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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2019  |  Volume : 30  |  Issue : 1  |  Page : 45-52
Study of contrast-induced oxidative stress in nondiabetic patients undergoing coronary angiography

1 Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
2 Department of Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Cardiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
4 Department of Nephrology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India

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Date of Submission02-Dec-2017
Date of Acceptance30-Dec-2017
Date of Web Publication26-Feb-2019


Administration of iodinated contrast media is associated with serious complications such as acute kidney injury (AKI). Oxidative stress is implicated as a major mechanism underlying the production of contrast-induced AKI (CI-AKI). There are very few human studies on oxidative stress occurring after contrast administration. Twenty-seven patients scheduled for coronary angiography were recruited. An average of 22.2 mL low-osmolal nonionic contrast was administered. Plasma conjugated dienes (CD), lipid hydroperoxides (LOOH), malondialdehyde (MDA), protein carbonyl (PC), protein thiols (PTs), ferric reducing ability of plasma (FRAP), erythrocyte super oxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase were estimated before, 30 min, 2 and 4 h after contrast administration. CD, LOOH, MDA, and PC increased (P <0.001), whereas PTs, FRAP, SOD, CAT (P <0.001), and GPx (P = 0.013) decreased in the first 4 h. Estimated glomerular filtration rate (eGFR) showed inverse association with MDA and positive association with GPx. The study provides evidence for oxidative stress following contrast administration even in the absence of predisposing factors. Association of eGFR with MDA and GPx indicate kidney as the source of oxidative stress. Hence, antioxidant therapy before contrast administration helps to prevent the development of oxidative stress, thereby reducing the risk of CI-AKI.

How to cite this article:
Manda P, Srinivasa Rao P V, Bitla AR, Vinapamula KS, Jeyaseelan L, Rajasekhar D, Vishnubhotla S. Study of contrast-induced oxidative stress in nondiabetic patients undergoing coronary angiography. Saudi J Kidney Dis Transpl 2019;30:45-52

How to cite this URL:
Manda P, Srinivasa Rao P V, Bitla AR, Vinapamula KS, Jeyaseelan L, Rajasekhar D, Vishnubhotla S. Study of contrast-induced oxidative stress in nondiabetic patients undergoing coronary angiography. Saudi J Kidney Dis Transpl [serial online] 2019 [cited 2021 Oct 21];30:45-52. Available from: https://www.sjkdt.org/text.asp?2019/30/1/45/252932

   Introduction Top

Iodinated contrast medium is widely used in various imaging techniques including coronary angiography. One of the serious complications of administration of iodinated contrast media is contrast-induced acute kidney injury (CI-AKI), which is also the third most common pre-renal cause of AKI. Risk factors for CI-AKI include pre-existing renal disease, hypotension, heart failure, anemia, old age, diabetes mellitus, and concomitant nephrotoxic medications.[1] Oxidative stress plays an important role in the generation of CI-AKI.[2] The basis for this concept is indirect and mainly comes from animal studies[3],[4],[5] and the preventive role of drugs having antioxidant properties.[5],[6],[7] There are very few human studies on oxidative stress following administration of contrast.[8],[9] More information on the effects of contrast on oxidative stress will help in better prevention strategies for CI-AKI. Hence, this study was taken up to understand the time course changes in the markers of oxygen radical production and markers of antioxidant defense following contrast administration.

