Effect of Coenzyme Q₁₀ on Biomarkers of Oxidative Stress and Cardiac Function in Hemodialysis Patients: The CoQ₁₀ Biomarker Trial

Abstract

Background

Oxidative stress is highly prevalent in patients with end-stage renal disease and is linked to excess cardiovascular risk. Identifying therapies that reduce oxidative stress has the potential to improve cardiovascular outcomes in patients undergoing maintenance dialysis.

Study Design

Placebo-controlled, three-arm, double-blind, randomized, clinical trial

Setting & Participants

65 patients undergoing thrice-weekly maintenance hemodialysis

Intervention

Patients were randomly assigned in a 1:1:1 ratio to receive once daily CoQ₁₀ (600 mg or 1200 mg) or matching placebo for 4 months.

Outcomes

The primary outcome was plasma oxidative stress, defined as plasma concentration of F₂-isoprotanes. Secondary outcomes included plasma isofurans, cardiac biomarkers, pre-dialysis blood pressure, and safety/tolerability.

Measurements

F₂-isoprostanes and isofurans were measured as plasma markers of oxidative stress, and N-terminal-pro-b-type natriuretic peptide and troponin T were measured as cardiac biomarkers at baseline, 1, 2, and 4 months.

Results

Among 80 patients randomized, 15 were excluded due to not completing at least one post-baseline study visit, and 65 were included in the primary intention-to-treat analysis. No treatment-related major adverse events occurred. Daily treatment with 1200 mg but not 600 mg CoQ₁₀ significantly reduced plasma concentrations of F₂-isoprostanes at 4 months compared to placebo (adjusted mean change −10.7 pg/ml [95% CI −7.1 to −14.3 pg/mL], P < 0.001; and −8.3 pg/ml [95% CI −5.5 to −11.0 pg/ml], P = 0.1, respectively). There were no significant effects of CoQ₁₀ treatment on plasma isofurans, cardiac biomarker concentrations, or pre-dialysis blood pressures.

Limitations

Study not powered to detect small treatment effects; difference in baseline characteristics among randomized groups.

Conclusions

In patients undergoing maintenance hemodialysis, daily supplementation with 1200 mg of CoQ₁₀ is safe and results in a reduction in plasma concentrations of F₂-isoprostanes, a marker of oxidative stress. Future studies are needed to determine whether CoQ₁₀ supplementation improves clinical outcomes for patients undergoing maintenance hemodialysis.

INTRODUCTION

Cardiovascular disease is the leading cause of death for patients undergoing maintenance hemodialysis (MHD) in the United States.¹ The prevalence of risk factors traditionally associated with cardiovascular disease, such as diabetes, hypertension, and lipid abnormalities, is higher in MHD patients than in the general population.² However, this increased prevalence of traditional risk factors does not explain most of the extraordinarily high cardiovascular disease risk in dialysis patients.^3,4 Increased oxidative stress is present in patients undergoing MHD, and has been identified as a key contributor to the pathogenesis of atherosclerosis and other cardiovascular disease in this population.^4–7 Identifying novel drug therapies that may reduce oxidative stress thus has the potential to improve clinical cardiovascular outcomes for patients undergoing MHD. F₂-isoprostanes and isofurans, two products of non-enzymatic peroxidation of arachidonic acid that have been shown to be reliable markers of oxidative stress, are elevated in MHD patients, and thus represent ideal targets for interventions aimed at reducing oxidative stress burden.^8–12

Coenzyme Q₁₀ (CoQ₁₀) is a potent lipophilic antioxidant that couples electron transport to oxidative phosphorylation in mitochondria, is readily available as a dietary supplement, and is used frequently as an alternative and complementary therapy. CoQ₁₀ supplementation has previously been shown to have potential clinical benefit in the treatment of heart failure and improves endothelial function in patients with type II diabetes.^13–16 Plasma concentrations of CoQ₁₀ are depressed in patients undergoing MHD, suggesting that CoQ₁₀ supplementation may represent an ideal antioxidant therapy for these patients.¹⁷

In this study, we hypothesized that administration of coenzyme Q₁₀ would lead to improvements in biomarkers of oxidative stress in patients undergoing MHD. We also assessed effects of the intervention on biomarkers of cardiac function and pre-dialysis blood pressures, and further monitored study agent safety and tolerability as secondary outcomes measures.

