Mitochondrial disorders share common cellular consequences: (1) decreased ATP production; (2) increased reliance on alternative anaerobic energy sources; and (3) increased production of reactive oxygen species. The purpose of the present study was to determine the effect of a combination therapy (creatine monohydrate, coenzyme Q(10), and lipoic acid to target the above-mentioned cellular consequences) on several outcome variables using a randomized, double-blind, placebo-controlled, crossover study design in patients with mitochondrial cytopathies. Three patients had mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), four had mitochondrial DNA deletions (three patients with chronic progressive external ophthalmoplegia and one with Kearns-Sayre syndrome), and nine had a variety of other mitochondrial diseases not falling into the two former groups. The combination therapy resulted in lower resting plasma lactate and urinary 8-isoprostanes, as well as attenuation of the decline in peak ankle dorsiflexion strength in all patient groups, whereas higher fat-free mass was observed only in the MELAS group. Together, these results suggest that combination therapies targeting multiple final common pathways of mitochondrial dysfunction favorably influence surrogate markers of cellular energy dysfunction. Future studies with larger sample sizes in relatively homogeneous groups will be required to determine whether such combination therapies influence function and quality of life.

Beneficial effects of creatine, CoQ10, and lipoic acid in mitochondrial disorders.

Polyunsaturated fatty acids (PUFAs) are important constituents in all organisms. They fulfil many functions, ranging from modulating the structure of membranes to acting as precursors of physiologically important molecules, such as the prostaglandins, which for a long time were the most prominent cyclic PUFA metabolites. However, since the beginning of the 1990s a large variety of cyclic metabolites have been discovered that form under autoxidative conditions in vivo to a much larger extent than do prostaglandins. These compounds--isoprostanes, neuroprostanes, phytoprostanes, and isofurans--proved subsequently to be ubiquitous in nature. They display a wide range of biological activities, and isoprostanes have become the currently most reliable indicators of oxidative stress in humans. In a relatively short time, the structural variety, properties, and applications of the autoxidatively formed cyclic PUFA derivatives have been uncovered.

Beyond Prostaglandins—Chemistry and Biology of Cyclic Oxygenated Metabolites Formed by Free‐Radical Pathways from Polyunsaturated Fatty Acids

The mitochondrial cocktail: Rationale for combined nutraceutical therapy in mitochondrial cytopathies

Aims Patients with paroxysmal atrial fibrillation (AF) often present with typical angina pectoris and mildly elevated levels of cardiac troponin (non ST-segment elevation myocardial infarction) during an arrhythmic event. However, in a large proportion of these patients, significant coronary artery disease is excluded by coronary angiography. Here we explored the potential underlying mechanism of these events.

Methods and results A total of 14 pigs were studied using a closed chest, rapid atrial pacing (RAP) model. In five pigs RAP was performed for 7 h (600 b.p.m.; n = 5), in five animals RAP was performed in the presence of angiotensin-II type-1-receptor (AT1-receptor) inhibitor irbesartan (RAP+Irb), and four pigs were instrumented without intervention (Sham). One-factor analysis of variance was performed to assess differences between and within the three groups. Simultaneous measurements of fractional flow reserve (FFR) and coronary flow reserve (CFR) before, during, and after RAP demonstrated unchanged FFR ( P = 0.327), but decreased CFR during RAP (RAP: 67.7 ± 7.2%, sham: 97.2 ± 2.8%, RAP+Irb: 93.2 ± 3.3; P = 0.0013) indicating abnormal left ventricular (LV) microcirculation. Alterations in microcirculatory blood flow were accompanied by elevated ventricular expression of NADPH oxidase subunit Nox2 ( P = 0.039), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1, P = 0.004), and F2-isoprostane levels ( P = 0.008) suggesting RAP-related oxidative stress. Plasma concentrations of cardiac troponin-I (cTn-I) increased in RAP (RAP: 613.3 ± 125.8 pmol/L vs. sham: 82.5 ± 12.5 pmol/L; P = 0.013), whereas protein levels of eNOS and LV function remained unchanged. RAP+Irb prevented the increase of Nox2, LOX-1, and F2-isoprostanes, and abolished the impairment of microvascular blood flow.

Conclusion Rapid atrial pacing induces AT1-receptor-mediated oxidative stress in LV myocardium that is accompanied by impaired microvascular blood flow and cTn-I release. These findings provide a plausible mechanism for the frequently observed cTn-I elevation accompanied with typical angina pectoris symptoms in patients with paroxysmal AF and normal (non-stenotic) coronary arteries.

