Abstract
Introduction Oxidative stress plays an important role in the pathogenesis of diabetic nephropathy (DN). This study examined if use of N-acetylcysteine for a month in moderate doses would reduce the oxidative stress in patients with DN and reduce the proteinuria.
Methods Fifteen volunteers with DN participated in the study. Participants took capsule form of N-acetylcysteine 1 gm twice a day for a month. Spot urines were collected and tested for protein/creatinine on days 1 and 30. Sera were collected on days 1, 15, 30, and 60 and tested for several oxidative stress biomarkers.
Results There was no significant change in proteinuria or any of the oxidant stress markers at any point: protein-creatinine ratio (day 1, 1.6 ± 1.8; day 30, 1.3 ± 1.3), 8-isoprostane (day 1, 5.9 ± 4.2 pg/mL; day 15, 4.67 ± 2.4 pg/mL; day 30, 5.1 ± 2.8 pg/mL; and day 60, 4.7 ± 1.9 pg/mL), total antioxidant status (day 1, 1.5 ± 0.1 mM; day 15, 1.6 ± 0.2 mM; day 30, 1.5 ± 0.1 mM; and day 60, 1.5 ± 0.2 mM), aconitase (day 1, 7.9 ± 5.9 mU/mL; day 15, 10.1 ± 5.9 mU/mL; day 30, 8.9 ± 6.2 mU/mL; and day 60, 7.8 ± 5.5 mU/mL), glutathione peroxidase (day 1, 261.4 ± 56.4 mU/mL; day 15, 263.9 ± 57.2 mU/mL; day 30, 269.2 ± 66.0 mU/mL; and day 60, 257.5 ± 48.2 mU/mL), and superoxide dismutase (day 1, 242.6 ± 79.3 mU/mL; day 15, 252.1 ± 68.1 mU/mL; day 30, 262.0 ± 73.3 mU/mL; and day 60, 255.7 ± 61.5).
However, 4 patients with initial high isoprostane levels showed nonsignificant decline at each subsequent time point.
Conclusions N-acetylcysteine in moderate doses given over a month did not have significant effect on the overall oxidative stress in patients with DN and did not reduce proteinuria.
Oxidative stress plays a major role in the pathogenesis of diabetic complication especially diabetic nephropathy.1-4The reactive oxygen species may be generated in diabetics from potentially diverse sources, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase accentuated by renin-angiotensin system, advanced glycation end products, mitochondrial overproduction in response to hyperglycemia, and the uncoupling of nitric oxide synthase. Our primary hypotheses was that an antioxidant drug like N-acetylcysteine (NAC) given orally over a period will reduce the oxidative stress in diabetic patients with nephropathy, and the reduction will be reflected in the oxidant stress marker level in the serum along with possible reduction in the proteinuria.
The study was designed therefore to investigate primarily the effectiveness of oral NAC given daily for 30 days in patients with diabetic proteinuria on the oxidative stress reduction in the patients, as measured by serum oxidant stress markers. One secondary outcome of interest was the effect of NAC on proteinuria as measured by spot urine protein-creatinine ratio.
N-acetylcysteine was chosen because it is the only antioxidant drug that has been clinically useful in ameliorating a renal condition whose pathogenesis may also involve increased oxidative stress. Several clinical studies have shown that oral NAC in moderate doses given as prophylaxis before radio-contrast study ameliorates contrast nephropathy.5,6On the other hand, use of other common antioxidants like vitamin E or vitamin C had not been successful in clinical trials.
N-acetylcysteine is a cysteine prodrug, and its antioxidant activity is due to its ability to replenish intracellular glutathione (GSH) level.7N-acetylcysteine in very high doses is Food and Drug Administration (FDA) approved for treatment of acute acetaminophen toxicity. It had also been used orally for several years in chronic obstructive pulmonary disease patients without major toxicity.8However, the oral capsule form of NAC is not approved by FDA, although it is available in Canada and Europe. The Mucomyst or Acetadote form of NAC is a liquid preparation with a strong disagreeable flavor. It can be used for short-term therapy like contrast prophylaxis, but it is not suitable for long-term oral use. We could not obtain permission from our VA hospital pharmacy to use non-US brand NAC for this study and had to obtain the capsule form from a reputed neutriceutical manufacturer.
METHODS
Fifteen adult patients with type 2 diabetes mellitus and varying levels of proteinuria were recruited for the study from the renal clinic at VA Medical Center, (Dayton, OH). The patients who were on dialysis or who had known allergy to NAC were excluded. The study was approved by the institutional review board of Wright State University, Dayton, OH, and all participants gave informed consent before joining the study.
