Beneficial effects of creatine, CoQ10, and lipoic acid in mitochondrial disorders.
Beneficial effects of creatine, CoQ10, and lipoic acid in mitochondrial disorders.
Author M Christine Rodriguez;Jay R MacDonald;Douglas J Mahoney;Gianni Parise;M Flint Beal;Mark A Tarnopolsky
Publication title Muscle & nerve
Publication date 2007
Volume Vol.35
Pages 235-242
doi10.1002/mus.20688
Impact factor 2.456 (2007)? 2.283 (2015)< /p>
Abstract: Mitochondrial diseases have distinct cellular consequences: (1) reduced ATP production; (2) increased reliance on alternative anaerobic energy sources; (3) increased production of reactive oxygen species. The purpose of this study was to determine the effect of a combination therapy (creatine monohydrate, coenzyme Q?10, and lipoic acid on the cellular consequences mentioned above, using a randomized, double-blind, placebo-controlled, crossover study design in patients with mitochondrial cytopathies, Targeting multiple outcome variables. 3 patients had mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS), 4 had mitochondrial DNA deletions (3 patients with chronic progressive external ophthalmoplegia and 1 patient with Kearns-Sayre syndrome) patients) and 9 patients with other non-mitochondrial diseases divided into the first two groups. Combination therapy reduced resting plasma lactate and urinary 8-isoprostane and attenuated the decline in peak ankle dorsiflexion strength in all patient groups but only in MELAS. group observed higher fat-free mass. Together, these results suggest that combination therapies targeting multiple ultimately identical pathways of mitochondrial dysfunction may favorably affect surrogate markers of cellular energy dysfunction in a relatively uniform manner. A larger study in this population is needed to determine whether this combination therapy affects function and quality of life.
Mitochondrial diseases represent a group of disorders that affect mitochondrial energy conduction and are characterized clinically, biochemically, and Genetic heterogeneity. 18 Although phenotypic expression varies widely, most patients have concomitant lactic acidosis, stroke or seizures, headache, retinitis pigmentosa, ptosis, poor exercise tolerance, ophthalmoplegia, myocardial infarction disease, neuropathy, and vision loss. 16 , 29 , 38
Mitochondrial dysfunction leads to many cellular consequences, including: (1) reduced ATP production; (2) increased reliance on alternative anaerobic energy sources; (3) ) increases the production of reactive oxygen species (ROS). 16 , 37 There are no single reports of therapeutic strategies for treating mitochondrial diseases that are aimed at mitigating the above cellular consequences 16 , 18 . The effects of compounds such as coenzyme Q?10 (Coenzyme Q?10) 2 , 4 , 21 or creatine (CRM) 13 , ?14 , ?38 are based on the concept that mitochondrial dysfunction leads to a number of cellular pathophysiological consequences. ?33 Most treatment strategies for mitochondrial diseases involve the use of combination therapies (or treatment “cocktails”) versus monotherapies. However, some studies have evaluated the efficacy of combination therapies targeting more than one of the above three approaches. , these were reported in either case, ?8, ?25 open trials, ?1, ?19, ?20, ?27, ?32 or retrospective studies.
26
Based on evidence of potential efficacy from human trials in mitochondrial disease or evidence from human trials or in vitro studies that the proposed compound may alleviate one or more of the final common pathways of mitochondrial dysfunction, we recommend that the combination be evaluated. The following compounds have potential therapeutic effects: (1) CrM (alternative energy source 36 and antioxidant 30); (2) alpha-lipoic acid (antioxidant 17 and can increase the absorption of CrM 6); (3) Coenzyme Q?10? [as an antioxidant oxidant 21 and bypasses the electron transport chain (ETC) 19 of complex I]. We report here the results of a randomized, double-blind, placebo-controlled, crossover trial investigating the efficacy of this targeted combination therapeutic cocktail combining CrM, CoQ?10, and alpha-lipoic acid in patients with mitochondrial cytopathies. Influence.
