Plasma and urinary free 3-nitrotyrosine following cardiac angiography procedures with non-ionic radiocontrast media

Enrico Fiaccadori1, Umberto Maggiore1, Carlo Rotelli1, Roberto Giacosa1, Maria Lombardi1, Sibilla Sagripanti1, Silvia Buratti2, Diego Ardissino2 and Aderville Cabassi1

1Dipartimento di Clinica Medica, Nefrologia e Scienze della Prevenzione, Università degli Studi di Parma and 2Dipartimento del Cuore, Azienda Ospedaliera-Universitaria, Parma, Italy

Correspondence and offprint requests to: Enrico Fiaccadori, MD, PhD, Dipartimento di Clinica Medica, Nefrologia e Scienze della Prevenzione, Università degli Studi di Parma, Via Gramsci 14, 43100 Parma, Italy. Email: enrico.fiaccadori{at}unipr.it



   Abstract
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 Subjects and methods
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Background. Radiocontrast media (RCM) administration is a common cause of hospital-acquired acute renal failure, especially in high-risk patients, but mechanisms of nephrotoxicity have not been fully elucidated. Reactive oxidant species recently have been shown to play a role in experimental RCM nephropathy, while there is clinical evidence that acetylcysteine, an antioxidant drug, has a protective effect against RCM nephropathy in humans. However, no study has been published showing that RCM administration elicits oxidative stress in humans.

Methods. In an unselected series of patients undergoing elective cardiac catheterization for coronary artery angiography and/or angioplasty, we monitored the time course of plasma and urinary levels of free 3-nitrotyrosine (3-NT), a stable marker of peroxynitrite generation resulting from the in vivo reaction of superoxide and nitric oxide. Urinary 3-NT levels were measured as the ratio of urinary 3-NT to urinary creatinine. Measurements were taken at baseline, immediately after the procedure and at 24, 48 and 72 h.

Results. Twenty-six patients were studied (median age 67.5 years, range 42–86; baseline serum creatinine 1.0 mg/dl, 0.6–1.5; RCM dose 215 ml, 100–580). Plasma 3-NT levels slightly increased over the 72 h following the procedure (P<0.001), while urinary 3-NT levels peaked at the end of the procedure (P<0.001). Urinary 3-NT levels reached at the end of the procedure were proportional to the RCM dose administered (P = 0.017).

Conclusions. The present study provides indirect evidence that RCM administration in humans is associated with an increased production of 3-NT. Further studies are needed to ascertain whether oxygen- and nitrogen-derived radical species play a major role in the pathogenesis of RCM-associated nephrotoxicity in the clinical setting.

Keywords: nephrotoxicity; oxidative stress; peroxynitrite; radiocontrast media



   Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Radiocontrast media (RCM) nephrotoxicity is a common cause of hospital-acquired acute renal failure (ARF), especially in patients with pre-existing renal dysfunction and/or diabetes mellitus [1]. The pathogenesis of RCM nephropathy has been attributed to a combination of vasoconstriction and direct tubular toxicity, but the exact mechanisms are still not fully understood [1].

As the administration of N-acetylcysteine (a thiol-donating antioxidant) has been shown to reduce ARF incidence following RCM administration in some studies [26], though not in all [710], a possible role for reactive oxidants has been inferred in human RCM nephropathy. However, despite the expanding utilization of antioxidants to prevent RCM nephropathy, there is lack of evidence that administration of non-ionic RCM to humans elicits oxygen and nitrogen radical species generation.

Thus, to verify if RCM administration in humans is associated with increased levels of oxidant species, we monitored the time course of urinary and plasma 3-nitrotyrosine (3-NT) levels. 3-NT is a stable marker of generation of peroxynitrite, a highly reactive oxidative and nitrosative species which derives from the in vivo reaction of superoxide anion and nitric oxide [11].

The study was performed in an unselected series of patients undergoing RCM administration because of elective coronary artery angiography and/or angioplasty.