METHODS

Study Design and Participants

This was a pilot, double-blind, parallel group, randomized clinical trial comparing daily CoQ₁₀ antioxidant therapy at two different doses (600 mg daily and 1200 mg daily), with matching placebo (NCT01408680). Study subjects were recruited by study coordinators from outpatient dialysis facilities in the Seattle metropolitan area from September 2011 through April 2013. Criteria for study participation included: patients receiving thrice-weekly MHD for at least 90 days, age >18 and <85 years, life expectancy greater than 1 year, and the ability to provide informed consent. Exclusion criteria included history of poor adherence to dialysis or medications; kidney transplant less than 6 months before study enrollment; use of antioxidant nutritional supplements other than vitamin-C; hospitalization within 30 days, history of a major atherosclerotic event within six months; and current use of a hemodialysis catheter.

A total of 146 patients were assessed for eligibility, and 80 gave consent and were randomized (Figure 1). Subjects were randomly assigned to one of the three study groups in a 1:1:1 ratio by a permuted block randomization strategy using a block size of 12. Prior to starting study treatment, seven subjects voluntarily elected not to participate in the study and one subject was hospitalized. Additionally, one subject assigned to placebo, 3 subjects assigned to 600 mg CoQ₁₀, and 2 subjects assigned to 1200 mg CoQ₁₀ withdrew from the study prior to the first post-baseline study visit. One subject assigned to 600 mg CoQ₁₀ died prior to the first post-baseline visit. Thus, 65 subjects were included in the intention-to-treat analysis. Of these, 58 (89%) completed the study (600 mg CoQ₁₀ group; 16; 1200 mg CoQ₁₀ group, 19; placebo group, 23) and were included in the per-protocol analysis (Figure 1). Among subjects included in the intention-to-treat analysis who did not complete all four study visits, two assigned to the placebo group underwent kidney transplant, one assigned to the placebo group and one assigned to 1200 mg CoQ₁₀ were lost to follow-up, one assigned to 600 mg and one assigned to 1200mg CoQ₁₀ withdrew consent, and one assigned to 1200mg CoQ₁₀ stopped the study drug due to an extended hospitalization (Figure 1). A data safety monitoring board oversaw the safety profile of the study, and no interim efficacy analyses were planned or conducted. The study was performed in accordance with the Declaration of Helsinki and was approved by the University of Washington Institutional Review Board (Human Subjects Application #40428). All subjects provided written informed consent before study enrollment.

Figure 1. Flow chart of study population, including the number of patients who were screened, gave consent, underwent randomization, completed the study treatment and were analyzed for the primary outcome.

Study Procedures

Subjects were screened for eligibility criteria within 28 days prior to beginning the study. The duration of study treatment was 4 months, and subjects underwent in-person pre-dialysis assessments at a dialysis facility at baseline, 2 weeks, and monthly thereafter. Fasting blood sample collection was performed at baseline, 1, 2 and 4 months. CoQ₁₀ was provided by study coordaintors to patients as a chewable wafer (Vitaline Formulas, Wilsonville, OR), and matching wafers were provided to the placebo group (CuliNex, LLC, Seattle, WA; and College Pharmacy, Colorado Springs, CO). Throughout the course of the study, patients received usual dialysis care prescribed by each patient’s nephrologist.

Outcomes

The primary outcome was change in the plasma marker of oxidative stress F₂-isoprotanes from baseline. Secondary outcomes included change in plasma isofurans concentration, plasma CoQ₁₀ concentration and CoQ₁₀ redox ratio (defined as the ratio of reduced CoQ₁₀H₂ [ubiquinol] to oxidized CoQ₁₀ [ubiquinone]), changes in biomarkers of cardiac function (troponin T and n-terminal pro-b-type natriuretic peptide [NT-proBNP]), and changes in pre-dialysis blood pressures. Safety and tolerability of the CoQ₁₀ preparations over the 4 month study period, defined as incidence of treatment-related adverse events and number of patients prematurely discontinuing study treatment, respectively, were also assessed as secondary outcomes. Adverse events were assessed by study coordinators at study visits using open-ended questions, and were attributed to study treatment by study investigators, who were blinded to assigned treatment.