All patients were provided with 500 mg of NAC capsules, to take 2 capsules by mouth twice a day for 30 days. The capsule form of NAC was obtained from Bluebonnet Nutrition Corporation (Sugar Land, TX).
Serum from the study participants were collected on days 1, 15, and 30 and then again at 30 days after stopping the study drug. Urine was collected only on days 1 and 30. Portions of the serum and urine were frozen immediately and saved at −70°C. At the end of the study, all frozen samples were mailed to the collaborative laboratory (Morgantown, WV) where oxidative stress markers were measured. The markers tested were as follows: serum 8-isoprostane, aconitase, glutathione peroxidase, superoxide dismutase, and total serum antioxidant stress. The assays carried out in this study are standard ones used in many studies of oxidative stress and antioxidant activity. Aconitase is a member of the citric acid cycle and considered to be a target of mitochondrial oxidant injury. Glutathione peroxidase is involved in the detoxification of peroxides and protection of cells against lipid peroxidation. Superoxide dismutase breaks down superoxide to oxygen and hydrogen peroxide.
Serum and urine were also tested locally (VA Medical Center, Dayton, OH) for kidney function tests and spot urine protein-creatinine ratio. Data were also collected from each participant regarding weight, blood pressure, body mass index, and concomitant medication use.
ASSAY OF OXIDANT STRESS MARKERS IN THE SERUM
8-Isoprostane
8-Isoprostane in the serum was measured using an enzyme-linked immunosorbent assay kit (Cayman Chemical, Ann Arbor, MI), according to the manufacturer's protocol. The intensity of color produced, which was inversely proportional to the amount of isoprostane produced, was calculated from a standard curve and expressed as picograms per milliliter of serum.
Total Antioxidant Status
Total antioxidant status was measured by monitoring radical cation formation from 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate) incubated with a peroxidase (metmyoglobin) and H2O2 to produce a radical cation with a stable blue color, which was measured at 600 nm. The colorimetric method was programmed into a Cobas Mira auto analyzer, using a Randox kit (Randox Laboratories, San Francisco, CA) according to a protocol reported previously. Data were expressed as millimoles per liter.
Aconitase
Aconitase activity was measured in serum samples using a Bioxytech Aconitase-340 assay kit, according to the manufacturer's protocol (Oxis Research, Portland, OR). The assay is based on the isomerization of citrate to isocitrate and the measurement of NADPH formed from NADP+, which is proportional to aconitase activity). Measurements were performed with a Cobas Mira auto analyzer (Roche Diagnostics Inc, Indianapolis, IN) at 340 nm for 5 minutes at 37°C. The concentration of aconitase was expressed as milliunits per milliliter of serum.
Glutathione Peroxidase
Glutathione peroxidase in serum was measured with a Cobas Mira auto analyzer (Roche), using a detection kit programmed according to the manufacturer's protocol as reported previously. Glutathione peroxidase was expressed as the amount of enzyme that transformed 1 μmol of NADPH to NADP per minute at 37°C.
Manganese Superoxide Dismutase
Activity of manganese superoxide dismutase in serum samples was measured with a superoxide dismutase assay kit (Cayman Chemical) according to the manufacturer's protocol. For analysis, 10-μL serum samples were treated with 190 μL of tetrazolium and 10 μL of 1 mM potassium cyanide to inhibit both Cu/Zn-SOD and extracellular SOD. The reaction was initiated by adding 20 μL of xanthine oxidase followed by incubation for 20 minutes at room temperature. Absorbance change was read at 450 nm, using a microplate reader. The activity of manganese superoxide dismutase (expressed as units per milliliter) was calculated from a standard curve constructed with known amounts of standards processed with samples. Manganese superoxide dismutase activity was defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical and was expressed as units per milliliter of serum.
STATISTICAL ANALYSIS
Sample size planning for a given power was not calculated because variability in the oxidant stress markers at different points of time was unknown. Also, limited funding prevented budgeting for large number of patients. Because each patient was his own control and measurements were taken at 4 time points, we agreed that 15 patients would be satisfactory for our exploratory study.
For statistical analysis, means and SDs are reported for continuous variables, and frequencies and percentages are reported for categorical variables. Repeated-measures analysis of variance was used for the oxidant stress markers and Wilcoxon signed-rank test was used for the proteinuria data. Inferences were made at the 0.5 level of significance.
The study was registered at ClinicalTrials.gov with protocol NCT00493727.