   Subjects and methods
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All adult patients consecutively referred to the Heart Disease Department for elective coronary artery angiography and/or angioplasty during a 3-week period (from January to February 2002) were considered for inclusion in the study. For logistic reasons, only patients undergoing cardiac catheterization on the morning of the first 3 days of the week were enrolled. We excluded patients with unstable angina, myocardial infarction in the previous 5 days, NYHA class IV, uncontrolled hypertension, those undergoing an emergency procedure, and those with a history of prior RCM reactions. Patients were not required to discontinue any drug during the course of the study. No drug was administered during the procedure, except for lidocaine hydrochloride for local anesthaesia. No patient was taking vitamin C, vitamin E or N-acetylcysteine. Written informed consent to participate in the study was obtained from each subject.

Before cardiac catheterization, we recorded data regarding demographics, medical history, medications, physical examination and baseline laboratory tests, on ad hoc pre-printed forms. As per routine, intravenous hydration with hypotonic saline (sodium chloride 80 mEq/l) was maintained at 1.5 ml/kg/h before (10 h), during and after (10 h) the procedure. During the procedure, haemodynamic data, cardiac rhythm, symptoms and signs of systemic reactions to RCM administration and total volume of contrast used were collected. A low-osmolality, non-ionic iodinated contrast medium was used (iopromide, Ultravist 370 Schering AG, Berlin Germany; iopromide 769 mg/ml, iodium 370 mg/ml, osmolality 770 mOsm/kg H2O).

Serum specimens at baseline were analysed for creatinine, blood urea nitrogen (BUN), glucose, cholesterol and blood cell count.

Urine samples were obtained by voiding immediately before the start of the procedure, at the end of the procedure in the haemodynamic laboratory, and at 24, 48 and 72 h in the ward. Blood samples were collected at the same time points, as 4 ml aliquots in EDTA-K3-containing vials which were centrifuged at 4°C at 2000 r.p.m. for 10 min. Plasma was then collected and stored at -20°C until analysis, in tubes containing 1/10 in volume of 4 mM butylated hydroxytoluene (BHT) to avoid amplification of oxidation during storage (-20°C) and the assay procedure. Urine samples were collected in 5 ml aliquots with 500 µl of 4 mM BHT, and subsequently stored at -20°C.

Solid-phase extraction coupled with high-performance liquid chromatography (HPLC) with UV detection at 274 nm was used to measure plasma and urine free 3-NT as previously described [12]. Free 3-NT was also checked after dithionite pre-treatment of the samples to exclude non-specific absorbance. Plasma samples underwent a pre-filtration step before analysis by two subsequent centrifugation procedures (Centricon YM-30, 30 000 MW and YM-3, 3000 MW, Millipore Co., Bedford, MA).

The detection limit for 3-NT was 0.12 µM with this method. Within- and between-assay coefficients of variation were 6.4 and 9.7 %, respectively. 3-NT in the urine was also confirmed by enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Nitrotyrosine ELISA kit, HyCult Biotechnology b.v., Uden, The Netherlands) and following the procedure indicated by the manufacturer and according to the method described by ter Steege et al. [13]. The detection limit for this solid-phase ELISA based on the sandwich principle is ~0.016 µM and the measurable range is from 0.016 to 1.2 µM. The intra- and interassay coefficients of variation were 5.2 and 10.1%, respectively. As urine in our patients did not contain relevant amounts of protein (negative urine dipstick), the ELISA detected predominantly free 3-NT in the urine samples. Values of urinary 3-NT levels were normalized by the urinary creatinine content in order to correct for urinary concentration. We further carried out in vitro experiments in order to exclude analytical interference among iopromide, 3-NT and creatinine in urine. For urinary RCM concentrations in the clinical range (0, 25, 50, 75, 100 and 125 mg of iodine/ml in the urine; n = 3 for each step), we measured dose–response curves and time–response curves which relate time of measurement to iopromide and creatinine urinary concentrations (before the administration of RCM, at the end of the procedure, at 24–48 and 72 h); no interference was found.

Six measurements of urinary 3NT and three measurements of plasma were missing because of insufficient sample volume (n = 4) or incorrect labelling of tubes during the collection and storage (n = 5).

ARF was defined either as an increase of at least 0.5 mg/dl of creatinine over baseline values at 48–72 h, or as an increase of at least 0.7 mg/dl of creatinine to a value >2 mg/dl [14].

