Secondary coenzyme Q10 deficiency and oxidative stress in cultured fibroblasts from patients with riboflavin responsive multiple Acyl-CoA dehydrogenation deficiency.

Coenzyme Q10 (CoQ10) is essential for the energy production of the cells and as an electron transporter in the mitochondrial respiratory chain. CoQ10 links the mitochondrial fatty acid ß-oxidation to the respiratory chain by accepting electrons from electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Recently, it was shown that a group of patients with the riboflavin responsive form of multiple acyl-CoA dehydrogenation deficiency (RR-MADD) carrying inherited amino acid variations in ETF-QO also had secondary CoQ10 deficiency with beneficial effects of CoQ10 treatment, thus adding RR-MADD to an increasing number of diseases involving secondary CoQ10 deficiency. In this study, we show that moderately decreased CoQ10 levels in fibroblasts from six unrelated RR-MADD patients were associated with increased levels of mitochondrial reactive oxygen species (ROS). Treatment with CoQ10, but not with riboflavin, could normalize the CoQ10 level and decrease the level of ROS in the patient cells. Additionally, riboflavin-depleted control fibroblasts showed moderate CoQ10 deficiency, but not increased mitochondrial ROS, indicating that variant ETF-QO proteins and not CoQ10 deficiency are the causes of mitochondrial ROS production in the patient cells. Accordingly, the corresponding variant Rhodobacter sphaeroides ETF-QO proteins, when overexpressed in vitro, bind a CoQ10 pseudosubstrate, Q10Br, less tightly than the wild-type ETF-QO protein, suggesting that molecular oxygen can get access to the electrons in the misfolded ETF-QO protein, thereby generating superoxide and oxidative stress, which can be reversed by CoQ10 treatment.

Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2.

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

Novel C12orf65 mutations in patients with axonal neuropathy and optic atrophy.

OBJECTIVE: Charcot-Marie Tooth disease (CMT) forms a clinically and genetically heterogeneous group of disorders. Although a number of disease genes have been identified for CMT, the gene discovery for some complex form of CMT has lagged behind. The association of neuropathy and optic atrophy (also known as CMT type 6) has been described with autosomaldominant, recessive and X-linked modes of inheritance. Mutations in Mitofusin 2 have been found to cause dominant forms of CMT6. Phosphoribosylpyrophosphate synthetase-I mutations cause X-linked CMT6, but until now, mutations in the recessive forms of disease have never been identified. METHODS: We here describe a family with three affected individuals who inherited in an autosomal recessive fashion a childhood onset neuropathy and optic atrophy. Using homozygosity mapping in the family and exome sequencing in two affected individuals we identified a novel protein-truncating mutation in the C12orf65 gene, which encodes for a protein involved in mitochondrial translation. Using a variety of methods we investigated the possibility of mitochondrial impairment in the patients cell lines. RESULTS: We described a large consanguineous family with neuropathy and optic atrophy carrying a loss of function mutation in the C12orf65 gene. We report mitochondrial impairment in the patients cell lines, followed by multiple lines of evidence which include decrease of complex V activity and stability (blue native gel assay), decrease in mitochondrial respiration rate and reduction of mitochondrial membrane potential. CONCLUSIONS: This work describes a mutation in the C12orf65 gene that causes recessive form of CMT6 and confirms the role of mitochondrial dysfunction in this complex axonal neuropathy.

Poor maternal nutrition followed by accelerated postnatal growth leads to alterations in DNA damage and repair, oxidative and nitrosative stress, and oxidative defense capacity in rat heart.