Analytic Procedures

Heparinized blood samples were transported to the lab on ice and centrifuged within one hour of collection at 2500 RPM, 4°C, for 15 minutes. Plasma was removed, stored at −80°C, and thawed immediately prior to analysis. For measurement of CoQ₁₀, 100 µL aliquots were mixed with 200 µL ice cold 1-propanol containing Coenzyme Q₉ (CoQ₉) and CoQ₉H₂ as internal standards. Precipitated proteins were removed by cold centrifugation, and the supernatant analyzed using an Agilent 6400-series liquid chromatography-tandem mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA) equipped with a Zorbax SB-C₁₈ column maintained at 40°C.¹⁸

For F₂-isoprostanes and isofurans measurements, internal standard [²H4]-15-F2T-isoprostane was added to plasma and followed by C-18 and silica solid phase extraction, thin layer chromatography, and derivatization to penta-fluorobenzyl ester, trimethylsilyl ether in preparation for gas chromatography/negative ion chemical ionization/mass spectrometry analysis (Agilent 5973, Agilent Technologies, Inc., Santa Clara, CA) using 15 cm × 0.25 µm thick fused silica capillary columns.¹⁹ Cardiac troponin T and NT-proBNP were measured using electrochemiluminescent-based immunoassays with calibrators and external controls run prior to samples (cobas^® E411, Roche Diagnostics, Indianapolis, IN).

Statistical Analyses

Sample size was computed on the basis of change in F₂-isoprostanes concentrations from baseline to 4 months using a t-test approach. Previously published observational data among MHD patients showed a mean F₂-isoprostane concentration of 96.2 pg/mL, with a standard deviation of 48.8 pg/mL.⁸ In the absence of available data from interventional studies from which to estimate an effect size, a reduction in F₂-isoprostane concentrations by 50% was postulated a priori to be clinically significant.²⁰ Given these assumptions, a sample size of 22 in each study arm (total, 66) was estimated to achieve 86% power to detect a 50% reduction in the primary outcome with a two-sided 5% significance level, assuming a drop-out rate of 10%.

Primary analyses were performed on an intention-to-treat basis, including all randomized participants who completed at least one post-baseline study visit, regardless of adherence or compliance status. Secondary analyses were performed on a per-protocol basis, including only patients who reported adherence to the assigned study agent and who completed all study visits. Multiple imputation for missing laboratory and blood pressure outcome data among study participants who did not complete all study follow-up visits was performed using chained equations with 5 imputations, with estimates combined using Rubin’s rules.^21–24 Missing values were modelled as continuous variables using linear regression. Imputation regressions included fixed independent variables for age, sex, race, etiology of end-stage renal disease, and also included oxidative stress, cardiac biomarker, and blood pressure values from prior study visits. Using this approach, laboratory and blood pressure outcome data for the third study visit were imputed for one subject, and for the fourth study visit for 7 subjects. Least squares means for study outcomes at each time point were determined using linear regression with robust standard errors adjusted for age, sex, race, and etiology of end-stage renal disease. The effect of randomized treatment assignment on primary and secondary outcomes over follow-up was assessed using generalized estimating equations with respect to intervention arm and time, with an unstructured correlation matrix. Changes from baseline to 1, 2, and 4 months of follow-up for each study outcome were also assessed using linear regression. All models included baseline values, age, sex, race, and etiology of end-stage renal disease as covariates due to observed imbalance among treatment groups, an approach that was pre-specified in the study protocol. CoQ₁₀ levels, oxidative stress markers, and cardiac biomarkers were log-transformed prior to analysis due to right-skewed distributions. One value for NT-proBNP of >350,000 pg/ml was greater than 4-fold higher than the next highest value and was excluded from analyses. All analyses were performed using Stata, version 11 (StataCorp LP, College Station, TX; www.stata.com). The nominal level of significance was defined as P<0.05.

RESULTS

Participant Characteristics

Overall, subjects had a mean age of 54 ± 13 years; 22 subjects (34%) were female, 17 (26%) were black, and 25 (40%) had diabetes (Table 1). Over 90% of subjects dialyzed using an arteriovenous fistula. Patients assigned to receive 600 mg CoQ₁₀ daily were younger, more likely to be black, and less likely to have diabetes compared to patients assigned to the placebo group. Patients assigned to receive 1200 mg CoQ₁₀ daily were less likely to be black and more likely to have diabetes and cardiovascular disease compared to patients assigned to placebo. The proportion of patients assigned to each treatment group did not differ between patients who dropped out and those who did not (Supplemental Table S1).

Table 1.