Patients: Seventeen patients with definite or probable mitochondrial disease were recruited from the Neuromuscular and Neurometabolic Clinic at McMaster University. Combined with clinical symptoms, fasting serum lactate concentration, muscle biopsy results (red fibrillar or cytochrome c oxidase negative fibers) and mitochondrial DNA (mtDNA) analysis. Only patients 8, 9, and 13 had no DNA mutations identified, and only confirmatory testing (increased thymidine, decreased thymidine phosphorylase activity) was performed in patients with mitochondrial neurogastrointestinal encephalopathy; however, their lactate concentrations increased High, histological abnormalities, low exercise tolerance, low aerobic capacity, and considered to have "possible mitochondrial cytopathology." One patient did not complete part of the study due to personal reasons; therefore, this patient's data were excluded from the analysis. The final analysis was based on 16 patients (10 women and 6 men) divided into three groups according to their diagnosis. Characteristics of the patient population are shown in Table 1 . The first group included three patients with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS group). The second group included three patients diagnosed with chronic progressive external ophthalmoplegia (CPEO) and one patient diagnosed with Kearns-Sayre syndrome (KSS), all of whom were detected in muscle-derived mtDNA Missing (CPEO/KSS group). The third group included patients with various mitochondrial diseases: six patients with mitochondrial cytopathies, two patients with Leber hereditary optic neuropathy and one patient with mitochondrial neurogastrointestinal encephalopathy (other group). The study received ethical approval from our institutional ethics committee, and all patients provided informed written consent.
CPEO, chronic progressive external ophthalmoplegia; cytopathy, mitochondrial cytopathy; KSS, Kearns–Sayre syndrome; LHON, Leber's hereditary optic neuropathy; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like episodes; MNGIE, mitochondrial neurogastrointestinal encephalopathy (no thymidine phosphorylase activity, high thymidine levels).
Design/Intervention.
Patients participated in a randomized, double-blind, placebo-controlled, crossover study in which each participant received 2 months of treatment and placebo, with 5 weeks between trials clearance period. The treatment phase consisted of 3 g CrM + 2 g glucose + flavoring (Avicena; Avicena, Palo Alto, CA), 300 mg alpha-lipoic acid (Tishcon, Westbury, NY), and 120 mg CoQ?10 (Qgel; Tishcon) 0:900 and 21:00 every day. During the placebo phase, identical-looking and tasting powder (5 g glucose + flavoring; Avicena) and gel capsules (soybean oil; Tishcon) were used as placebos.
After fasting for 4 hours, patients in both trials completed testing before and after each intervention session at approximately the same time each day (within 2-3 hours).
Measurement.
Participants' height and weight were recorded only at the first visit. All other outcome measures were taken at all other visits. Participants performed grip strength, ankle dorsiflexion (joint angle at 90°), and knee extension strength tests using a custom-made force sensor device, and data were entered directly into a computer containing data acquisition and analysis software as described previously.
38 For all strength measurements, participants were tested on the right side and were individualized for hand size and held constant between visits. To reach peak intensity, participants performed three 5-s trials, approximately 30 s apart. Record the experimental value with the best result. Participants also performed a 1-minute isometric grip strength and ankle dorsiflexion fatigue test (9 seconds work: 1 second rest period). Pulmonary function tests, including forced vital capacity and forced expiratory volume in 1 s, were performed using a spirometer (Koko; PDS Instrumentation, Louisville, CO). Each patient completed at least two spirometry measurements per visit to ensure that the values ??were consistent with their first attempt. Bioelectrical impedance (Prism BIA 101A; RJL Systems, Clinton Twp, MI) was performed to determine body composition.
Venous blood sampling and urine collection.