Low birth weight and accelerated postnatal growth lead to increased risk of cardiovascular disease. We reported previously that rats exposed to a low-protein diet in utero and postnatal catch-up growth (recuperated) develop metabolic dysfunction and have reduced life span. Here we explored the hypothesis that cardiac oxidative and nitrosative stress leading to DNA damage and accelerated cellular aging could contribute to these phenotypes. Recuperated animals had a low birth weight (P<0.001) but caught up in weight to controls during lactation. At weaning, recuperated cardiac tissue had increased (P<0.05) protein nitrotyrosination and DNA single-stranded breaks. This condition was preceded by increased expression of DNA damage repair molecules 8-oxoguanine-DNA-glycosylase-1, nei-endonuclease-VIII-like, X-ray-repair-complementing-defective-repair-1, and Nthl endonuclease III-like-1 on d 3. These differences were maintained on d 22 and became more pronounced in the case of 8-oxoguanine-DNA-glycosylase-1 and nei-endonuclease-VIII-like. This was accompanied by increases in xanthine oxidase (P<0.001) and NADPH oxidase (P<0.05), major sources of reactive oxygen species (ROS). The detrimental effects of increased ROS in recuperated offspring may be exaggerated at 22 d by reductions (P<0.001) in the antioxidant enzymes peroxiredoxin-3 and CuZn-superoxide-dismutase. We conclude that poor fetal nutrition followed by accelerated postnatal growth results in increased cardiac nitrosative and oxidative-stress and DNA damage, which could contribute to age-associated disease risk.

Coenzyme Q10 quantification in muscle, fibroblasts and cerebrospinal fluid by liquid chromatography/tandem mass spectrometry using a novel deuterated internal standard.

RATIONALE: Neurological dysfunction is common in primary coenzyme Q10 (2,3-dimethoxy, 5-methyl, 6-polyisoprene parabenzoquinone; CoQ10 ; ubiquinone) deficiencies, the most readily treatable subgroup of mitochondrial disorders. Therapeutic benefit from CoQ10 supplementation has also been noted in other neurodegenerative diseases. CoQ10 can be measured by high-performance liquid chromatography (HPLC) in plasma, muscle or leucocytes; however, there is no reliable method to quantify CoQ10 in cerebrospinal fluid (CSF). Additionally, many methods use CoQ9 , an endogenous ubiquinone in humans, as an internal standard. METHODS: Deuterated CoQ10 (d6 -CoQ10 ) was synthesised by a novel, simple, method. Total CoQ10 was measured by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using d6 -CoQ10 as internal standard and 5 mM methylamine as an ion-pairing reagent. Chromatography was performed using a Hypsersil GOLD C4 column (150 × 3 mm, 3 µm). RESULTS: CoQ10 levels were linear over a concentration range of 0-200 nM (R(2) = 0.9995). The lower limit of detection was 2 nM. The inter-assay coefficient of variation (CV) was 3.6% (10 nM) and 4.3% (20 nM), and intra-assay CV 3.4% (10 nM) and 3.6% (20 nM). Reference ranges were established for CoQ10 in CSF (5.7-8.7 nM; n = 17), fibroblasts (57.0-121.6 pmol/mg; n = 50) and muscle (187.3-430.1 pmol/mg; n = 15). CONCLUSIONS: Use of d6 -CoQ10 internal standard has enabled the development of a sensitive LC/MS/MS method to accurately determine total CoQ10 levels. Clinical applications of CSF CoQ10 determination include identification of patients with cerebral CoQ10 deficiency, and monitoring CSF CoQ10 levels following supplementation.

A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy.

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.

Assessment of mitochondrial electron transport chain function in a primary astrocyte cell model of hyperhomocystinaemia.

Elevated plasma homocysteine (Hcy) has been detected in patients with various neurodegenerative conditions. Studies on neurones and cerebral tissue have revealed that hyperhomocystinaemia may inhibit mitochondrial electron transport chain (ETC) enzyme activity resulting in neuronal morbidity. As astrocytes convey a protective and supportive role towards neurones, we postulated that Hcy-induced astrocytic ETC inhibition may contribute to neurological dysfunction. In order to investigate this hypothesis, we established a cellular model of hyperhomocystinaemia using primary rat astrocytes. Which were incubated were incubated with 200 µM, 500 µM Hcy and the Hcy metabolite, thiolactone (10 µM). Following 96 h of incubation with 200 µM and 500 µM Hcy, an approximate two-fold (1.11 nmol/mg) and three-fold (1.45 nmol/mg) increase in mitochondrial levels of Hcy, respectively, were detected compared to control levels (0.54 nmol/mg). However, on exposure to Hcy (200 or 500 µM) and Hcy-thiolactone (10 µM), the activities of astrocytic ETC complex I, II-III and IV were found to be comparable to control levels. In addition, the extracellular lactate:pyruvate ratio and the intracellular glutathione status of primary rat astrocytes were not significantly different between Hcy (200 or 500 µM) treated and controls. In conclusion, the results of this study suggest that Hcy induced impairment of astrocytic ETC function may not contribute to the pathophysiology of hyperhomocystinaemia.