RESULTS
The demographic and other clinical data of the 15 patients are shown in Table 1. All of our patients were obese type 2 diabetic male with mean age of 64.7 (±7.37) years. Thirteen patients were white, and 2 were African Americans. Most patients had hypertension, and half of them were on insulin. Eighty-seven percent were on a statin, and 73% were on either angiotensin-converting enzyme inhibitor (ACEI) or angiotensin receptor blocker (ARB) drug. One third used some form of vitamins regularly, and 2 (13%) of 15 were smokers.
There was no significant change in any of the oxidative stress markers at any point (Table 2). However, in 4 patients with high isoprostane levels at the beginning, the levels declined progressively at each subsequent time points (Fig. 1). Mean 8-isoprostane on these 4 patients were as follows: 11.2 ± 5.3 (day 1), 8.2 ± 1.2 (day 15), 7.9 ± 2.3 (day 30), and 6.4 ± 1.2 (day 60). However, this decline was not statistically significant (P = 0.12).
The mean serum creatinine of all the patients was 2.13 (±0.8) mg/dL. The mean urine protein-creatinine ratio was 1.6 (±1.8) at the beginning and 1.3 (±1.3) at the end of 30 days. The difference was not significant. The results of spot urine protein-creatinine in individual patients are shown in Figure 2.
DISCUSSION
N-acetylcysteine is a potent antioxidant, and many cellular and animal studies have confirmed that both in vitro and in vivo use NAC ameliorates the oxidant stress.9,10Intravenous infusion of NAC improved total antioxidant status in rats in a lung injury model.11Superoxide dismutase content in heart muscle was increased in diabetic rats fed NAC in their drinking water.12However, there have been few human studies of NAC that measured oxidative stress levels. Van Schooten et al.13have studied oral NAC in smoking volunteers and found that total antioxidant scavenging capacity increased in plasma but not in bronchoalveolar lavage fluid. Medved et al.14investigated oral NAC in healthy subjects and found that exercise capacity improved in the subjects and the GSH levels increased in muscle tissue but not in whole blood.
The results of this pilot study did not support our hypothesis that N-acetylcysteine used orally for a month would reduce the serum biomarkers of oxidant stress in a cohort of patients with diabetic proteinuria. However, there were several limitations in the study that cautions against concluding against the hypothesis.
We had to choose the dosing and length of exposure arbitrarily because there had not been any previous study to guide our choice. We chose a dose which was twice that used in prophylaxis against contrast nephropathy. However, because this study was initiated, others noted that the use of vitamin E reduces serum biomarkers, for example, 8-isoprostane only at a dose exceeding 1600 units per day and only after use for 16 weeks.15A higher dose of NAC used for longer period may have shown decrease in the oxidative biomarkers.
The response of serum biomarkers also depend on the baseline level of the marker. In one study, vitamin C reduced 8-isoprostane in serum only when the baseline level was greater than 50 μg/mL.16The baseline levels in most of our patients were very low, and that may have precluded the demonstration of a significant reduction. The 4 patients who had higher baseline levels showed a trend in reduction over the period of the study, although it did not reach statistical significance because of the small number. Most of our patients were on ACEI or ARB and a statin, drugs known to lower baseline oxidative marker levels. The sites of sera collection and assay performance were physically distant; so the sera were stored at −70°C, and the assay were done many months later at the end of the study. Consequently, we cannot preclude some technical problem, which may have affected the accuracy of the results.
For logistical reason, we had to use an NAC formulation that is not FDA approved. Although the company where we acquired NAC was reputable, we cannot be sure about the actual amount of NAC available in each capsule or its in vivo dissolution characteristics. Unfortunately, we could not measure the NAC serum level in the participants to determine absorption and actual dose compliance and could not measure the isoprostane in urine. In this population of patients with proteinuria, urinary levels may be more physiologically significant than the serum levels.
Lastly, as previously mentioned, serum biomarkers may be a poor measure for the cellular or mitochondrial oxidative status and thus may not have any correlation with the biological effects of antioxidant drugs. N-acetylcysteine may have a long-term benefit in chronic renal disease, which is not evident from this short-term study.
Of note, a recently published study by Renke et al.17also did not find any reduction in proteinuria after 8 weeks of oral NAC 1200 mg per day in a cohort of nondiabetic chronic renal failure patients with proteinuria ranging from 0.4 to 6.36 g/d. Like our study, these patients were also already on ACEI or ARB therapy. These investigators did not measure the oxidant stress markers but measured tubular injury markers and renal fibrosis and did not find any difference with NAC therapy versus control.
Measuring the oxidative stress in vitro in humans is a new area of research. Better markers and assay systems may become available in the future to help us understand the biological and clinical effects of antioxidant drugs.