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Acute atrial tachyarrhythmia induces angiotensin II type 1 receptor-mediated oxidative stress and microvascular flow abnormalities in the ventricles

The mutation 3243A→G is the most common heteroplasmic pathogenic mitochondrial DNA (mtDNA) mutation in humans, but it is not understood why the proportion of this mution decreases in blood during life. Changing levels of mtDNA heteroplasmy are fundamentally related to the pathophysiology of the mitochondrial disease and correlate with clinical progression. To understand this process, we simulated the segregation of mtDNA in hematopoietic stem cells and leukocyte precursors. Our observations show that the percentage of mutant mtDNA in blood decreases exponentially over time. This is consistent with the existence of a selective process acting at the stem cell level and explains why the level of mutant mtDNA in blood is almost invariably lower than in nondividing (postmitotic) tissues such as skeletal muscle. By using this approach, we derived a formula from human data to correct for the change in heteroplasmy over time. A comparison of age-corrected blood heteroplasmy levels with skeletal muscle, an embryologically distinct postmitotic tissue, provides independent confirmation of the model. These findings indicate that selection against pathogenic mtDNA mutations occurs in a stem cell population.

Selection against Pathogenic mtDNA Mutations in a Stem Cell Population Leads to the Loss of the 3243A→G Mutation in Blood

Iso[4]-levuglandin E2 (iso[4]-LGE2, 5-acetyl-6-formyl-9-hydroxy-7(E),11(Z)-heptadecadienoic acid) is a structural isomer of levuglandin E2 (8-acetyl-9-formyl-12-hydroxy-5(Z),10(E)-heptadecadienoic acid) that we postulate to be generated during autoxidation of arachidonic acid or arachidonate esters. To facilitate detection and identification of iso[4]-LGE2 in biological samples, a succinct total synthesis was developed that depends on conjugate addition of a higher order vinyl cyanocuprate to a γ-alkoxy enone to provide a key carbon−carbon bond-forming step. Bismethoxime pentafluorobenzyl ester trimethylsilyl ether derivatives of LGE2 and iso[4]-LGE2 were prepared and shown to be suitable for mass spectral identification of these highly oxidized lipids.

Total Synthesis of Iso[4]-levuglandin E2

Mitochondria are the source of most oxygen-derived free radicals. Mutations in mitochondrial DNA can impair mitochondrial electron transport resulting in decreased ATP production and increased free radical-induced oxidant injury. The specific mitochondrial DNA mutation A8344G alters the TCC loop or the mitochondrial tRNA for lysine. We investigated a large five-generational family harboring this mutation to determine whether the degree of heteroplasmy (proportion of mutated mitochondrial genomes) for the mtA8344G mutation correlated with a marker of oxidant damage. We measured F2-isoprostanes because they are specific and reliable markers of oxidant injury formed when free radicals attack esterified arachidonate in cell membranes. Family members with high heteroplasmy (N40%) had significantly higher F2isoprostane levels (62 F 39 pg/ml) than those with lower heteroplasmy (33 F 13 pg/ml, P b 0.001). The degree of heteroplasmy for the mtA8344G mutation in this family correlated positively with F2-isoprostane levels (P = 0.03). This study highlights the underappreciated role freeradicalsplayinthecomplex pathophysiologyofinheritedmitochondrialDNAdisorders.Themostimportantnovelfindingfromthisfamily is that some currently asymptomatic individuals with moderate heteroplasmy have evidence of ongoing free-radical mediated oxidant injury.

Original Contribution Degree of heteroplasmy reflects oxidant damage in a large family with the mitochondrial DNA A8344G mutation

Quantification of Isoprostanes as Indicators of Oxidant Stress in Vivo

Compounds for use in methods of treating, preventing, or ameliorating pulmonary hypertension; reducing glutamine metabolism in a patient in need thereof, and/or increasing SIRT3 activity in a patient in need thereof.

L. Jackson Roberts

Papers

Total Synthesis of Iso[4]-levuglandin E2

Original Contribution Degree of heteroplasmy reflects oxidant damage in a large family with the mitochondrial DNA A8344G mutation

Quantification of Isoprostanes as Indicators of Oxidant Stress in Vivo

Use of 2-hydroxybenzylamine in the treatment and prevention of pulmonary hypertension

Quantification of Isoprostanes as an Index of Oxidant Stress Status In Vivo