Baseline Patient Characteristics by Treatment Group

	Placebo (N = 26)	600 mg Coenzyme Q10 (N = 18)	1200 mg Coenzyme Q10 (N = 21)
Age, years	57± 13	49 ± 16	56 ± 12
Male gender, n (%)	19 (69)	12 (67)	13 (62)
Race, n (%)
White	18 (69)	8 (44)	12 (57)
Black	6 (23)	7 (38)	4 (18)
Asian	1 (4)	1 (6)	2 (10)
Other/Unknown	1 (4)	2 (12)	3 (15)
Height, centimeters	171 ± 10	174 ± 11	170 ± 9
Weight, kilograms	86 ± 28	93 ± 31	85 ± 22
Body Mass Index	29 ± 8	31 ± 10	29 ± 7
Months since initiation of dialysis	31 (17, 68)	67 (33, 88)	38 (14, 71)
Cause of end-stage renal disease, n (%)
Diabetes	8 (31)	4 (22)	10 (47)
Hypertension	9 (35)	2 (11)	6 (29)
Glomerular Disease	3 (12)	4 (22)	0
Other	6 (23)	8 (45	5 (24)
Comorbidities, n (%)
Diabetes	8 (35)	7 (39)	10 (48)
Hypertension	22 (85)	17 (94)	18 (90)
Cardiac Disease	6 (23)	3 (17)	6 (32)
Stroke	3 (12)	3 (17)	1 (6)
Peripheral Vascular Disease	4 (17)	3 (17)	5 (26)
Dyslipidemia	5 (19)	5 (31)	8 (40)
Cancer	5 (19)	3 (18)	2 (11)
Tobacco Use, n (%)
Current smoker	3 (12)	3 (17)	2 (10)
Ever smoker	11 (42)	10 (56)	10 (48)
Never smoker	15 (58)	8 (44)	11 (52)
Vascular Access Type, n (%)
Arteriovenous fistula	24 (92)	15 (83)	19 (90)
Arteriovenous graft	2 (8)	3 (17)	2 (10)
Medications, n (%)
ACE inhibitor or ARB	6 (23)	8 (44)	9 (43)
Anti-platelet agent	10 (38)	3 (17)	13 (62)
Statin	8 (31)	4 (22)	7 (33)
Insulin	6 (23)	3 (17)	8 (38)
Beta-blocker	10 (38)	11 (61)	7 (33)

Summary statistics are mean ± SD, median (interquartile range) or number (%) as appropriate.

Abbreviations: ACE, angiotensin converting enzyme; ARB, angiotensin receptor blocker

Change in Plasma CoQ₁₀ Concentrations

Compared to patients assigned to placebo, those assigned to either 600 mg or 1200 mg CoQ₁₀ had significantly higher mean plasma CoQ₁₀ concentrations over study follow-up (P = 0.001, and P < 0.001, respectively) (Table 2). There was no significant difference in the mean CoQ₁₀ redox ratio among patients assigned to placebo versus 600 mg daily CoQ₁₀ supplementation. In contrast, after adjustment for baseline values, patients assigned to 1200 mg of CoQ₁₀ had a significantly higher mean redox ratio over study follow-up, (P < 0.001).

Table 2.

Mean Total Plasma Coenzyme Q₁₀ Concentrations and Coenzyme Q₁₀ Redox Ratios over Study Period, by Treatment Group

	Placebo (N = 26)	600 mg Coenzyme Q10 (N = 18)	1200 mg Coenzyme Q10 (N = 21)
Total CoQ10, ng/ml
Baseline	624 (516, 731)	720 (568, 872)	490 (375, 604)
1 month	737 (489, 984)	1901 (1354, 2448)	2875 (2308, 3443)
2 months	642 (445, 840)	1878 (1329, 2427)	2938 (2247, 3629)
4 months	681 (426, 936)	1901 (1280, 2521)	1974 (1328, 2621)
p-valuea	Reference	0.001	<0.001
CoQ10redox ratiob
Baseline	13.6 (11.1, 16.2)	14.4 (11.5, 17.4)	12.8 (10.7, 15.0)
1 month	15.5 (11.7, 17.4)	15.5 (12.7, 18.2)	20.4 (16.8, 24.1)
2 months	13.9 (10.9, 16.9)	15.4 (12.8, 18.1)	18.5 (15.2, 21.8)
4 months	16.0 (12.8, 19.1)	14.1 (11.9, 16.2)	16.8 (13.4, 20.1)
p-valuea	Reference	0.34	<0.001

Note: Results reported are from the primary intention-to-treat analysis. Values are least squares mean (95% confidence interval), adjusted for age, sex, race, and etiology of end-stage renal disease.

