Beneficial effects of inosine, coenzyme Q 10 and lipoic acid on mitochondrial diseases
Author: M. Cristina Rodriguez; Jay R. MacDonald; Douglas J. Mahoney; Jenny Pariser; Mr flint beale; Mark Tano Polschi
Magazine name muscle &；; nerve
Publication date 2007
Volume 35
Page code 235-242
00. 1002/ mu
Impact factor 2.456(2007)? 2.283(20 15)
Abstract: Mitochondrial diseases have the same cellular consequences: (1)ATP production decreases; (2) increasing the dependence on alternative anaerobic energy; (3) Increase the production of reactive oxygen species. The purpose of this study is to determine the effect of combined therapy (creatine monohydrate, coenzyme Q? The effects of 10 and lipoic acid on the above-mentioned cell consequences were studied in a randomized, double-blind, placebo-controlled and cross-over design for patients with mitochondrial cell diseases, with multiple outcome variables as the goal. Three patients suffered from mitochondrial encephalopathy, lactic acidosis and stroke-like attack (MELAS), four patients had mitochondrial DNA deletion (3 cases of chronic progressive extraocular muscle paralysis, Kearns-Sayre syndrome 1 case), and the other nine non-mitochondrial diseases were divided into the first two groups. Combined therapy can reduce the resting plasma lactic acid and urine 8- isoprostadil in all patients, and reduce the peak value of ankle dorsiflexion muscle strength, but only MELAS group observed a higher amount of fat removal. In a word, these results show that multiple combination therapies aimed at mitochondrial dysfunction in the same way can favorably affect the surrogate markers of cell energy dysfunction. In the future, it is necessary to carry out a larger sample size study in a relatively homogeneous population to determine whether this combination therapy affects function and quality of life.
Mitochondrial diseases represent a group of diseases affecting mitochondrial energy conduction, which are characterized by clinical, biochemical and genetic heterogeneity. Although the phenotypic expression of 18 is quite different, most patients are complicated with lactic acidosis, stroke or seizure, headache, retinitis pigmentosa, ptosis, low exercise endurance, ophthalmoplegia, cardiomyopathy, neuropathy and decreased vision. 16 , 29 , 38
Mitochondrial dysfunction leads to many cellular consequences, including: (1) decreased ATP production; (2) increasing the dependence on alternative anaerobic energy; (3) Increase the production of reactive oxygen species. 16,37 has no curative effect on mitochondrial diseases, and most of the strategies are aimed at alleviating the above-mentioned honeycomb consequences. Report on treatment strategies for patients with mitochondrial diseases 16, 18 The effects of single compounds, such as coenzyme Q, have been tested. 10 (coenzyme q? 10) 2,4,21or creatine (CRM). 13 , ? 14 , ? Based on the concept that mitochondrial dysfunction leads to pathophysiological consequences of some cells, compared with monotherapy, most mitochondrial diseases have combined therapy (or cocktail therapy). Some studies have evaluated the efficacy of combination therapy with more than one of the above three methods. However, these are reports anyway. 8 , ? Of the 25 public tests, 1,? 19 , ? 20 , ? 27 , ? 32 or retrospective study. 26
Based on the evidence of potential efficacy in human trials of mitochondrial diseases or from human trials or in vitro studies, we suggest to evaluate the potential efficacy of the combination drugs: (1)CrM (alternative energy source 36 and antioxidant 30); (2)α- lipoic acid (antioxidant 17 and increasing the absorption of CrM 6); (3) coenzyme q? 10? [Complex I as antioxidant 2 1 and bypass electron transfer chain (ETC) 19]. We report here the results of a randomized, double-blind, placebo-controlled, crossover trial, which studied the combination of this targeted therapy cocktail with CrM, CoQ? Effects of 10 and α -lipoic acid on patients with mitochondrial cytopathy.
