Oxidant stress in nephrotic syndrome: comparison of F2-isoprostanes and plasma antioxidant potential

Gursharan Dogra1,, Natalie Ward1, Kevin D. Croft1, Trevor A. Mori1, P. Hugh R. Barrett1, Susan E. Herrmann1, Ashley B. Irish2 and Gerald F. Watts1

1 Department of Medicine and Western Australian Heart Research Institute, University of Western Australia, and 2 Department of Nephrology, Royal Perth Hospital, Perth, Western Australia



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The nephrotic syndrome (NS) is associated with an increased risk of coronary heart disease. Increased oxidant stress may contribute to this by means of hyperlipidaemia and/or hypoalbuminaemia. In this study we assessed the contributory role of oxidant stress, as measured by F2-isoprostanes and plasma oxygen radical absorbance capacity (ORAC), in subjects with NS.

Methods. We studied 14 subjects with NS and 17 age- and sex-matched healthy non-proteinuric controls. Measurement of plasma and urinary F2-isoprostanes was carried out using a combination of silica and reverse-phase cartridges, high-performance liquid chromatography, and gas chromatography mass spectrometry using electron-capture negative ionization. The plasma ORAC assay measured the decrease in fluorescence of phycoerythrin added to plasma in the presence of a free-radical generator. The ORAC value (µM) was calculated as the ratio of the area under the fluorescence decay curve for plasma to the area under the fluorescence decay curve for a Trolox standard.

Results. Plasma ORAC was significantly lower in NS patients compared with controls: mean (standard error) NS patients 3306 µM (286); controls 4882 µM (496), P=0.011. In univariate linear regression analysis, plasma albumin was significantly positively correlated with plasma ORAC (r=0.40, P=0.03). Plasma and urinary F2-isoprostanes did not differ significantly between NS and control groups.

Conclusions. This study demonstrates that in the NS there is decreased free-radical trapping capacity of plasma that is inversely correlated with hypoalbuminaemia, but no increase in plasma and urinary F2-isoprostanes. Decreased total plasma antioxidant potential in combination with hyperlipidaemia may contribute to the increased risk of cardiovascular disease seen in NS.

Keywords: anti-oxidant defence; dyslipidaemia; F2-isoprostanes; nephrotic syndrome; oxidant stress; oxygen radical absorbance capacity (ORAC)



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Nephrotic syndrome (NS) is characterized by heavy proteinuria (>3.5 g/day) associated with peripheral oedema, hypoalbuminaemia, and hyperlipidaemia [1], and an increased risk of coronary heart disease [2]. Elevated plasma low-density lipoprotein (LDL) cholesterol is likely to contribute to this increased cardiac risk [3]. However, hypoalbuminaemia is also independently associated with increased risk of cardiovascular disease [4], possibly by increasing oxidant stress. Albumin is a non-enzymatic protein antioxidant that inhibits LDL peroxidation in vitro [5]. Although increased oxidant stress has been described in animal models of glomerulonephritis with and without nephrotic proteinuria [6], oxidant stress and its association with serum albumin levels and other water-soluble antioxidants has not been reported in patients with glomerulonephritis and associated nephrosis.

In-vivo measurement of free radical generation and consequent peroxidative damage has been difficult to quantify [7]. The F2-isoprostanes, free-radical oxidation products of arachidonic acid, have been quantified in human models of increased oxidative stress [8,9] and are a useful measure of in-vivo lipid peroxidative damage [10]. We have recently described an improved method for the measurement of urinary and plasma F2-isoprostanes, specifically 8-isoprostaglandin F2{alpha} (8-iso-PGF2{alpha}) [11]. In this study we compared oxidant stress between patients with NS and healthy non-proteinuric controls to test the hypothesis that oxidant stress is increased in NS as a consequence of hypoalbuminaemia and/or hyperlipidaemia. We have measured plasma and urinary F2-isoprostanes, as well as plasma total antioxidant potential using the ORAC assay (oxygen radical absorbance capacity) [12].