^ap-value for comparison of average treatment effect from generalized estimating equation model, adjusted for age, sex, race, etiology of end stage renal disease, and baseline measurements; reference group: placebo. Outcome measurements log transformed prior to analysis.

^bCoQ₁₀ redox ratio calculated as ratio of CoQ₁₀H2 (reduced) to CoQ₁₀ (oxidized)

Change in Plasma F₂-Isoprostane Concentrations

Compared to patients assigned to placebo, patients assigned to 1200 mg daily CoQ₁₀ supplementation had significantly lower mean plasma concentrations of F₂-isoprostanes over the study treatment period (P = 0.002) (Table 3). Adjusted mean change from baseline for plasma F₂-isoprostanes in patients assigned to this higher dose of CoQ₁₀ was −7.7 pg/ml (95% confidence interval [CI] −2.4 to −13.0 pg/ml) at 2 months and −10.7 pg/ml (95% CI −7.1 to −14.3 pg/ml) at 4 months (P = 0.005 and P < 0.001, respectively) (Figure 2). For patients assigned to 600 mg daily CoQ₁₀, there was a decrease in F₂-isoprostane concentrations from baseline to the 4-month follow-up time point of −8.3 pg/ml (95% CI −5.5 to −11.0 pg/ml), but this change was not significantly different compared to the placebo group (P = 0.10). Mean F₂-isoprostanes concentrations over the 4-month study duration were also not significantly different for patients assigned to 600 mg CoQ₁₀ compare to those assigned to placebo.

Table 3.

Mean Plasma Markers of Oxidative Stress over Study Period, by Treatment Group

	Placebo (N = 26)	600 mg Coenzyme Q10 (N = 18)	1200 mg Coenzyme Q10 (N = 21)
F2-isoprostanes, pg/ml
Baseline	49.3 (40.6, 58.0)	45.6 (35.1, 56.1)	62.4 (43.5, 81.3)
1 month	54.0 (44.8, 63.2)	47.1 (38.7, 55.6)	48.5 (36.8, 60.3)
2 months	50.4 (41.1, 59.7)	42.1 (32.6, 51.6)	51.9 (37.2, 66.7)
4 months	45.1 (36.6, 53.6)	39.2 (30.9, 47.6)	47.5 (33.2, 61.8)
p-valuea	Reference	0.34	0.002
Isofurans, pg/ml
Baseline	64.8 (56.4, 73.1)	68.1 (60.1, 76.2)	94.2 (77.7, 110)
1 month	86.2 (62.5, 110)	84.5 (67.5, 102)	73.2 (57.4, 88.9)
2 months	75.6 (62.5, 86.7)	90.0 (57.9, 122)	115 (74.8, 155)
4 months	69.8 (60.0, 79.6)	73.5 (62.9, 84.0)	82.8 (66.7, 98.9)
p-valuea	Reference	0.71	0.07

Note: Results reported are from the primary intention-to-treat analysis. Values are least squares means (95% confidence interval), adjusted for age, sex, race, and etiology of end-stage renal disease.

^ap-value for comparison of average treatment effect from generalized estimating equation model, adjusted for age, sex, race, etiology of end stage renal disease, and baseline measurements; reference group: placebo. Outcome measurements log transformed prior to analysis.

Figure 2. Mean Change in Plasma F₂-Isoprostanes and Isofurans Concentrations from Baseline to Months 1, 2 and 4 of Follow-up, by Treatment Group.

Values are least squares means adjusted for baseline measurements, age, sex, race, etiology of end-stage renal disease. Error bars represent ± standard error of the least squares mean. Asterisk indicates P < 0.01 for comparison of mean change from baseline with reference group of patients assigned to placebo.