Patients: 17 patients with definite or possible mitochondrial diseases were recruited from the neuromuscular and neurometabolic clinic of McMaster University. Combined with clinical symptoms, fasting serum lactic acid concentration, muscle biopsy results (red fibers or cytochrome c oxidase negative fibers) and mitochondrial DNA(mtDNA) analysis. No DNA mutation was found in patients No.8, No.9 and 13, and only the patients with mitochondrial neurogastrointestinal encephalopathy (thymidine increased and thymidine phosphorylase activity decreased) were confirmed. However, they have increased lactic acid concentration, abnormal histology, low exercise endurance and low aerobic capacity, so they are considered as "possible mitochondrial cytopathy". One patient did not complete part of the study for personal reasons; Therefore, the patient's data were excluded from the analysis. The final analysis was based on 16 patients (10 women and 6 men), and they were divided into three groups according to their diagnosis. The table shows the characteristics of the patient population 1. The first group included three patients with mitochondrial encephalopathy, lactic acidosis and stroke-like attack (MELAS group). The second group included three patients diagnosed as chronic progressive extraocular muscle paralysis (CPEO) and one patient diagnosed as Kans-Searle syndrome (KSS), all of whom had detected deletions in myogenic mtDNA (CPEO/KSS group). The third group included patients with various mitochondrial diseases: 6 patients with mitochondrial cytopathy, 2 patients with Leber hereditary optic neuropathy and 1 patient with mitochondrial neurogastrointestinal encephalopathy (other groups). The study was approved by the Ethics Committee of our institution, and all patients provided informed written consent.
CPEO, chronic progressive extraocular muscle paralysis; Cytopathy, mitochondrial cytopathy; KSS, Kearns-Searle syndrome; LHON and Leber hereditary optic neuropathy; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like attacks; MNGIE, mitochondrial neurogastrointestinal encephalopathy (no thymidine phosphorylase activity and high thymidine level).
Design/intervention.
Patients participated in a randomized, double-blind, placebo-controlled, crossover study, in which each participant received two months of treatment and placebo treatment, with a five-week clearance period between the two trials. The treatment stage includes 3 g CrM+2 g glucose+flavoring agent (neoalkali; Avicena, Palo Alto, California), 300 mg α -lipoic acid (Tishcon, Westbury, new york) and 120 mg CoQ? 10(Qgel； Tishcon) at 0: 900 and 2 1:00 every day. In the placebo stage, the powder with the same appearance and taste (5 g glucose+flavoring agent; Avicena) and gel capsules (soybean oil; Tishcon) was used as a placebo.
After fasting for 4 hours, the patients in the two experiments completed the tests before and after each intervention stage at about the same time every day (within 2-3 hours).
Measure.
Participants' height and weight were recorded only at the first visit. All other interviews used all other outcome indicators. Participants used customized force sensor equipment to test grip strength, ankle dorsiflexion (joint angle is 90) and knee extension force, and the data were directly input into the computer containing data acquisition and analysis software, as mentioned above. For all strength measurements, participants were tested on the right side and personalized according to the size of their hands, which remained unchanged between each visit. In order to reach the peak intensity, participants took three 5s tests with an interval of about 30 s. Record the test value with the best result. Participants also performed 1 min isometric grip strength and ankle flexion fatigue test (9 seconds working time: 1 sec rest period). Use a spirometer (Koko；; PDS instrument in Louisville, Colorado), including forced vital capacity and forced expiratory volume within 65438 0 seconds. Every patient should complete the vital capacity measurement at least twice at each visit to ensure that the value is consistent with the first attempt. Bioelectrical impedance (prism bia101a; ; RJL system, Clinton Twp, Michigan) to determine body composition.
Venous blood sampling and urine collection.
Whole blood was collected from the anterior elbow vein and put into precooled heparin (for lactic acid analysis) or EDTA (for coenzyme Q determination? 10) and centrifuge at 2500 rpm 10 minute. Plasma was stored at -80℃. Each patient provided a urine sample, about 10 ml, which was quickly frozen and stored at-80 C for subsequent analysis of creatine, creatinine, 8- hydroxy -2'- deoxyguanosine (8-OHdG) and 8- isoprostadin (8-IsoP).
lactic acid
Plasma lactic acid concentration was measured by YSI 2300 Stat Plus lactic acid analyzer (YSI, Huangquan, Ohio). The intra-batch and intra-batch variation coefficients of lactic acid were 2.65438 0% and 65438 0.7%, respectively.