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
Fourteen patients aged 18–75 years who had nephrotic-range proteinuria (>3.5 g/day) with a primary glomerular disorder were recruited from renal clinics over a 2-year period. Patients with a serum creatinine >150 µmol/l, diabetes mellitus, malignancy, hypothyroidism, secondary cause for proteinuria, excess ethanol consumption, active smoking history, macrovascular atherosclerotic disease, or patients on aspirin, immunosuppressive therapy, including steroids, allopurinol, fish oils, lipid-lowering therapy, or multivitamin and anti-oxidant vitamin preparations were excluded from the study. Patients were not excluded if they were taking angiotensin-converting enzyme inhibitors (ACE-I) or a stable dose of any other anti-hypertensive agent. Nephrotic patients on lipid-lowering therapy were studied after a 6-week period off treatment. Seventeen healthy controls, matched for age, sex, and body mass index (BMI), were recruited from the community. The Ethics Committee at Royal Perth Hospital approved the study, and all volunteers gave written consent. The research was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association.

Study design and laboratory methods
The study was a cross-sectional comparison of nephrotic (NS) and control (CS) subjects. Blood pressure was measured using a Dinamap 1846 SX/P monitor (Critikon Ltd., Tampa, Florida) after resting the patient for 10 min in the supine position. Venous blood samples were obtained after a 12-h fast for the following variables, which were measured by standard laboratory methods unless otherwise stated: creatinine, albumin, uric acid, bilirubin, total cholesterol, high-density lipoprotein (HDL)-cholesterol and triglycerides. Low-density lipoprotein (LDL)-cholesterol was calculated using the modified Friedewald formula, except if triglyceride levels >4.5 mmol/l, when it was directly determined by an enzymatic, colorimetric assay using reagents from Boehringer Mannheim (LDL-C, Boehringer Mannheim GmBH, Mannheim, Germany) on a Hitachi 917 analyser. Glomerular filtration rate was calculated using the Cockcroft and Gault equation. The inter-assay coefficient of variation (CV) of these analytical assays was <6%. Nephrotic subjects provided 24-h urine collections for assessment of protein, creatinine and F2-isoprostanes excretion, while control subjects provided overnight urine collections. Urinary protein and F2-isoprostanes concentrations were corrected for creatinine excretion to allow for the difference in collection methods.

Plasma ORAC assay
Citrated blood samples, collected after a 12-h fast, were centrifuged immediately at 1000 g for 15 min at 4°C. Plasma was stored at -80°C until analysis. As previously described, the ORAC assay measures the decrease in fluorescence of phycoerythrin added to plasma in the presence of a free radical generator [12] and measures the ability of plasma components to trap free radicals. It is measured against a Trolox standard and phosphate-buffer blank. Citrated plasma was diluted 150-fold with phosphate buffer, and phycoerythrin was added. Free-radical generation was facilitated by addition of AAPH (160 mmol/l), a peroxyl radical generator. Fluorescence (excitation 540 nm; emission 565 nm) was measured immediately and every 5 min for 70 min. Area under the fluorescence decay curve (AUC) was calculated and compared to AUC for the Trolox standard. The ORAC value (µM) was calculated as a ratio of the plasma AUC to the Trolox AUC. The inter assay CV is <7% [23].

F2-isoprostanes
Blood samples for plasma F2-isoprostanes were collected into cold tubes containing EDTA and reduced glutathione and centrifuged immediately at 1000 g for 15 min at 4°C. Plasma was protected from oxidation by the addition of 20 µg of butylated hydroxytoluene (BHT) per millilitre of plasma and stored at -80°C until analysis. Urine collected for F2-isoprostanes was stored in 10 ml aliquots containing 200 µg of BHT at -80°C until analysis. Measurement of plasma and urine F2-isoprostanes was carried out using a combination of silica and reverse-phase cartridges, high-performance liquid chromatography, and gas chromatography mass spectrometry using electron-capture negative ionization, as previously described [11]. Urinary F2-isoprostane levels were corrected for creatinine excretion. The inter assay CV for urinary and plasma F2-isoprostanes is 3.7 and 5.6% respectively [11]. Plasma fatty acid composition and plasma arachidonic acid levels were measured as the methyl ester derivatives by gas chromatography using heptadecanoic acid as an internal standard.