Collect whole blood from the antecubital vein into pre-cooled vacuum tubes containing heparin (for lactate analysis) or EDTA (for determination of CoQ?10) and centrifuge at 2500 rpm for 10 minutes . Store plasma at -80°C. Each patient provided a urine specimen sample, approximately 10 ml of which was snap frozen and stored at -80°C for creatine, creatinine, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8 - Subsequent analysis of isoprostane (8-IsoP).
Lactate
Plasma lactate concentration was measured using a YSI 2300 Stat Plus lactate analyzer (YSI, Yellow Springs, OH). The intra-batch and intra-batch coefficients of variation for lactate were 2.1% and 1.7%, respectively.
Coenzyme Q?10.
Plasma CoQ?10 concentrations were determined by high performance liquid chromatography (HPLC) using an electrochemical detector. Aliquot plasma (0.5 ml) into a 10 ml vacuum container containing 1 ml 1-propanol and 0.5 ml CoQ9, mix for 5 min, and centrifuge at 300 μg for 5 min. Filter the sample using a 0.22 μM syringe filter and transfer it to a chromatography vial for direct HPLC analysis. Coenzyme Q?9 was added to the mixture as an internal standard, as levels of Coenzyme Q?9 are insignificant in human blood. The resulting sample was injected into a reversed-phase stainless steel column (150 × 3 mm) RP‐C18 packed with 3 μm packing and equipped with an electrochemical detector (ESA, Bedford, MA). Connect to a guard chamber with a single electrode (Model 5020; E = +350 mV) and a coulometric analysis cell with dual electrodes (Model 5011; E1 = -400 mV, E2 = +300 mV). Using a mobile phase of mixed and degassed methanol, 1-propanol and ethanol (70:20:10) containing 50 mM lithium acetate as conductivity salt at a flow rate of 0.5 ml/min and a total run time of less than 15 min? First Coenzyme Q?10 is measured by reducing ubiquinone (E = -400 mV) and then oxidizing the resulting ubiquinol (E = +300 mV). Coenzyme Q?10 and Coenzyme Q?10?H?2 were detected with the highest sensitivity on the last electrode. The correlation coefficient of the standard curve is 0.997. The coefficient of variation was determined to be <2%.
Creatine and Creatinine.
Determination of creatine concentration, creatinine and creatine:creatinine ratio in urine using HPLC. Aliquot urine (1 ml) into microcentrifuge tubes and centrifuge at 10,000 rpm for 10 min. Dilute the urine supernatant to one-tenth dilution using ddH?O? (0.1 ml supernatant to 0.9 ml ddH?O). Maintain the diluted urine supernatant at 10 °C using a refrigerated autosampler. A Hewlett Packard LC1100 series HPLC (Agilent, Mississauga, Ontario) was used with the UV detector set to λ = 210 nm and the sample was injected into a 250 × 4.6 mm C18 Phenomenex10-μHydro-RP 80 column.
The Hewlett Packard LC1100 data analysis program generates calibration curves and analyzes the resulting data. The mobile phase was potassium dihydrogen phosphate (20 mM) adjusted to pH 5.0 using potassium hydroxide at a flow rate of 1.0 ml/min. The coefficient of variation is 3.1%.
8-IsoP.
Urinary 8-IsoP concentrations were determined using a commercial enzyme-linked immunosorbent assay (MediCorp, Montreal, Quebec) following the manufacturer's instructions. The correlation coefficient of the standard curve is 0.988. The coefficient of variation is 10.5%. 8-IsoP values ??are expressed relative to creatinine (g).
8-OHdG.
The concentration of 8-OHdG in urine was determined using HPLC as previously described. 3 ?8-OHdG values ??are expressed relative to creatinine (g).
Statistics.
Statistical analysis was performed using a three-way (group × treatment × time) or two-way (group × treatment) repeated measures analysis of variance (ANOVA). Given the previous hypothesis that combination therapy would reduce lactate and reduce oxidative stress due to the antioxidant properties of each of the three components, we used one-tailed tests for oxidative stress markers. When significant results are found, Tukey HSD post-mortem tests are run. All analyzes were performed using Statistica v. 5 software (StatSoft, Tulsa, Oklahoma). Values ??of P ?<0.05 were considered statistically significant. All data are given as mean ± SD.