Coenzyme q 10.
Plasma coenzyme Q was determined by high performance liquid chromatography with electrochemical detector. 10 concentration. Plasma (0.5 ml) was divided into 1 ml 1- propanol and 0.5 ml coenzyme q? 9 in 10 ml vacuum container, mix for 5 minutes, and then mix at 300? G centrifuge for 5 minutes. The sample was filtered with a 0.22μM syringe filter, then transferred to a chromatographic bottle for direct analysis by HPLC. Do you know coenzyme Q? 9 was added to the mixture as internal standard, coenzyme Q? The content of 9 in human blood is negligible. Inject the obtained sample into a reversed-phase stainless steel chromatographic column (150×3 mm)RP‐C 18, with a packing of 3μm, equipped with an electrochemical detector (ESA, Bedford, Massachusetts) and connected to a single-electrode guard room (model 5020); E = +350 mV) and a coulomb battery with double electrodes (50 1 1 type; E 1 = -400 mv, E2 = +300 mv). The mobile phase degassed by methanol, 1- propanol and ethanol (70:20: 10) is used, which contains 50 mM lithium acetate as conductive salt, the flow rate is 0.5 ml/min, and the total running time is less than 15 min? First, coenzyme Q was measured by reducing ubiquinone (E = -400 mV) and then oxidizing the obtained panthenol (E = +300 mV). 10。 Coenzyme q 10 and coenzyme q? 10? h？ 2 Detect at the last electrode with the highest sensitivity. The correlation coefficient of standard curve is 0.997. The coefficient of variation is determined by the following formula
Creatine and creatinine.
The concentrations of creatine and creatinine in urine and the ratio of creatine to creatinine were determined by HPLC. Urine (1 ml) was divided into microcentrifuge tubes and centrifuged at 1000 rpm for10 minute. Use ddH? 2? o？ Dilute the urine supernatant to one tenth (0. 1 ml supernatant to 0.9 ml ddH? 2O). Keep the diluted urine supernatant at10 C with a refrigerated autosampler. Hp LC 1 100 series high performance liquid chromatograph (Agilent, Mississauga, Ontario) was used, the ultraviolet detector was set to λ= 2 10 nm, and the sample was injected with 250× 4.6 mm c18 phenenex10-. HP LC 1 100 data analysis program will generate calibration curves and analyze the obtained data. The mobile phase is to adjust the pH value of potassium dihydrogen phosphate (20 mM) to 5.0 with potassium hydroxide, and the flow rate is 65438 0.0 ml/min. The coefficient of variation is 3. 1%.
8- isoprene.
According to the manufacturer's instructions, the concentration of 8-IsoP in urine was determined by commercial enzyme-linked immunosorbent assay (MediCorp, Montreal, Quebec). The correlation coefficient of standard curve is 0.988. The coefficient of variation is 10.5%. 8-IsoP value is expressed relative to creatinine (g).
8-OHdG .
As described above, the concentration of 8-OHdG in urine was determined by HPLC. 3 ? The value of 8-OHdG is expressed relative to creatinine (g).
Statistics.
Three-way (group× treatment× time) or two-way (group× treatment) repeated measurement analysis of variance (ANOVA) was used for statistical analysis. In view of the previous hypothesis that combination therapy can reduce lactate and oxidative stress, because each of the three components has antioxidant properties, we used a single-tailed test of oxidative stress markers. When important results are found, Tukey HSD post-test will be run. Statistica v was used for all analyses. 5 Software (StatSoft, Tulsa, Oklahoma). p？ A value of < 0.05 is considered statistically significant. All data are given as mean standard deviation.
Coenzyme q 10 and creatine: creatinine.