Detection of Borrelia miyamotoi and Powassan Virus Lineage II (Deer Tick Virus) from Odocoileus virginianus Harvested Ixodes scapularis in Oklahoma.

Odocoileus virginianus (white-tailed deer) is the primary host of adult Ixodes scapularis (deer tick). Most of the research into I. scapularis has been geographically restricted to the northeastern United States, with limited interest in Oklahoma until recently as the I. scapularis populations spread due to climate change. Ticks serve as a vector for pathogenic bacteria, protozoans, and viruses that pose a significant human health risk. To date, there has been limited research to determine what potential tick-borne pathogens are present in I. scapularis in central Oklahoma. Using a one-step multiplex real-time reverse transcription-PCR, I. scapularis collected from white-tailed deer was screened for Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, and deer tick virus (DTV). Ticks (n = 394) were pooled by gender and life stage into 117 samples. Three pooled samples were positive for B. miyamotoi and five pooled samples were positive for DTV. This represents a minimum infection rate of 0.8% and 1.2%, respectively. A. phagocytophilum, B. burgdorferi, and B. microti were not detected in any samples. This is the first report of B. miyamotoi and DTV detection in Oklahoma I. scapularis ticks. This demonstrates that I. scapularis pathogens are present in Oklahoma and that further surveillance of I. scapularis is warranted.

Rosuvastatin lowers coenzyme Q10 levels, but not mitochondrial adenosine triphosphate synthesis, in children with familial hypercholesterolemia.

OBJECTIVE: To investigate whether statin therapy affects coenzyme Q10 (CoQ10) status in children with heterozygous familial hypercholesterolemia (FH). STUDY DESIGN: Samples were obtained at baseline (treatment naïve) and after dose titration with rosuvastatin, aiming for a low-density lipoprotein cholesterol level of 110 mg/dL. Twenty-nine patients were treated with 5, 10, or 20 mg of rosuvastatin for a mean period of 29 weeks. RESULTS: We found a significant (32%) decrease in peripheral blood mononuclear cell (PBMC) CoQ10 level (P = .02), but no change in PBMC adenosine triphosphate synthesis (P = .60). Uncorrected plasma CoQ10 values were decreased significantly, by 45% (P < .01). In contrast, ratios of plasma CoQ10/total cholesterol and CoQ10/low-density lipoprotein cholesterol remained equal during treatment. CONCLUSIONS: In children with FH, rosuvastatin causes a significant decrease in cellular PBMC CoQ10 status but does not affect mitochondrial adenosine triphosphate synthesis in children with FH. Further studies should address whether (rare) side effects of statin therapy could be explained by a deterioration in CoQ10 status.

Pyridoxal 5'-phosphate deficiency causes a loss of aromatic L-amino acid decarboxylase in patients and human neuroblastoma cells, implications for aromatic L-amino acid decarboxylase and vitamin B(6) deficiency states.

Pyridoxal 5'-phosphate, the active form of vitamin B(6), is an essential cofactor for multiple enzymes, including aromatic l-amino acid decarboxylase that catalyses the final stage in the production of the neurotransmitters dopamine and serotonin. In two patients with inherited disorders of vitamin B(6) metabolism, we observed reductions in plasma aromatic l-amino acid decarboxylase activity. In one patient, this change was related to an increase in K(m) for pyridoxal 5'-phosphate. Furthermore, pyridoxal 5'-phosphate-deficient human SH-SY5Y neuroblastoma cells were found to exhibit reduced levels of aromatic l-amino acid decarboxylase activity and protein but with no alteration in expression. Further reductions in activity and protein were observed with the addition of the vitamin B(6) antagonist 4-deoxypyridoxine, which also reduced aromatic l-amino acid decarboxylase mRNA levels. Neither pyridoxal 5'-phosphate deficiency nor the addition of 4-deoxypyridoxine affected aromatic l-amino acid decarboxylase stability over 8 h with protein synthesis inhibited. Increasing extracellular availability of pyridoxal 5'-phosphate was not found to have any significant effect on intracellular pyridoxal 5'-phosphate concentrations or on aromatic l-amino acid decarboxylase. These findings suggest that maintaining adequate pyridoxal 5'-phosphate availability may be important for optimal treatment of aromatic l-amino acid decarboxylase deficiency and l-dopa-responsive conditions.

