Lipid Peroxidation and Total Antioxidant Capacity in Patients with Chronic Renal Failure

INTRODUCTION

Oxygen free radicals (OFR) play a significant role in the pathogenesis of many chronic diseases such as diabetes mellitus, cancer, chronic renal failure etc. (Psotova et al., 2001). Studies in patients with varying degrees of kidney impairment suggests that patients with chronic renal disease are in a state of oxidative stress compared with healthy controls. Chronic Renal Failure (CRF) is a pro-oxidant state and the degree of intracellular and extracellular oxidative stress is related to the severity of renal failure (Massy and Khoa, 2002). Studies have shown that OFR are involved in progressive renal injury. Attack of OFR on polyunsaturated fatty acids initiate lipid peroxidation which leads to alterations in biological membranes and causes progressive renal injury. A team of endogenous and exogenous antioxidants representing the Total Antioxidant Capacity (TAC) of extracellular fluids provides greater protection against attack by OFR (Oka et al., 2001). In the literature, there are limited and conflicting data on the relationship between lipid peroxidation and total antioxidant capacity in CRF patients (Mircescu et al., 2005). Evaluating oxidative stress in CRF patients by measuring lipid peroxidation and TAC can lead to a better understanding of free radical damage in CRF patients. The comparison of serum MDA and serum FRAP may find its use as an indicator of the progressive follow up in the CRF patients and for antioxidant therapy.

MATERIALS AND METHODS

The study was carried out at MS Ramaiah Medical Teaching Hospital, Bangalore, in the year 2006, by taking forty patients of either sex and within the age group of 25-70 years as cases. Patients with clinically diagnosed chronic renal failure (on conservative management before dialysis) due to chronic glomerulonephritis and other glomerular diseases, chronic pyelonephritis and obstructive uropathy with serum creatinine levels more than 3.0 mg dL^-1 were included in this study. Patients with CRF due to diabetes mellitus, essential hypertension, liver diseases, coronary artery disease, vasculitis and other autoimmune disorders were excluded. The clinical history and other necessary details were obtained from their case sheets. The study was conducted after informed consent was obtained from them and approved by the ethical committee of the institution. Control group consisted of forty healthy males and females with age group of 25-70 years.

Under aseptic precautions 5 mL of fasting venous blood samples were collected. Clotted blood was subjected to centrifugation. The clear serum was separated and used for the following biochemical investigations: Malondialdehyde, Ferric Reducing Antioxidant Power (FRAP) assay, Serum creatinine and Blood Urea Nitrogen (BUN). All the chemicals used were of highest analytical grade available in India.

Lipid peroxidation was measured by serum MDA estimation according to the colorimetric method of Satoh (1978).Lipoproteins are precipitated from the specimen by adding TCA. 0.05 M sulphuric acid and 0.67% TBA in 2 M sodium sulphate are added to this precipitate and the coupling of lipid peroxide with TBA is carried out by heating in a boiling water bath for 30 min. The resulting chromogen is extracted in n-butanol, which is measured colorimetrically at 530 nm.

Total antioxidant capacity was measured by Ferric Reducing Antioxidant Power (FRAP) assay according to the method of Iris Benzie and Strain (1996).At low pH, when a ferric tripyridyltriazine (Fe^III-TPTZ) complex is reduced to the ferrous (Fe^II) form, an intense blue color with absorption maximum at 593 nm develops. FRAP directly analyzes total low molecular weight antioxidants.

Serum creatinine and BUN were estimated by standard clinical chemistry methods (Newman et al., 1999).

RESULTS AND DISCUSSION

Statistically significant increase in MDA levels were found in CRF patients as compared to controls (p<0.001) and statistically significant decrease in TAC in CRF patients as compared to controls (p<0.001). MDA showed significant positive correlation with serum creatinine (r = 0.794) (p<0.01) and BUN (r = 0.492) (p<0.01). TAC (FRAP assay) showed significant negative correlation with serum creatinine, r = -0.76, (p<0.01) and BUN, r = -0.398, (p<0.05). Significantly negative correlation was observed between MDA and TAC, r = -0.752, (p<0.01) in CRF patients (Table 1, 2, Fig. 1).