The time change in urinary and plasma 3-NT was analysed using a repeated-measures mixed model. Both plasma 3-NT and the ratio of 3-NT to creatinine were transformed, taking the natural logarithm in order to improve normality. Normality assumption was verified using normal plots and the Shapiro–Wilk test.

We also performed one additional analysis. Because we found that urinary 3-NT levels peaked at the end of the procedure, we examined, using ordinary regression analysis, whether at this time point the increase of urinary 3-NT level (dependent variable) was proportional to the dose of RCM administered (independent variable).

Statistical analysis was performed with GenStat software (Release 6.1, 2002, VSN International, UK).



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Twenty-six patients were enrolled. Their demographic and clinical characteristics are shown in Table 1. The highest baseline creatinine level was 1.5 mg/dl. Four patients had diabetes mellitus. The median dose of RCM administered was 215 ml (range 100–580). No patient experienced adverse reactions during RCM administration. Only one patient underwent angioplasty. The median levels of plasma creatinine at baseline and after the procedure were 1.0 mg/dl, whereas at 24, 48 and 72 h, they were 1.1 mg/dl. Only one patient developed acute renal dysfunction; he was an 86-year-old, non-diabetic male with baseline serum creatinine of 1.5 mg/dl.


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Table 1. Baseline characteristics of the study population

 
Plasma levels of 3-NT are shown in Figure 1 (upper panel): a slight but sustained increase from baseline levels (P<0.001) throughout the observation period was evident. The urinary 3-NT to creatinine ratio rose significantly at the end of the procedure (an increase of ~180%, P<0.001) but returned to baseline levels 24 h later (lower panel). As shown in Figure 2, this increase was proportional to the dose of RCM administered (P = 0.017); no relationship was found between RCM dose and plasma 3-NT levels. The increase in plasma creatinine levels was not statistically related either to the dose of RCM or to urinary levels of 3-NT at 24 h (data not shown). The patient developing ARF received 150 ml of RCM, and his urinary levels of 3-NT at 24 h were just above the median levels for the study population.



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Fig. 1. Time course of 3-nitrotyrosine (3-NT) levels in plasma (upper panel, left), of the 3-NT to creatinine ratio in urine (lower panel, left), and of either 3-NT or creatinine concentration in urine (small panels, right). Measurements were taken before starting coronary angiography, at the end of this procedure, and after 24, 48 and 72 h. At each time point, plasma 3-NT measurements were available in 26, 25, 25, 25 and 26 subjects, respectively. Urinary 3-NT measurements were available for 26, 26, 25, 24 and 23 subjects. Bars represent mean±SE. Data are log transformed. In each panel, the reported P-value refers to the test for any time change during the study. The symbol above the error bar indicates whether the test for a change from baseline at that time point was significant. +P<0.05; °P<0.01; *P<0.001.

 


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Fig. 2. Relationship between the change in the urinary 3-nitrotyrosine (3-NT) to creatinine ratio and the dose of contrast media administered. The change was computed as the difference between the measure taken after and that taken before coronary angiography. The urinary 3-NT to creatinine ratio is log transformed.

 


   Discussion
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Our study shows that the administration of iopromide, a low-osmolality, non-ionic RCM, for cardiac angiography is followed by an increased 3-NT appearance in plasma and urine, and that the pattern of 3-NT increase in plasma is different from that in urine: plasma 3-NT levels increased over many hours, whereas urinary 3-NT levels increased after the end of the procedure, proportionally to the amount of RCM dose administered.

Some limitations of our study must be taken into account. First of all, it can be argued that the increase of urinary 3-NT to creatinine ratio observed after the angiographic procedure could have been determined by a transiently reduced excretion of creatinine, rather than by an increased production of 3-NT. As a matter of fact, we observed a reduction in urinary creatinine concentration after the procedure. Since we did not obtain a timed urine collection, we cannot provide direct proof to refute this alternative explanation. However, we believe this hypothesis unlikely; in fact, contrast media are known to induce transient osmotic diuresis and poliuria, thus lowering the urinary creatinine concentration. In any case, even the concentration of urinary 3-NT was significantly increased after the procedure.