   Materials and Methods Top

Patients scheduled to undergo coronary angiography (CAG) in the Department of Cardiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, from December 2014 to March 2015 were considered for recruitment into the study. Six hundred and fifty-seven patients underwent elective coronary angiography during this period. Of these, 30 consecutive patients who met the inclusion criteria and willing to participate were recruitted for the study. Patients with normal serum creatinine levels who were found suitable and scheduled for elective CAG by the cardiologist were included in the present study. Patients with history of preexisting renal disease, diabetes mellitus, hypertension, hypotension, thyroid disorders, cardiogenic shock, those on glucocorticoid therapy, or allergic to contrast media and patients unwilling to participate were excluded from the study. The study was approved by the Institutional Ethics Committee. Written informed consent was obtained from all the patients. Patients were advised liberal oral fluids before angiography as part of patient management protocol of the department. The contrast used was low-osmolal nonionic contrast agent, Iohexol (Omnipaque 350 mg I/mL, Wipro GE Healthcare, China). Of the 30 patients included, three patients who developed CI-AKI as per the modified European Society of Urogenital Radiology guidelines[10] were not included for data analysis to maintain homogeneity of the study groups which will strengthen the conclusions of the study. The average dose of the contrast administered was of 22.2 mL (range 15–50 mL). CAG was performed by using standard techniques.

Five mL of peripheral venous blood sample was collected in nitrogen filled heparinized tubes before, 30 min, 2 and 4 h after administration of contrast. One sample for creatinine was collected at the end of 48 h for all the patients. Plasma was separated and hemolysate obtained by washing erythrocytes four times with saline before lysing with equal volume of cold distilled water. The separated plasma and hemolysate were stored at -80°C until analysis. The oxidant and antioxidant markers were assayed using the following methods:

Conjugated dienes (CD) with alternating double and single bonds between carbon atoms absorb wavelengths of 230–235 nm in the UV region and the value is expressed as optical density.[11] Lipid hydroperoxides (LOOH) which are products of fatty acid oxidation abstract hydrogen from a methylene group, forming a carbon-centered radical that reacts with molecular oxygen to form lipid peroxide that is detected spectrophotometrically using Fox reagent at 560 nm.[12] Malondialdehyde (MDA), which is another indicator of lipid peroxidation, was measured spectrophoto-metrically as thiobarbituric acid reactive substances (TBARS) after precipitating the proteins with trichloroacetic acid (TCA).[13] Protein carbonyl (PC) content was measured based on the method described by Levine et al, in which 2, 4-dinitro phenyl hydrazine reacts with PCs forming a Schiff base to produce the corresponding hydrazone that is analyzed spectrophotometrically at 370 nm.[14] Assay for thiols was performed using Ellman's reagent or 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB). The procedure is based on the reaction of thiols with DTNB to give a mixed disulfide and 2-nitro-5-thiobenzoic acid (TNB) which is quantified by the absorbance of the anion (TNB) at 412 nm.[15] Ferric reducing ability of plasma (FRAP) assay was performed as per the method described by Benzie and Strain. The antioxidant power reduces ferric ions to ferrous ions at low pH resulting in the formation of a colored ferrous-tripyridyltriazine complex. FRAP values are obtained by comparing the change in absorbance of test reaction mixtures at 593 nm with ferrous standards.[16] Super oxide dismutase (SOD) activity measurement is based on the ability of SOD to inhibit the oxidation of adrenaline to form a pink-colored adrenochrome. SOD activity was determined by monitoring the rate of adrenochrome formation spectrophotometrically at 470 nm. One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of adrenaline oxidation.[17] Catalase (CAT) activity was measured by continuous spectrophotometric rate determination. One unit of CAT will decompose 1.0 μM of H2O2 per min at pH 7. The rate of disappearance of H2O2 is observed as rate of decrease in absorbance at 240 nm.[18] Glutathione peroxidase (GPx) activity was estimated by an enzymatic reaction initiated by the addition of terbutyl hydroperoxide (TBHP) to a reaction mixture containing reduced glutathione (GSH), reduced nicotinamide adenine dinucleotide (NADPH) and glutathione reductase (GR). The change in absorbance was monitored spectrophotometrically at 340 nm.[19] Activities of holo and apo forms of glutathione reductase (GR) in hemolysate are measured with and without addition of FAD and by spectrophotometric determination of NADP formed.[20] All the analyses were performed using Lambda 25 UV-visual double beam spectrophotometer (Perkin Elmer, Singapore). Within-run imprecision for the parameters ranged from 0.9% to 3.9%. Creatinine was estimated by standard methods using comercial kits on CX5 autoanalyser (Synchron Cx5 Beckman Coulter, Inc., Galway, Ireland). The estimated glomerular filtration rate (eGFR) was calculated using Cockcroft Gault formula.[21]