Table 1:	Comparison of the parameters in controls and CRF patients

Table 2:	Correlation between serum creatinine, serum MDA and serum FRAP in CRF patients

Serum MDA-nmol mL^-1, Serum creatinine-mg dL^-1, Serum FRAP-μmol L^-1


Fig. 1:	Correlation between MDA and FRAP values in CRF patients

Oxidative stress is defined as an imbalance between formation of reactive oxygen species and antioxidative defence mechanisms. ROS can damage proteins, lipids, carbohydrates and nucleic acids. Plasma membranes are critical targets of free radical reactions. ROS can easily produce injuries to cell membranes by initiation of polyunsaturated fatty acid peroxidation, inactivation of membrane enzymes and receptors and protein crosslinking and fragmentation (Oka et al., 2001; McCall and Frei, 1999). Thus responsible for pathogenesis of a variety of diseases like diabetes mellitus, cancer or chronic renal failure (Massy and Khoa, 2002; Halliwell, 1991).

There are conflicting results regarding the status of antioxidant systems in patients with CRF. Also, there is a wide variation in the published levels of malondialdehyde (MDA) in CRF patients (Annuk et al., 2001; Luciak and Trznadel, 1991; Bolton et al., 2001). Several studies suggest that concentration of MDA, a byproduct of lipid peroxidation is significantly increased in CRF patients before initiation of dialysis and renal replacement therapy when compared with the control group (Fiorillo et al., 1998; Mircescu et al., 2005; Leonardo et al., 2005).In the present study, there was a significant increase in MDA levels in CRF patients. MDA concentrations significantly increase with the severity of kidney dysfunction. This suggests increased lipid peroxidation in these patients (Leonardo et al., 2005; Martin-Mateo et al., 1999). Total Antioxidant Capacity (TAC) parameter summarizes the overall activity of antioxidants and antioxidant enzymes (Psotova et al., 2001; Haugen and Nath, 1999). It should be noted that co-operation between different antioxidant pathways provides greater protection against attack by Reactive Oxygen Species (ROS) or nitrogen radicals, compared to any single compound. Thus, the overall antioxidant capacity may give more relevant biological information compared to that obtained by the measurement of individual biomarkers (Malliaraki et al., 2003). The FRAP assay is presented as a novel method for assessing antioxidant power/TAC (Iris Benzie and Strain, 1996). This parameter is able to give information regarding the total charge of antioxidants present in the serum. The index thus obtained is considered as a measure of the system`s ability to regulate the damage due to the ROS production (Psotova et al., 2001; Iris Benzie and Strain, 1996). Studies of TAC in patients with chronic renal failure have shown varying results (McCall and Frei, 1999; Gazdikova et al., 2000). Increased levels of TAC indicate absorption of stock organ antioxidants eg. uric acid and the induction or activation of antioxidant enzymes as an adaptation to the oxidative stress, but at a later phase of oxidative stress, the TAC falls due to depletion of antioxidants (Psotova et al., 2001)^. In addition, high concentration of a number of metabolites, including uric acid can lead to pro-oxidant effects, introducing a further decrease of the antioxidant capacity. This could be the consequence of increased exposure to the oxidative attacks or lipid peroxidation and its over consumption as an antioxidant (Malliaraki et al., 2003). A number of enzymes and enzymatic pathways contribute to the detoxification of free radicals and a decreased efficiency of these pathways including NADPH, NADH and GSH can also readily explain for the reduced activity of antioxidant systems. Imbalances in the intracellular redox systems may impair detoxification mechanisms (Pias and Aw, 2002). The results of this study reflect the significant increase in serum MDA levels and depletion of antioxidative defence mechanisms with progressive renal injury as evidenced by significant decrease in FRAP levels in CRF patients. The significant negative correlation between MDA and FRAP levels in CRF patients supports the mechanism of oxidative stress caused by an imbalance between formation of ROS and attenuation of antioxidative defence mechanisms.