Statistical analysis
Results are expressed as mean±SEM. Independent t-test was used for between group comparisons (Statistical Package for Social Sciences, SPSS Inc., Chicago, Illinois). Skewed data (serum creatinine, serum triglyceride, plasma and urinary F2-isoprostanes) are described as geometric mean and 95% confidence interval and log transformed prior to analyses. Correlations were tested using linear regression methods. Categorical variables were compared using Chi-square and Fisher's exact tests.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The clinical characteristics of the NS and CS groups are shown in Table 1Go. Median disease duration in the NS patients was 7 months (range 1–156 months). Seven of the 14 NS patients were on an ACE-I, two were on angiotensin II receptor antagonists, and seven patients received diuretics. Of the 14 NS patients, renal histology showed minimal-change disease (n=4), membranous glomerulonephritis (n=3), focal and segmental glomerulonephritis (n=3), IgA glomerulonephritis (n=1), IgA-negative mesangioproliferative glomerulonephritis (n=1), and two patients had not had a renal biopsy.


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Table 1. Clinical characteristics of nephrotic (NS) and control (CS) Subjects

 
Serum creatinine and calculated GFR were similar in both groups (Table 2Go). As expected, NS group had statistically higher urinary protein:creatinine ratio, total and LDL-cholesterol and triglyceride concentrations, and a significantly lower serum albumin compared with controls. In the NS group, serum uric acid was significantly higher (P<0.0001) and serum bilirubin was significantly lower (P=0.001) as well as being positively correlated with serum albumin (r=0.53, P=0.03).


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Table 2. Biochemical characteristics of the nephrotic (NS) and control (CS) subjects

 
Plasma total antioxidant potential as assessed by the ORAC assay was significantly lower in the NS group compared with controls (P=0.011) (Table 3Go). In total group analysis, using univariate regression, ORAC was significantly positively correlated with serum albumin only (r=0.40, P=0.03). No significant correlation was shown between ORAC and serum bilirubin, uric acid, LDL-cholesterol, blood pressure, and ACE-I status. Additionally, ORAC values were not significantly correlated with either urinary or plasma F2-isoprostanes (r=-0.22, P=0.25 and r=-0.04, P=0.84).


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Table 3. Plasma ORAC assay, plasma and urinary F2-isoprostanes, and arachidonic acid in the nephrotic (NS) and control (CS) subjects

 
The NS group had statistically higher plasma arachidonic acid levels (P<0.001) (Table 3Go), but plasma fatty acid composition was not different. Plasma arachidonic acid levels were significantly positively correlated with total cholesterol (r=0.71, P=0.003). Plasma F2-isoprostane levels were not significantly different between NS and CS groups (Table 3Go), even after correcting for plasma arachidonic acid content (NS 32.1±4.0; CS 45.9±9.6, P=0.163). In multiple regression analysis, plasma F2-isoprostane levels in the NS group were inversely associated with serum albumin (regression coefficient-0.06, standard error 0.02, P=0.031) independent of arachidonic acid and serum cholesterol. Plasma F2-isoprostane levels did not correlate with LDL-cholesterol, blood pressure, serum bilirubin, or uric acid in either group, or with ACE-I status and disease duration in the NS group.

Urinary 8-iso PGF2{alpha} and 2,3-dinor-8-iso PGF2{alpha} levels corrected for creatinine excretion were not significantly different between the two groups (Table 3Go). Using univariate regression, neither urinary metabolite showed significant associations with serum albumin, ACE-I usage, disease duration, or any of the serum lipids and lipoproteins.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We have shown that in the NS there is reduced free-radical trapping by plasma as measured by the ORAC assay. However, products of peroxidative damage, as measured by F2-isoprostanes, were not significantly increased in NS compared with controls, even after correcting for arachidonic acid, the main substrate for F2-isoprostanes. We also found that plasma F2-isoprostane levels were inversely correlated with serum albumin in the NS group.