Secondly, we could not determine the main site of peroxynitrite generation after RCM administration. In this case, both vascular wall and tubular structures could be involved in the peroxynitrite production, leading to increased plasma and urinary 3-NT generation. However, the observation of an early and relevant increase in urinary 3-NT levels, as compared with the slower and continuous increase observed in plasma, seems to suggest a more direct and relevant effect of RCM on the kidney.

Thirdly, we were unable to demonstrate that the renal outcome and/or serum creatinine course was related to 3-NT level variations. Even though urinary 3-NT levels were increased at the end of the procedure, only one patient suffered from ARF following RCM administration, and his urinary 3-NT levels at 24 h were just above the median levels for the study population. However, our study was designed in a small sample of patients to verify the hypothesis that the administration of RCM is associated with increased oxidative stress marker generation in humans, and not to examine the relationship between oxidative stress and the incidence of RCM nephropathy. In any case, the increased plasma and urinary 3-NT following RCM administration represents important evidence of an in vivo rise in oxidative stress.

Fourthly, we cannot exclude that the increase in 3-NT levels could represent a non-specific response to an acute increase in plasma osmolarity due to RCM administration. The infusion of hyperosmolar substances can be associated with increased oxidative stress, at least in experimental conditions [15].

Finally, despite the fact that it has been suggested that 3-NT could also be generated via peroxynitrite-independent mechanisms, the in vivo relevance of non-peroxynitrite-mediated tyrosine nitration pathways still remains to be demonstrated [16].

Our results thus provide indirect evidence that the administration of RCM in humans is associated with increased generation of peroxynitrite. In fact, 3-NT is a stable marker of the in vivo generation of peroxynitrite, a highly reactive species derived from superoxide anion and nitric oxide [11]. An increased formation of peroxynitrite has already been demonstrated in experimental conditions of renal ischaemia/reperfusion not related to RCM administration, and is thought to be, at least in part, responsible for renal parenchymal damage, due to peroxynitrite-associated oxidative and nitrosative effects on several targets, such as sulfhydryl groups and aromatic rings of proteins, cell membrane lipids and nucleic acids [11].

RCM administration causes intrarenal vasoconstriction, as renal blood flow decreases within 1 h in humans [17]. Peroxynitrite might contribute to acute RCM vasoconstrictive effects through the reduction of the nitric oxide availability and also by the nitrosation of tyrosine residues of enzymes, such as prostacyclin synthase and nitric oxide synthase, involved in the synthesis of medullary vasodilators; the latter are likely to play a critical role in the control of vascular tone in the external medulla, where the ischaemic damage induced by RCM seems to occur [18].

Taken together, these studies make plausible the hypothesis that the effects of RCM administration could involve oxygen- and nitrogen-derived radical species, whose peroxynitrite is a combined expression: even though animal models of RCM nephropathy support the involvement of free radical species [19], up to now the evidence in humans was only indirect, as it was derived from the observation that the administration of N-acetylcysteine (a thiol donor) may reduce the incidence of RCM nephropathy [26].

Our findings also support the notion that the dose of RCM might play an important role in the genesis of RCM nephropathy. In fact, it has been shown recently that an RCM dose above the threshold calculated on the basis of a weight- and creatinine-adjusted maximum RCM dose is a strong predictor of RCM nephropathy requiring dialysis [20]. Moreover, taking into account the dose of RCM administered might also help reconcile some apparently discordant findings in studies performed on patients undergoing cardiac angiography: in fact, in five studies, the prophylactic administration of an antioxidant (N-acetylcysteine) was definitely protective [26], in another study it was protective only in patients receiving <140 ml of RCM [7], whereas the administration of the drug brought no benefit whatsoever to patients receiving low RCM doses [9].

In conclusion, our study documents that the administration of RCM for cardiac angiography studies is associated with increased urinary and plasma 3-NT levels, and that the increase in the urine is proportional to the dose of RCM used.

The demonstration that frequently observed clinical situations at risk for ARF, such as RCM administration, are associated with increased levels of reactive oxygen and nitrogen species could further encourage the use of antioxidant therapies. However, future studies are necessary to determine whether or not increased radical species following RCM administration lead to renal damage in humans, and whether a preventive strategy aimed to tailor the dose of the antioxidant to the amount of RCM could add further benefit, especially in high-risk patients.

Conflict of interest statement. None declared.



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 4.03
Accepted in revised form: 20.11.03