High-Dose N-Acetylcysteine in Cardiac Surgery

Background

Renal impairment following cardiopulmonary bypass is common. 11.4% (1) to 42% (2) of patients with previously normal renal function show a postoperative rise in serum creatinine. While most of these patients do not require either short or long term renal replacement, the mortality of patients with acute renal failure is substantially greater than those who do not develop renal dysfunction1.

Cardiopulmonary bypass activates components of the non-specific immune system, which leads to the generation of compounds containing oxygen free radicals. A study of 14 patients undergoing cardiac surgery found increased levels of serum lipid peroxidation products (thiobarbituric acid reactive substances) within 15 minutes of the commencement of cardiopulmonary bypass, which returned to preoperative levels by the following morning. The total serum antioxidative capacity was correspondingly decreased intraoperatively, and remained decreased at 24 hours postoperatively (3). A similar study of total plasma antioxidant status showed decreased levels up to 72 hours postoperatively(4). It is clear that cardiopulmonary bypass causes oxidative stress and depletion of antioxidant capacity.

N-acetylcysteine is routinely used in the treatment of paracetamol overdose. Paracetamol is metabolised by the liver by cytochrome p450 to form a toxic reactive oxygen compound. This metabolite is normally detoxified by conjugation with hepatic reduced glutathione (GSH). In overdose, the GSH stores are depleted, leading to liver cell necrosis through oxidative damage. N-acetylcysteine is a sulfydryl group donor, which allows regeneration of GSH, thus augmenting the antioxidant defence of the liver. Treatment of paracetamol overdose is currently the only approved indication for N-acetylcysteine. Possible adverse reactions include an urticarial rash, nausea and vomiting, and an anaphylactoid reaction (involving hypotension, tachycardia, bronchospasm, and facial oedema). These reactions occur most commonly either during, or at the end of, the period of the loading dose infusion, and may be concentration related (5).

Oxidative stress can be produced experimentally using hypertonic glycerol. Intramuscular injection of hypertonic glycerol in rats precipitated acute renal failure associated with a marked decrease in renal reduced glutathione (GSH) levels. Pretreatment with N-acetylcysteine largely prevented these changes (6). During reperfusion after renal ischaemia in rats, renal blood flow was reduced compared to pre-ischaemic values. GFR was also reduced, and plasma peroxynitrite (a marker of nitric oxide synthesis, a component of oxidative stress) was increased. Pretreatment with N-acetylcysteine partially prevented these changes (7). N-acetylcysteine (combined with a nitric oxide donor and an endothelin converting enzyme inhibitor) also decreased the effect of renal ischaemia/reperfusion injury in dogs, in terms of improved renal function as well as reduced interstitial pro-inflammatory cytokines and inducible nitric oxide synthase (8).

Radiocontrast commonly causes renal dysfunction, in part through oxidative stress in the kidney. While not an approved indication, intravenous N-acetylcysteine has been used successfully to attenuate radiocontrast induced nephropathy, and was more effective than standard intravenous fluid prophylaxis (relative risk 0.28) (9). While not an approved product indication, N-acetylcysteine is currently used in our hospital for this purpose.

Infrarenal abdominal aortic aneurysm repair commonly causes ischaemia/reperfusion injury to the kidney, producing a similar oxidative stress to that of radiocontrast. A mixture of antioxidants including N-acetylcysteine given to patients undergoing infrarenal abdominal aortic aneurysm repair produced a significantly better creatinine clearance (compared to placebo controls) 48 hours postoperatively (10).

While never investigated for its effects on renal function after cardiac surgery, the effect of perioperative N-acetylcysteine on other systems has been studied. The oxidative burst response of neutrophils from patients undergoing cardiopulmonary bypass was significantly attenuated by infusion of N-acetylcysteine into the bypass circuit (11).