Defences against free-radical-mediated oxidative damage consist of anti-oxidative enzymes and the free-radical scavengers such as albumin [13]. In nephrotic subjects, decreased ORAC values represent a decreased ability to trap free radicals that is positively correlated to serum albumin. In response to reduced anti-oxidant defences, one may expect increased evidence of oxidative damage. However, this was not evident in our study. One explanation for this discrepancy could be that measures of oxidative damage in plasma, such as F2-isoprostanes, may not reflect tissue changes accurately, particularly in glomerulonephritis, where changes in oxidant stress may be confined to the cellular components of the glomerulus [6]. Furthermore, compared with studies showing that hypercholesterolaemia increases F2-isoprostane production [14,15], the lipid disorder in our nephrotic patients was a mixed hyperlipidaemia of relatively recent onset, and increase in peroxidative damage may be a longer-term consequence of sustained hypercholesterolaemia.

Bilirubin, a water-soluble antioxidant, was significantly lower in NS patients, reflecting reduction in albumin-bound unconjugated bilirubin. It is unlikely to contribute to changes in plasma anti-oxidant capacity significantly, particularly since bilirubin levels were not correlated with ORAC values. Uric acid, another water-soluble antioxidant, was significantly elevated in NS patients. Although, hyperuricaemia has been considered a marker of cardiovascular risk [16], in our NS patients elevated serum uric acid was not correlated with serum albumin or ORAC values, and is more likely to be a consequence of tubular dysfunction, hypertension, or the use of anti-hypertensives [17].

Platelets from hypoalbuminaemic, nephrotic plasma have a significantly higher affinity for arachidonic acid, and demonstrate increased cyclo-oxygenase (COX)-mediated prostaglandin synthesis [18]. Although this mechanism could provide an explanation for the inverse association between plasma F2-isoprostanes and serum albumin, COX inhibition with aspirin does not depress total urinary isoprostane excretion significantly [19], suggesting minimal contribution from this synthetic pathway. Accordingly, we did not find increased urinary F2-isoprostanes in the nephrotic group.

Limitations
Firstly, the potential confounding effect of ACE-I therapy in seven of our 14 NS patients needs to be considered. Angiotensin II increases vascular superoxide production [20] and inhibition of the renin–angiotensin system using ACE-I potentially reduces oxidant stress. However, urinary and plasma F2-isoprostanes did not differ between patients according to ACE-I usage. Secondly, additional measures of the oxidant/anti-oxidant system, such as vitamin E and C and protein oxidation products, would have strengthened our observations. Finally, a cross-sectional study design is not a rigorous test of a causal relationship. Anti-oxidant intervention trials examining cardiovascular end-points would further test our hypothesis, and a larger, longitudinal study would be required to explore the time-dependent effects of glomerulonephritis on oxidant stress.

In conclusion, this study provides evidence for decreased anti-oxidant defences in NS, and this may primarily be a consequence of hypoalbuminaemia. However, plasma markers of actual peroxidative damage were not increased. Decreased total plasma antioxidant potential in association with hyperlipidaemia may contribute to the progression of coronary artery disease and glomerulopathy in NS.



   Acknowledgments
 
This work was supported by research grants from the NHMRC, Medical Research Foundation at Royal Perth Hospital and the Australian Kidney Foundation. We thank Dr John Burnett for direct measurement of LDL-cholesterol. We are grateful to the renal physicians in Perth for referral of nephrotic patients for the study. Dr G. Dogra was in receipt of a Young Investigator's Award from the Australian Atherosclerosis Society for research related to this work (Dogra G, Herrmann SM, Playford DA et al. Endothelial dysfunction in nephrotic range proteinuria: relative roles of dyslipidaemia and oxidative stress. (Abstract; Clinical and Experimental Pharmacology and Physiology 2000; 27: A29.)



   Notes
 
Correspondence and offprint requests to: Dr G. Dogra, University Department of Medicine, Royal Perth Hospital, Box X2213 GPO, Perth, Western Australia 6001, Australia. Back



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 28. 9.00
Revision received 20. 3.01.



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