the Simon Broome Register Group. Risk of fatal coronary heart disease in familial hypercholesterolemia. BMJ 1991;303: 893–6. [3] Goldstein JL, Hobbs H, Brown MS. Familial hypercholesterolemia. In: Scriver C, Beaude A, Sly W, Vall D, editors. The methabolic and molecular basis of inherited disease. New York, USA: McGraw-Hill; 2001. p. 2863–913. [4] Rader DJ, Cohen J, Hobbs HH. Monogenic hypercholesterolemia: new insights in pathogenesis and treatment. J Clin Invest 2003;111:1795–803. [5] Hartvigsen K, Chou MY, Hansen LF, Shaw PX, Tsimikas S, Binder CJ, Witztum JL. The role of innate immunity in atherogenesis. J Lipid Res 2009:S388–93. [6] Duarte MMMF, Rocha JBT, Moresco RN, Duarte T, Da Cruz IBM, Loro VL, Schetinger MRC. Association between ischemia-modified albumin, lipids and inflammation biomarkers in patients with hypercholesterolemia. Clin Biochem 2009;42:666–71. [7] Vassalle C, Pratali L, Boni C. An oxidative stress score as a combined measure of the pro-oxidant and anti-oxidant counterparts in patients with coronary artery disease. Clin Biochem 2008;41:1162–7. [8] Cederberg J, Basu S, Eriksson UJ. Increased rate of lipid peroxidation and protein carbonylation in experimental diabetic pregnancy. Diabetologia 2001;44:766–74. [9] Kirkova M, Ivancheva E, Russannova E. Lipid peroxidation and antioxidant enzyme activity in aspirin-treated rats. Gen Pharmacol 1995;26:613–7.
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