Coenzyme Q?10 and Creatine: Creatinine.
As expected, plasma CoQ?10 and urinary creatine:creatinine ratios were significantly higher in the combination treatment compared with the placebo phase. Plasma CoQ?10 concentrations were 172% higher after combination therapy (1.94±0.89 μg/ml) than placebo (0.71±0.24 μg/ml) (P<0.05; n=14), and the creatine:creatinine ratio was higher 600% (2.45±2.08) versus placebo (0.35±0.20) (P<0.05).
Plasma lactate.
A significant treatment × time interaction was found in plasma lactate (P?<0.05, one-tailed), with lower plasma lactate concentrations during the combination treatment phase and no effect observed during the placebo phase (Fig. 1).
*? P ?<0.05, one-tailed. COMB, combination therapy; CPEO, chronic progressive external ophthalmoplegia; KSS, Kearns–Sayre syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes. Black column, combination therapy; column, placebo.
A significant three-way interaction (group × treatment × time) (P?<0.05) was observed for FFM, TBW and %BF (Fig. 2), with increases in FFM and TBW and decreases in %BF only To MELAS GROUP.
(A) Fat-free mass (FFM), (B) total body water (TBW) and (C) body fat percentage (%BF) before and after each treatment phase in the three groups. *?P?<0.05; **?P?<0.05, one-tailed. COMB, combination therapy; CPEO, chronic progressive external ophthalmoplegia; KSS, Kearns–Sayre syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes. Black column, combination therapy; column, placebo.
Lung function.
No effects of treatment, group or time on forced vital capacity or forced expiratory volume were observed within 1 s (Table 2).
Table 2. Lung function (n = 11).
CPEO, chronic progressive external ophthalmoplegia; FEV?1, forced expiratory volume 1 s; FVC, forced vital capacity; KSS, Kearns–Sayre syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like attacks.
Strength measures.
Although there was a non-significant trend toward a decrease in peak grip strength at the end of each phase regardless of treatment ( P ? = 0.054), there was no significant decrease in peak grip strength by treatment, group or time. Influence. There were also no treatment, group, or time effects on grip or ankle dorsiflexion fatigue (expressed as peak fatigue or zone fatigue) or peak knee extension strength. However, a significant two-way interaction (treatment × time) in peak ankle dorsiflexion strength was observed, with peak ankle dorsiflexion strength significantly decreasing after placebo (from 31.16±13.68 Nm to 29.06±13.31 Nm), but not Combination treatment (from 29.32 ± 13.78 Nm to 29.31 ± 12.05 Nm) was observed (P < 0.05, n = 16).
Urine 8-OHdG and 8-IsoP.
There were no treatment or group effects for urinary 8-OHdG; however, there was no statistically significant trend toward lower 8-OHdG/creatinine after combination treatment compared with placebo (3,472.05 ± 1,883.06 ng/g creatinine, respectively) vs. 4,165±1,985.00 ng/g creatinine; P = 0.065). The therapeutic effect of 8-IsoP was observed such that lower urinary 8-IsoP/creatinine content was observed after combined treatment compared with placebo (6,572.47 ± 3,356.64 ng/g creatinine vs. 7,463.43 ± 3,155.23 ng/g creatinine, respectively). ; P <0.05).
Combined treatment with CrM, CoQ?10, and lipoic acid reduces resting lactate concentration, prevents loss of peak ankle dorsiflexion strength, and reduces oxidative stress, which is excreted via 8-IsoP in the urine and urine reflected in a significant reduction. Directional trends in 8-OHdG excretion in all groups. Furthermore, in the MELAS group, patients experienced positive changes in body composition (increase in FFM and TBW, decrease in %BF). The combination therapy had no effect on lung function, peak grip strength or knee extension strength, or grip strength or ankle dorsiflexion percentage, or regional fatigue.