Fungal endophytes of native grasses decrease insect herbivore preference and performance.

Endophytic fungal symbionts of grasses are well known for their protective benefit of herbivory reduction. However, the majority of studies on endophyte-grass symbioses have been conducted on economically important, agricultural species-particularly tall fescue (Lolium arundinaceum) and perennial ryegrass (Lolium perenne)-raising the hypothesis that strong benefits are the product of artificial selection. We examined whether fungal endophytes found in natural populations of native grass species deterred insect herbivores. By testing several native grass-endophyte symbiota, we examined phylogenetic signals in the effects of endophytes on insects and compared the relative importance of herbivore and symbiotum identity in the outcome of the interactions. Preference was assessed using three herbivore species [Spodoptera frugiperda (Lepidoptera), Schistocerca americana (Orthoptera), Rhopalosiphum padi (Hemiptera)] and ten native symbiota, which spanned seven grass genera. We also assessed herbivore performance in a no choice experiment for five native symbiota against S. frugiperda. We compared greenhouse and laboratory trials with natural levels of herbivory measured in experimental field populations. In all cases, we included the agronomic grass species, L. arundinaceum, to compare with results from the native grasses. Both in the field and in experimental trials, herbivores showed a significant preference for endophyte-free plant material for the majority of native grasses, with up to three times lower herbivory for endophyte-symbiotic plants; however, the degree of response depended on the identity of the herbivore species. Endophyte presence also significantly reduced performance of S. frugiperda for the majority of grass species. In contrast, the endophyte in L. arundinaceum had few significant anti-herbivore effects, except for a reduction in herbivory at one of two field sites. Our results demonstrate that the mechanisms by which native symbionts deter herbivores are at least as potent as those in model agricultural systems, despite the absence of artificial selection.

Oxidative stress and mitochondrial dysfunction in neurodegeneration; cardiolipin a critical target?

Oxidative stress and subsequent impairment of mitochondrial function is implicated in the neurodegenerative process and hence in diseases such as Parkinson's and Alzheimer's disease. Within the brain, neuronal and astroglial cells can display a differential susceptibility to oxidant exposure. Thus, astrocytes can up regulate glutathione availability and, in response to mitochondrial damage, glycolytic flux. Whilst neuronal cells do not appear to possess such mechanisms, neuronal glutathione status may be enhanced due to the trafficking of glutathione precursors from the astrocyte. However, when antioxidants reserves are not sufficient or the degree of oxidative stress is particularly great, mitochondrial damage occurs, particularly at the level of complex IV (cytochrome oxidase). Whilst the exact mechanism for the loss of activity of this enzyme complex is not know, it is possible that loss and/or oxidative modification of the phospholipid, cardiolipin is a critical factor. Consequently, in this short article, we also consider (a) cardiolipin metabolism and function, (b) the susceptibility of this molecule to undergo oxidative modification following exposure to oxidants such as peroxynitrite, (c) loss of mitochondrial cardiolipin in neurodegenerative disorders, (d) methods of detecting cardiolipin and (e) possible therapeutic strategies that may protect cardiolipin from oxidative degradation.

A new perspective on the treatment of aromatic L-amino acid decarboxylase deficiency.

The final step in production of the neurotransmitters dopamine and serotonin is catalyzed by aromatic l-amino acid decarboxylase (AADC). AADC deficiency is a debilitating genetic condition that results in a deficit in these neurotransmitters, and manifests in infancy as a severe movement disorder with developmental delay. Response to current treatments is often disappointing. We have reviewed the literature to look for improvements to the current treatment strategy and also for new directions for AADC deficiency treatment. There may be differences in the mode of action, side-effect risk and effectiveness between different dopamine agonists and monoamine oxidase inhibitors currently used for AADC deficiency treatment. The range of these drugs used requires re-evaluation as some may have greater efficacy than others. Pyridoxal 5'-phosphate, the AADC cofactor may stabilize AADC and could increase AADC activity. Pyridoxal 5'-phosphate could have advantages as a treatment instead of pyridoxine. Atypical neuroleptics and peripheral AADC inhibitors both increase AADC activity in vivo and could be a future direction for AADC deficiency treatment and related conditions. Parkinson's disease gene therapy to deliver and express the human AADC gene in striatum is being tested in humans. Consequently gene therapy for AADC deficiency could be a realistic aim however an animal model of AADC deficiency is important for further progression.