Secondary Outcomes

In contrast to the findings for the primary outcome measure, there was no significant change from baseline in plasma concentrations of isofurans with either either dose of CoQ₁₀ (Figure 2). There was a trend toward lower mean concentrations of isofurans over the 4-month study period for patients assigned to 1200 mg CoQ₁₀ compared to placebo, though this trend was not statistically significant (P = 0.07) (Table 3). There were also non-significant trends toward lower mean serum concentrations of NT-proBNP and cardiac troponin T for patients assigned to the higher CoQ₁₀ dose compared to placebo (P = 0.10 and P = 0.09, respectively) (Table 4). These trends were not evident for patients assigned to the lower 600 mg dose. There were no significant differences in mean pre-dialysis systolic or diastolic blood pressures comparing patients assigned to either dose of CoQ₁₀ versus placebo (Table 4).

Table 4.

Mean Pre-Dialysis Blood Pressure and Serum Concentrations of Cardiac Troponin T and N-terminal-proBNP, by Treatment Group

	Placebo (N = 26)	600 mg Coenzyme Q10 (N = 18)	1200 mg Coenzyme Q10 (N = 21)
Troponin T, ng/ml
Baseline	0.05 (0.04, 0.07)	0.11 (0.07, 0.15)	0.09 (0.04, 0.14)
1 month	0.08 (0.04, 0.11)	0.11 (0.07, 0.16)	0.08 (0.03, 0.13)
2 months	0.05 (0.04, 0.08)	0.10 (0.07, 0.14)	0.08 (0.02, 0.15)
4 months	0.07 (0.04, 0.10)	0.11 (0.07, 0.15)	0.06 (0.02, 0.10)
p-valuea	Reference	0.71	0.10
N-terminal-proBNP, pg/ml
Baseline	8412 (2672, 13,151)	8862 (4262, 13,461)	6794 (2668, 10,920)
1 month	11,294 (5033, 17,555)	8857 (2958, 14,757)	7768 (2443, 13,094)
2 months	10,740 (5579, 15,901)	11,169 (4714, 17.624)	6062 (0, 12,353)
4 months	9562 (2803, 16,321)	13,510 (5752, 21,268)	2601 (0, 5558)
p-valuea	Reference	0.92	0.09
Pre-dialysis systolic blood pressure, mmHg
Baseline	133 (126, 140)	144 (134, 155)	138 (131, 146)
1 month	136 (127, 145)	144 (136, 152)	143 (131, 154)
2 months	141 (130, 151)	146 (135, 157)	143 (131, 154)
4 months	138 (127, 150)	140 (131, 150)	128 (112, 143)
p-valuea	Reference	0.87	0.90
Pre-dialysis diastolic blood pressure, mmHg
Baseline	73 (67, 79)	78 (68, 89)	75 (69, 81)
1 month	73 (66, 79)	78 (72, 84)	77 (68, 85)
2 months	77 (69, 85)	79 (70, 87)	77 (69, 85)
4 months	75 (68, 82)	77 (71, 84)	70 (62 (78)
p-valuea	Reference	0.99	0.93

Note: Results reported are from the primary intention-to-treat analysis. Values are least squares means (95% confidence interval), adjusted for age, sex, race, and etiology of ESRD.

^ap-value for comparison of average treatment effect from generalized estimating equation model, adjusted for age, sex, race, etiology of ESRD, and baseline measurements; reference group: placebo. Outcome measurements log transformed prior to analysis.

Sensitivity Analyses

Three sets of sensitivity analyses were performed. In the first, per-protocol analyses were performed among the pre-specified group of patients who completed all four study visits and who adhered to the assigned study treatment (N = 58). In these per-protocol analyses, the non-significant trends seen in the primary analysis toward lower mean concentrations of isofurans, troponin T and NT-proBNP among patients assigned to 1200 mg CoQ₁₀ compared to placebo were found to be significant (P < 0.05) (Supplementary Tables S2 and S3). In a second set of sensitivity analyses, statistical comparisons were performed adjusting only for baseline outcome values, and were similar to the primary results. Finally, analyses performed replacing missing laboratory values with the last observed value rather than using multiple imputation also showed similar results.

Safety and Tolerability

Adverse events in study participants are reported in Table 5. One patient assigned to 600 mg CoQ₁₀ died two weeks after enrollment, prior to the second study visit. Six patients were hospitalized, including 1 patient assigned to placebo, 3 patients assigned to 600 mg CoQ₁₀, and 2 patients assigned to 1200 mg CoQ₁₀. One hospitalization for abdominal pain in a patient assigned to 1200 mg CoQ₁₀ was deemed possibly related to study treatment. Study agents were generally well tolerated. One patient assigned to 600 mg CoQ₁₀ elected to discontinue treatment after the second study visit due to difficulty chewing the study agent wafers. One patient assigned to 600 mg and one assigned to 1200 mg CoQ₁₀ withdrew prior to the second study visit due to gastrointestinal discomfort. The overall adverse event rates were 0.25, 1.3, and 0.45 per patient-year for placebo, 600 mg and 1200 mg CoQ₁₀, respectively.