Mitochondrial diseases that result from mutations result in defects in oxidative phosphorylation, resulting in increased dependence on nonaerobic energy sources 16 , 38 and an elevated plasma lactate concentration. 16 , 29 , 38 Either the phosphocreatine (PCR) system, adenylate kinase/AMP deaminase, or glycolysis/glycogenolysis can be used to provide ATP; however, due to the negative impact on glycolysis/glycogen Increased dependence on glycolysis, resulting in elevated lactate 38 CrM was included in the combination therapy used in this study to enhance the PCr system. The increase in urinary creatine:creatinine and the decrease in plasma lactate concentration after combined treatment indirectly suggested that the CrM component in combined treatment may provide another anaerobic energy source for muscle contraction.
Lower levels of total creatine 36 and PCr? 14 were observed in muscle from patients with mitochondrial disease, further supporting the potential benefit of CrM supplementation in such patients. Recent research by Kornblum et al. 14 studied the effect of CrM supplementation on intramuscular PCr in patients with CPEO or KSS. In contrast, previously observed results in healthy subjects 6 , 11 did not cause CrM to work upon supplementation despite significant increases in intramuscular creatine concentrations as measured by phospho-31 NMR spectroscopy. . 14 A limitation of the current study is that creatine or PCr content was not measured in brain or skeletal muscle. However, Burke et al. 6 showed that in healthy volunteers, when CrM was combined with lipoic acid, muscle PCr and total creatine concentrations were significantly higher than when CrM was supplemented alone. Therefore, lipoic acid may have increased CrM uptake in our patients, resulting in the observed decrease in resting plasma lactate concentrations.
An alternative or additional explanation for the lower lactate concentrations could be that combination treatment improves mitochondrial ATP production. Coenzyme Q?10 is the electron acceptor in ETC, which transfers electrons from complexes I and II to complex III. 16 , 18 , 33 The goal of CoQ 10 supplementation is to bypass defects in ETC to maximize ATP production.
16 A study using cultured lymphocytes from patients with mitochondrial cytopathies found that combination therapy with CoQ?10 increased mitochondrial ATP production, approximately 49% of which was attributable to CoQ?10. 19 In contrast, the results of human studies are not conclusive, with some reports reporting beneficial effects of CoQ in reducing plasma resting lactate concentrations in patients with mitochondrial disease, 1 , 2 and others not. 19 , 20 , 38 Unlike previous reports, patients in our study were also given lipoic acid. Lipoic acid occurs naturally in mitochondria and is an important cofactor for pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. 33 Lipoic acid acts as a potent antioxidant 31 , 33 and reduces markers of oxidative stress in healthy volunteers. 17 Increased scavenging of ROS by lipoic acid may slow the “vicious cycle” in mitochondrial disease, in which ROS production leads to mtDNA mutations that exacerbate defects in oxidative phosphorylation, leading to more ROS generation. 16 Therefore, Coenzyme Q?10, in combination with lipoic acid, may have the ability to increase ATP production, resulting in decreased utilization of alternative energy sources and lower plasma lactate concentrations.
Combination therapy attenuated the decline in peak ankle dorsiflexion strength after placebo treatment. Presumably, the components of CRM in combination therapy would result in improved strength values ??compared to placebo, as CRM has been shown to improve strength in patients with mitochondrial disease 35 , 38 or Duchenne muscular dystrophy 34 and in older healthy volunteers. . 5 Given that we did not directly measure creatine or PCr content in the muscle, we cannot conclude that the CrM component of the combination therapy caused a decrease in peak ankle dorsiflexion strength. Other studies show that taking CoQ improves strength in people with mitochondrial disease 10 supplements. 4, 9
Previous studies have shown that CrM supplementation can improve body composition. 5,34 The MELAS group in this study demonstrated improved combination therapy, increased FFM and TBW, and decreased %BF following combination therapy; however, these improvements were not seen in patients in the CPEO/KSS or other groups. Patients with MELAS exhibited a more severe clinical phenotype compared with patients with other forms of mitochondrial disease represented in the other two groups of this study. Therefore, patients with MELAS had greater room for improvement in all variables measured in this study, including body composition.