A study of dogs undergoing cardiopulmonary bypass found preload-recruitable stroke work was maintained at pre-bypass levels in those animals given N-acetylcysteine, whereas it fell in control animals. N-acetylcysteine significantly enhanced myocardial oedema resolution, and prevented the rise in plasma 8-isoprostane (a marker of oxidative stress) seen in control animals (12). Similar results were found in a study of 40 patients undergoing cardiac surgery: left ventricular biopsy specimens showed less 8-isoprostane content and less nitrotyrosine (a marker of nitric oxide production; nitric oxide can act as an oxygen free radical). There were no differences in haemodynamics or clinical outcomes noted, but unfortunately indices of renal function were not examined in this study (13).

Ascorbate is an antioxidant which might be expected to have a similar action to N-acetylcysteine. Supplemental ascorbate was given to 43 patients before, and for 5 days after, coronary artery bypass surgery. Patients receiving ascorbate had a 16.3% incidence of postoperative AF, compared to 34.9% in control subjects, perhaps by reducing oxidative damage to the myocardium (14).

Pulmonary endothelium-dependent vasodilation is usually impaired after cardiopulmonary bypass, a process thought to be related to reactive oxygen species. Pulmonary vasodilation induced by acetylcholine following cardiopulmonary bypass was better maintained in 12 patients given a cocktail of antioxidants (including N-acetylcysteine) compared to that in 10 control patients (15).

There is thus evidence that in the immune, cardiovascular and respiratory systems, N-acetylcysteine may be of benefit to patients undergoing cardiac surgery.

Clinical sepsis or experimental exposure to lipopolysaccharide stimulate cells of the inflammatory system to form oxygen free radicals. N-acetylcysteine decreases cytokine and adhesion molecule gene expression and NF-kB activation in vitro. In animal models of sepsis N-acetylcysteine reduces inflammatory cell chemotaxis and improves survival (as reviewed by ref. 16). In patients suffering oxidative stress due to sepsis, N-acetylcysteine results in decreased NF-kB activation and decreased IL-8 production (16).

Hypotheses N-acetylcysteine administered from the time of induction of anaesthesia prior to cardiac surgery and for 24 hours postoperatively results in decreased change in serum creatinine from baseline to peak level within first 5 postoperative days.

Other secondary outcomes which will be measured include:

- better creatinine clearance over the first postoperative day;

- shorter ICU stay;

- shorter hospital stay;

- lower serum creatinine levels on day 2 post-op;

- greater plasma antioxidant activity;

- less oxidative stress;

- less NF-kB activation in the cellular components of blood;

- less pro-inflammatory cytokine response; and

- less activation of the nitric oxide synthase pathway.

Study Design - overview and rationale

Patients will be randomised to receive N-acetylcysteine from the induction of anaesthesia until 24 hours postoperatively, or a placebo (5% glucose).

Serum creatinine is the most commonly used clinical indicator of renal function along with urine output. Both will be measured for 48 hours postoperatively - the time period during which renal impairment is most likely to develop. A more sensitive indicator of renal dysfunction is creatinine clearance. This will be measured over the first 24 hours postoperatively, and will form the primary end point of the study. As patients in our intensive care unit are routinely given frusemide to maintain a postoperative urine output of >0.5ml/kg/hr (once fluid volume, inotropy and vascular tone have been manipulated into what is judged to be acceptable ranges), the dose of frusemide required is also an indicator of renal function. Rarely are other diuretics given, though where this is the case it will also be noted.

The efficacy of N-acetylcysteine in preventing oxidative stress will be assessed using a measure of total plasma antioxidant activity (the bathocuproine assay) (17) and by quantification of the 8-isoprostane levels (18). Total antioxidant activity and 8-isoprostane have previously been shown to be affected by cardiac surgery and N-acetylcysteine.

Any renal effect of N-acetylcysteine will be correlated with levels of plasma pro-inflammatory cytokines (IL-1, IL-6 and TNF-alpha), which are known to be associated with oxidative-stress induced renal failure (19, 20). Activation of inducible nitric oxide production is also associated with renal failure (21), and the effect of N-acetylcysteine on nitric oxide synthase mRNA expression in the cellular components of blood will be assayed by real-time PCR. Nitric oxide production will be assessed by measurement of by plasma nitrotyrosine concentration. Assay of nitrotyrosine is superior to the traditional Greiss reaction (which measures nitrate and nitrite derivatives of nitric oxide), as nitrate and nitrite undergo renal excretion, and many of these patients will have altered renal function.