Table 5.

Adverse Events in Study Participants

	Placebo (N = 28)	600 mg Coenzyme Q10 (N = 26)	1200 mg Coenzyme Q10 (N = 26)	P-value
Serious adverse eventsa	2 (7)	5 (19)	3 (12)	0.4
Death	0 (0)	1 (4)	0 (0)	0.3
Initial or prolonged hospitalizationb	1 (4)	3 (12)	2 (8)c	0.5
Important medical events	1 (4)	1 (4)	1 (4)	0.9
Other adverse events	0 (0)	2 (8)	1 (4)	0.3
Abdominal discomfort	0 (0)	1 (4)	1 (4)	0.6
Hyperkalemia not requiring hospitalization	0 (0)	1 (4)	0 (0)	0.3

Note: Values are given as number (percentage). Adverse events assessed among all randomized subjects, and defined as any undesirable medical event occurring to a subject, whether or not it is related to the study agent.

^aSerious adverse event (SAE) defined as: death, life-threatening experience, inpatient hospitalization or prolongation of existing hospitalization, persistent or significant disability/incapacity, congenital anomaly/birth defect, or an important medical events that may jeopardize the subject and may require medical or surgical intervention

^bOne hospitalization for abdominal pain in a patient assigned to 1200 mg CoQ₁₀ was deemed possibly related to study treatment. All other adverse events were deemed unlikely related to study treatment.

DISCUSSION

In this randomized, double-blind, placebo-controlled study of patients receiving MHD, the provision of daily CoQ₁₀ as antioxidant therapy was safe, well tolerated, and resulted in a significant and dose dependent increase in plasma CoQ₁₀ levels compared to placebo. In addition, CoQ₁₀ supplementation using a 1200 mg daily dose significantly decreased plasma concentrations of F₂-isoprostanes, a robust plasma marker of oxidative stress. In the primary intention-to-treat analysis, there was no significant effect of CoQ₁₀ on plasma isofurans, troponin-T, and NT-proBNP, or on pre-dialysis systolic or diastolic blood pressures. However, trends toward reductions in isofurans, troponin-T, and NT-proBNP with 1200 mg daily CoQ₁₀ were evident, and were significant in a per-protocol analysis.

We chose to study CoQ₁₀ in this clinical trial for a number of reasons. First, CoQ₁₀ is a readily available dietary supplement that has shown a favorable safety profile and has been well tolerated at high doses in previous clinical trials of patients with congestive heart failure, diabetes, and a variety of neurological disorders.^25–28 Second, patients undergoing MHD are known to have low plasma CoQ₁₀ levels compared to age- and sex-matched healthy individuals, and that these levels are associated with reduced coronary blood flow.^17,29 Third, previous data from a non-randomized CoQ₁₀ dose escalation study in MHD patients confirmed that doses as high as 1800mg per day were safe and well-tolerated, and suggested that CoQ₁₀ supplementation may be associated with improvements in oxidative stress burden in these individuals.³⁰ Finally, prior studies have shown that in patients with kidney disease, perturbations in cell metabolism lead to preferential dysregulation of mitochondrial respiratory machinery that is closely associated with the enhanced oxidative stress seen in these individuals.^31,32 CoQ₁₀, which exerts antioxidant activity by coupling electron transport to oxidative phosphorylation locally in mitochondria, thus represents an ideal antioxidant agent for the dialysis population.

In our study, we found that CoQ₁₀ supplementation significantly reduced plasma F₂-isoprostanes but not isofurans concentrations. Prior studies have demonstrated that relative formation of lipid peroxidation products is profoundly influenced by ambient oxygen concentration.¹⁰ Specifically, in highly oxygenated tissues such as the central nervous system, isofurans formation predominates, while F₂-isoprostanes formation is favored in cellular environments where oxygen concentrations are low. In advanced chronic kidney disease, rarefication of post-glomerular capillaries leads to substantial tissue hypoxia.³³ Additionally, uremia leads to accelerated atherogenesis, and recent research has suggested that atherosclerotic plaques are characterized by hypoxic cores.³⁴ Thus, in patients undergoing maintenance dialysis, production of F₂-isoprostanes as a marker of oxidative stress may predominate, and thus be preferentially amenable to reduction with antioxidant therapy. These observations together provide one possible explanation for the observed differential effect of CoQ₁₀ on F₂-isoprostanes compared to isofurans in our study.