   Statistical Analysis Top

Data were expressed as mean ± standard deviation. Data were converted to percentage of baseline value before performing analysis of variance (ANOVA) for repeated measures with bonferroni post hoc testing. Generalized estimating equations were used to study the association between repeated measures variables. Analysis of the data was performed using Microsoft Excel 2007 for Windows (Microsoft Corporation, Redmond, WA, USA), Statistical Package for the Social Sciences (SPSS) version 16.0 (SPSS Inc., Chicago, IL, USA). A value of P <0.05 was considered as significant.

   Results Top

The demographic and base line characteristics of patients are presented in [Table 1]. The changes in the oxidant markers observed are presented in [Table 2] and [Figure 1]. CD, LOOH, MDA and PC showed a significant increase (P < 0.001) in the first 4 h indicating production of reactive oxygen species (ROS) due to the administration of the contrast. There was a significant decrease in thiols, FRAP, SOD, CAT (P <0.001) and GPx (P = 0.013) demonstrating a decrease in antioxidant defense [Table 3] and [Figure 2]. eGFR was significantly lower in cases and showed significant inverse association with MDA (B = -2.398, P = 0.015) and positive association with GPx (P = 0.814, P<0.001) [Figure 3].
Table 1: Demographic and baseline characteristics of the patients (n=27).

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Table 2: Time course changes in the oxidant markers studied.

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Figure 1: Percentage time course changes in the oxidant markers studied.
A: ANOVA for repeated measures (0–4 h), B: 0 h versus 30 min, C: 0 h versus 2 h, D: 0 h versus 4 h, E: 30 min versus 2 h, F: 30 min versus 4 h, G: 2 h versus 4 h. ANOVA was performed after transforming the data to percentages of baseline (0 h) value to include only the changes observed during the study period in the analysis.

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Table 3: Time course changes in the antioxidant parameters studied.

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Figure 2: Percentage time course changes in the antioxidant parameters studied.
A: ANOVA for repeated measures (0–4 h), B: 0 h versus 30 min, C: 0 h versus 2 h, D: 0 h versus 4 h, E: 30 min versus 2 h, F: 30 min versus 4 h, G: 2 h versus 4 h. ANOVA was performed after transforming the data to percentages of baseline (0 h) value to include only the changes observed during the study period in the analysis.

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Figure 3: Regression curve analysis of eGFR with MDA (A) and GPx (B).
eGFR: estimated glomerular filtration rate, MDA: Malondialdehyde, GPx: Glutathione peroxidase eGFR showed inverse association with MDA and positive association with GPx.

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   Discussion Top

Oxidative stress has been implicated in CI-AKI.[1],[2] AKI is one of the important risk factors for progression to chronic kidney disease and hence needs to be identified and managed early. Experimental findings showed that administration of iodinated contrast induces renal hypoxia which enhances ROS production, mainly in the medullary thick ascending limb that has dense mitochondrial population. The protective effect of ROS scavenging drugs[6],[7],[8] supports this. As the patients undergoing coronary angiography will be having CAD, the baseline oxidative stress that may be present due to CAD is likely to confound the conclusions drawn on oxidative stress that occurs after the administration of contrast. To avoid this, the data were converted into percentages with baseline as 100% before inclusion into statistical analysis. CD and LOOH, followed by MDA are the products of action of free oxygen radicals on lipids. In the present study, plasma levels of CD and LOOH showed significant increase during the first 4 h after the administration of contrast (P <0.001). The levels increased in the first 2 h followed by a decline but were still higher than the baseline value at the end of 4 h [Table 2]. These findings provide evidence for production of ROS immediately after the contrast administration. There are no studies reporting on changes in CD, which is the earliest of the lipid peroxidation products, after contrast administration. Saitoh et al[8] reported increased urinary LOOH at 2 h after CAG, which was completely abolished with administration of glutathione prior to the procedure. This study also supports our findings. Animal studies[3] reported increased kidney tissue MDA levels after administration of contrast. In the present study, plasma MDA increased more than threefold during the first 4 h after contrast administration. PCs, the product of action of free oxygen radicals on proteins, showed 74% increase (P <0.001). Similar findings are reported by others for MDA[9] and PCs.[5]