At a molecular level, many of the genes responsible for stimulating oxidative stress are regulated by the promoter NF-kB. The cellular components of blood will be assayed for NF-kB using an established ELISA technique (22) thought to be more sensitive than the electrophoretic mobility shift assay used to demonstrate an effect of N-acetylcysteine in human sepsis (16). NF-kB in the cellular components of blood will also be assayed using real-time PCR.

Randomisation The randomisation will be based on random numbers generated by computer. Once consent is obtained, the allocation of either treatment with N-acetylcysteine or placebo will be organised by an independent person (clinical trials pharmacist) who will dispense the coded infusion bags. This will be delivered to the anaesthetic staff looking after the patient in theatre, and the ICU nurse caring for the patient postoperatively.

Detailed protocol Immediately following the induction of anaesthesia, prior to the first surgical incision, N-acetylcysteine will be administered in a dose of 150 mg/kg IV in 200 mL 5% glucose over 15 minutes followed by continuous IV infusion of 50 mg/kg in 500 mL 5% glucose over 4 hours, then 100 mg/kg in 1 L 5% glucose over 20 hours (total dose 300 mg/kg in 24 hours). This is the standard dose of N-acetylcysteine used clinically in paracetamol overdose (5.) Patients randomised to receive placebo will receive an equivalent volume of 5% glucose. The appearance of the 5% glucose and N-acetylcysteine solutions is similar, and there will be no marking on the infusion bag other than an identifying study number.

A 24 hour urine collection will begin immediately on arrival in ICU, to allow determination of creatinine clearance. This will be measured in the hospital clinical pathology laboratory. Creatinine clearance will be the primary endpoint of the study.

Clinical data will be recorded as detailed below by the investigators or the ICU research nurse.

Four 20 ml samples of heparinised blood will be taken from the arterial line for cytokine and molecular analysis. Samples will be taken immediately after the induction of anaesthesia, on arrival in the intensive care unit, and 6 and 24 hours postoperatively. Immediately following collection, the blood will be centrifuged at low speed to separate the plasma from the cellular components, both of which will be stored in aliquots at -70 degrees prior to batch analysis.

Analysis of plasma total antioxidant activity and 8-isoprostane, IL-1, IL-6, TNF-alpha and nitrotyrosine concentrations will be performed using commercially available ELISA reagent kits (Oxford Biomedical Research, Oxis Research, BioCore). The cellular components of blood will be assayed for NF-kB concentration using a commercially available ELISA kit (Oxford Biomedical Research), and for iNOS and NF-kB mRNAs using a real-time PCR machine and Applied Biosystems pre-developed assay reagents with 18S as the endogenous control. The principal investigator has experience of these or similar techniques.

Statistics and power calculation Using data available from our cardiac surgery database of over 2500 patients in the last 5 years, we expect a mean increase in serum creatinine from baseline to a peak value of 50 micromol/L in the control group, with a standard deviation of 30 micromol/L. Given these changes, 60 patients are needed to have a 90% power of detecting a 30 micromol/L difference between the control and the intervention group at an alpha of 0.05.

Data collection Data collection will be performed by the principal investigator, ICU research nurse and ICU nursing staff.

The following variables will be obtained:

Name gender, age, and medical record number Date of admission to ICU Operative procedure and time on cardiopulmonary bypass Preoperative assessment of left ventricular function Serum creatinine and urea preoperatively, immediately postoperatively, and every 24 hours thereafter (as measured for clinical purposes) Doses of frusemide administered (or rate of frusemide infusion) Use of inotropes Cardiac output whenever measured for clinical purposes in the first 24 hours postoperatively Urine output in each 6 hour period for the 24 hours postoperatively Date of discharge from ICU and hospital or death

Protocol violations All protocol violations will be recorded. It will then be decided whether the nature of such violation had been such that the patient should be excluded from primary data analysis. Such evaluation will be blinded to treatment.