In addition to the impact of CoQ₁₀ administration on F₂-isprostanes, we observed a reduction in troponin T and NT-pro-BNP concentrations with CoQ₁₀ supplementation in a pre-specified per-protocol analysis. Such a trend is consistent with previous investigations among healthy patients that have observed an inverse association between serum levels of CoQ₁₀ and NT-proBNP, and a reduction in NT-proBNP levels in individuals assigned to take CoQ₁₀ supplements.^35,36 Additionally, a large multi-center randomized trial conducted in patients with moderate to severe heart failure showed a trend toward decline in NT-proBNP with even low dose (300 mg per day) CoQ₁₀ supplementation.¹³ To our knowledge our findings represent the first report of a potential benefit of antioxidant therapy on markers of cardiac function in patients with kidney disease. Larger trials will be required for assessment of postulated cardiovascular and other clinical benefits.

This pilot study has a number of important strengths. First, we used a double-blind, randomized, placebo-controlled study design, and had low drop-out rates and high treatment adherence. Second, we used a CoQ₁₀ formulation with well-established bioavailability and purity that has been used previously in randomized clinical trials.^37,38 Third, the lipid peroxidation biomarkers (F₂-isoprostanes and isofurans) were analyzed in a reference quality laboratory using gas chromatography-mass spectrometry, and CoQ₁₀ concentrations were measured via a validated technique utilizing ultra-performance liquid chromatography-tandem mass spectrometry.

Despite its strengths, the results of our study should be interpreted in light of some limitations. First, this was a pilot study, and the small number of participants resulted in limited statistical power to demonstrate small treatment benefits. Thus, it is possible that the in a larger study, a CoQ₁₀ dose of 1200 mg may result in a significant reduction in troponin T and NT-proBNP. Second, there were some differences in baseline characteristics between the treatment groups due to chance in the setting of a small number of subjects randomized. We attempted to account for these differences through statistical adjustment, but residual confounding by measured or unmeasured factors may exist. If present, such residual confounding may have influenced the magnitude of the observed treatment effect, and the directionality of such influence may be unclear. Third, in our study we observed attrition of subjects following randomizing, including five subjects assigned to the placebo group, 10 subjects assigned to 600 mg CoQ₁₀ and 7 subjects assigned to 1200 mg CoQ₁₀. Although the a priori defined primary analysis pre-specified inclusion only of subjects who completed at least one post-baseline study visit, the differential attrition of subjects from the CoQ₁₀ groups compared to the placebo group may have led to baseline imbalance among groups. We have partially addressed this limitation by utilizing multiple imputation for missing outcome data among subjects who withdrew from the study after the second study visit and by adjusting for baseline characteristics in our statistical models, but we cannot exclude the possibility that differential attrition led to bias in our estimates of treatment effect. Fourth, in our study we measured only two products of lipid peroxidation (F₂-isoprostanes and isofurans) as markers of systemic oxidative stress, and did not assess other oxidative stress pathways such as protein or DNA oxidation. Although isoprostanes have been shown in previous studies to be the most accurate and reliable plasma measures of oxidative injury in vivo, it is possible that CoQ₁₀ supplementation may have had an effect on oxidative stress pathways that we did not assess.^39–41 Finally, there were differences in baseline concentrations of F₂-isoprostanes and isofurans among the treatment groups observed, again due to chance in the setting of the small number of study participants. In the primary analyses, we adjusted for baseline biomarker concentrations to ensure robust statistical comparisons, but we cannot exclude a possible contribution of regression toward the mean in the observed CoQ₁₀ treatment effect.

In conclusion, in patients undergoing MHD, daily CoQ₁₀ supplementation was safe and well tolerated, and resulted in a reduction in F₂-isoprostanes, a plasma marker of oxidative stress. Future studies are warranted to determine whether treatment with CoQ₁₀ among dialysis patients results in improvement in clinical outcomes such as rates of infection, hospitalization, cardiovascular events, and mortality.