As the increase in these initial lipid peroxidation products was observed at 30 min, we collected blood samples at 0 and 10, 15 and 20 min for another five patients inquisitive to know how early ROS production starts. We could find a significant increase in CD at 15 min (0.35 ± 0.02 vs. 0.36 ± 0.02; 2.6 % increase; P = 0.002) and in LOOH at 20 min (3.12 ± 1.7 vs. 4.18 ± 2.13; 9.5% increase; P = 0.028), whereas MDA levels showed no change (1.06 ± 0.4 vs. 1.06 ± 0.4; P = 0.228) when compared to baseline levels. Thus our findings of the present study provide strong evidence for ROS production due to contrast administration.

Plasma FRAP levels which reflect the overall antioxidant capacity decreased reaching 59.8% of the base line by the 4th h (P <0.001). Earlier studies reported a decrease[22] or no change[23] in FRAP levels after contrast administration. However, these were either animal studies or studies on the effect of antioxidants in patients undergoing interventions using contrast media. Protein thiol (PT), another marker of anti-oxidant defense, decreased reaching 65% of the baseline levels (P <0.001) at the end of 4 h after contrast administration. This finding is in line with that of Deng et al,[5] who reported a decrease in PTs after contrast administration that is prevented by prior administration of antioxidants.

There was a continuous fall in the activity of erythrocyte SOD resulting in 7% of the baseline levels (P <0.001) at the end of 4 h after contrast administration. Studies conducted on rats,[3],[4] and in humans[22] have shown a similar decrease in the SOD activity. However, Akgüllü et al[7] did not find change in SOD levels. Erythrocyte CAT activity and GPx activity also decreased during the study period after contrast administration resulting in 19% and 5% of the baseline levels respectively (P <0.001 and P <0.013 for CAT and GPx, respectively) at the end of 4 h after contrast administration. Earlier studies have also reported a decrease in CAT[3],[4] and GPx[3],[6] activity after contrast administration. There are no studies on GR in CI renal injury. In the present study also, erythrocyte GR did not alter significantly after contrast administration.

The degree of renal injury following contrast administration is dependent on baseline renal function, presence of risk factors such as diabetes, volume, and type of contrast used. All our patients had normal baseline renal function, were nondiabetic and received low-osmolal, nonionic contrast medium in low volumes (15–50 mL). Finding of oxidative stress even in these minimal renal injury conditions without any additional risk factors provide a strong evidence for oxidative stress as the main mechanism of renal injury due to contrast administration. Further supporting this concept, we observed significant negative association between eGFR and MDA (B = -2.398, P = 0.015) and positive association with GPx (B = 0.814, P <0.001) indicating that the source of oxidative stress is kidney. Findings of the present study indicate that contrast administration results in oxidative stress which is likely to contribute to the production of AKI. Our findings strengthen the concept of administration of antioxidants before contrast administration to reduce the iatrogenic renal injury and the associated morbidity and mortality.


We would like to thank Sri Venkateswara Institute of Medical Sciences and Tirumala Tirupati Devasathanams, Tirupati for providing financial support under Sri Balaji Aarogya Vara Prasadini Scheme (GRANT NO: SBAVP-RG/MD/22).

Conflicts of interest:

None declared.

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Correspondence Address:
P V. L. N Srinivasa Rao
Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati - 517 507, Andhra Pradesh
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DOI: 10.4103/1319-2442.252932

PMID: 30804266

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]


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