High levels of ROS and oxidative stress are related to the pathophysiology of mitochondrial diseases. Higher levels of oxidative stress have been reported in patients with mitochondrial disease compared with controls 21 , 39 and a higher degree of heterogeneity in mitochondrial DNA mutations in patients. 7 All three compounds in the combination therapy have oxidative stress-reducing properties. Creatine has direct antioxidant properties in cell-free systems 15 and provides cytoprotective effects to mammalian cells incubated with a variety of oxidants. 30 Coenzyme Q?10 acts as an antioxidant for lipids and mitochondrial membranes 10,33 and may also reduce electron leakage from the ETC by bypassing defects in oxidative phosphorylation. 10 Finally, healthy volunteers had lower urinary isoprostane levels after lipoic acid supplementation. 17 We observed lower 8-IsoP concentrations after combination treatment compared with placebo; however, only a trend toward lower 8-OHdG levels was observed. Isoprostanes are prostaglandin-like compounds formed from the peroxidation of arachidonic acid. 22?-?24 They are chemically stable, formed in vivo, and are a peroxidation-specific product detectable at steady-state levels in a variety of human tissues and body fluids;? All these characteristics make 8-IsoP Considered the most reliable marker for assessing oxidative stress in the body. 23 , 24 8-OHdG is formed by hydroxylation of guanosine residues and is often used as a biomarker for DNA damaging ROS. 28,39 Since 8-OHdG is a biomarker for oxidative damage to all DNA, not only mitochondrial DNA, it is possible that the presence of nuclear DNA may mask or dilute combination treatments used to reduce oxidative damage to mtDNA beneficial effects.
Few randomized controlled trials have examined the role of nutraceuticals in patients with mitochondrial disease.
Those that have been rigorously examined and whose only effects are single compounds such as CRM 12 , 13 , 38 or CoQ 10 , 9 have been reviewed. Other studies that examined the effects of combination treatments 1 , 19 , 20 , 26 , 27 , 32 did not use the same rigorous study design as our study. As a result, direct comparisons with these studies are difficult, especially when combined with the fact that different compounds, combinations, and outcome measures were examined in different mitochondrial disease populations. Given the nearly unlimited combinations, multiple screening methods must be employed to test potential therapies based on sound first principles before evaluation in future clinical trials. Methodologies, such as the use of transgenic animal models or hybrid animals, may prove useful in evaluating the many potential combinations of the dozen or so compounds currently used in "mitochondrial cocktails."
Our results show that combined therapy with CrM, CoQ?10, and lipoic acid, targeting three consequences of mitochondrial dysfunction, improves resting plasma lactate concentration, body composition, and ankle compared with placebo. Dorsiflexion strength and oxidative stress. However, one treatment strategy may not be universally applicable to all mitochondrial diseases because one patient group has greater benefit than others (MELAS > CPEO/KSS = other).
This research was supported by a generous donation from Warren Lammert and his family. Coenzyme Q?10 and lipoic acid were donated by Tishcon, and creatine monohydrate was donated by Avicena.
8-IsoP, 8-isoprostane; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; %BF, body fat percentage; Coenzyme Q 10, Coenzyme Q 10 ?;?CPEO, chronic progressive external ophthalmoplegia; CrM, creatine monohydrate; ETC, electron transport chain; FFM, fat-free material; HPLC; KSS, Kearns–Sayre syndrome; MELAS, mitochondria Encephalopathy, lactic acidosis and stroke-like episodes; mtDNA, mitochondrial DNA; PCr, creatine phosphate; ROS, reactive oxygen species; TBW, total body water
Omitted