As expected, compared with the placebo phase, the plasma coenzyme Q? The ratio of 10 to urinary creatine: creatinine is obviously higher. Plasma COQ (1.94 0.89 μ g/ml) after combined therapy? The concentration of 10 was higher than that of placebo (0.7 1.24 μ g/ml) 172%(P? & lt0.05; ? n？ = 14), and the creatine/creatinine ratio was 600% (2.45 2.08), which was higher than that of the placebo group (0.35 0.20) (P < 0.05).
Plasma lactate.
There was a significant interaction between plasma lactic acid and treatment × time (P? < 0.05, single tail), the plasma lactic acid concentration in the combined treatment stage was low, and no effect was observed in the placebo stage (figure 1).
*? p？ & lt0.05, single tail. Comb, comprehensive therapy; CPEO, chronic progressive extraocular muscle paralysis; KSS, Kearns-Searle syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like attacks. Black column, combined therapy; List, placebo.
It was observed that FFM, TBW and% BF had significant three-way interaction (group× treatment× time) (P? < 0.05) (Figure 2), FFM and TBW increased, and only% BF in MELAS group decreased.
(a) Fat-free mass (FFM), (b) Total body moisture (TBW) and (c) Body fat percentage (%BF) before and after each treatment stage. *? p？ & lt0.05; **? p？ & lt0.05, single tail. Comb, comprehensive therapy; CPEO, chronic progressive extraocular muscle paralysis; KSS, Kearns-Searle syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like attacks. Black column, combined therapy; List, placebo.
Lung function.
Within 65438 0 seconds, no influence of treatment, grouping or time on forced vital capacity or forced expiratory volume was observed (Table 2).
Table 2. ? Lung function (n? = 1 1)。
CPEO, chronic progressive extraocular muscle paralysis; FEV？ 1, forced expiratory volume1s; ; FVC, forced vital capacity; KSS, Kearns-Searle syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like attacks.
Strength measurement.
Although for the end of each stage, no matter what treatment method is adopted, the trend of peak grip strength is not obvious (P? = 0.054), but it has no effect on the peak grip strength by group or time. There is no treatment, group or time effect on grip strength or ankle flexion fatigue (peak fatigue or local fatigue) or peak knee extension strength. However, it is observed that there is a significant two-way interaction (treatment time) in the peak intensity of ankle dorsiflexion. After taking placebo, the peak intensity of ankle dorsiflexion decreased significantly (from 31.1613.68 nm to 29.06 13.3 1 nm), but it was not observed. 0.05，n？ = 16)。
Urine 8-OHdG and 8-IsoP.
Urine 8-OHdG has no treatment or group effect; However, compared with placebo, the decrease trend of 8-OHdG/ creatinine after combined treatment was not statistically significant (3,472.051,883.06 ng/g creatinine and 4,41651,985.00 ng/g creatinine, respectively; p？ = 0.065)。 The therapeutic effect of 8-IsoP was observed, so the urine 8-IsoP/ creatinine content was lower after combined treatment (6,572.47 3,356.64 ng/g creatinine and 7,463.43 3, 155.23 ng/g creatinine, respectively). ; P :CPEO/KSS = others), so one treatment strategy may not be universally applicable to all mitochondrial diseases.
The research was generously donated by Warren Lamert and his family. Auxiliary enzyme q? 10 and lipoic acid were donated by Tishcon and creatine monohydrate was donated by Avicena.
8- isoproterenol; ? 8-OHdG, 8- hydroxy -2'- deoxyguanosine; ? % BF, body fat percentage; Coenzyme q 10, coenzyme q? 10? ; ? CPEO, chronic progressive extraocular muscle paralysis; CrM, creatine monohydrate; Electronic transmission chain; FFM, fat-free substance; High performance liquid chromatography; KSS, Kearns-Searle syndrome; MELAS, mitochondrial encephalopathy, lactic acidosis and stroke-like attacks; Keywords MtDNA, mitochondrial DNAPCr, creatine phosphate; ROS, reactive oxygen species; TBW, you're covered in water.
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