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
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Abstract |
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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)
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Introduction |
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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 (8-iso-PGF2
) [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].
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Subjects and methods |
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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.
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Results |
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Urinary 8-iso PGF2 and 2,3-dinor-8-iso PGF2
levels corrected for creatinine excretion were not significantly different between the two groups (Table 3
). 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.
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Discussion |
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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 reninangiotensin 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.
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Acknowledgments |
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Notes |
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References |
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