Decreased ubiquinone availability and impaired mitochondrial cytochrome oxidase activity associated with statin treatment.

In order to investigate the potential involvement of mitochondrial electron transport chain (ETC) dysfunction in myotoxicity associated with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin) treatment, assessment was made of ETC activity and ubiquinone status in two patients experiencing myopathy following treatment with simvastatin (40 mg/day) and cyclosporin (patient 1) and simvastatin (40 mg/day) and itraconazole (patient 2). Analysis of skeletal muscle biopsies revealed a decreased ubiquinone status (77 and 132; reference range: 140-580 pmol/mg) and cytochrome oxidase (complex IV) activity (0.006 and 0.007 reference range: 0.014-0.034). To assess statin treatment in the absence of possible pharmacological interference from cyclosporin or itraconazole, primary astrocytes were cultured with lovastatin (100 microM). Lovastatin treatment resulted in a decrease in ubiquinone (97.9 +/- 14.9; control: 202.9 +/- 18.4 pmol/mg; p < 0.05), and complex IV activity (0.008 +/- 0.001; control: 0.011 +/- 0.001; p < 0.05) relative to control. These data, coupled with the patient findings, indicate a possible association between statin treatment, decreased ubiquinone status, and loss of complex IV activity.

Blood Mononuclear Cell Mitochondrial Respiratory Chain Complex IV Activity Is Decreased in Multiple Sclerosis Patients: Effects of ß-Interferon Treatment.

OBJECTIVES: Evidence of mitochondrial respiratory chain (MRC) dysfunction and oxidative stress has been implicated in the pathophysiology of multiple sclerosis (MS). However, at present, there is no reliable low invasive surrogate available to evaluate mitochondrial function in these patients. In view of the particular sensitivity of MRC complex IV to oxidative stress, the aim of this study was to assess blood mononuclear cell (BMNC) MRC complex IV activity in MS patients and compare these results to age matched controls and MS patients on ß-interferon treatment. METHODS: Spectrophotometric enzyme assay was employed to measure MRC complex IV activity in blood mononuclear cell obtained multiple sclerosis patients and aged matched controls. RESULTS: MRC Complex IV activity was found to be significantly decreased (p < 0.05) in MS patients (2.1 ± 0.8 k/nmol × 10-3; mean ± SD] when compared to the controls (7.2 ± 2.3 k/nmol × 10-3). Complex IV activity in MS patients on ß-interferon (4.9 ± 1.5 k/nmol × 10-3) was not found to be significantly different from that of the controls. CONCLUSIONS: This study has indicated evidence of peripheral MRC complex IV deficiency in MS patients and has highlighted the potential utility of BMNCs as a potential means to evaluate mitochondrial function in this disorder. Furthermore, the reported improvement of complex IV activity may provide novel insights into the mode(s) of action of ß-interferon.

The effect of HMG-CoA reductase inhibitors on coenzyme Q10: possible biochemical/clinical implications.

The HMG-CoA reductase inhibitors, also known as statins, have an enviable safety profile; however, myotoxicity and to a lesser extent hepatotoxicity have been noted in some patients following treatment. Statins target several tissues, depending upon their lipophilicity, where they competitively inhibit HMG-CoA reductase, the rate-limiting enzyme for mevalonic acid synthesis and subsequently cholesterol biosynthesis. HMG-CoA reductase is also the first committed rate-limiting step for the synthesis of a range of other compounds including steroid hormones and ubidecarenone (ubiquinone), otherwise known as coenzyme Q(10) (CoQ(10)). Recent interest has focused on the possible role CoQ(10) deficiency may have in the pathophysiology of the rare adverse effects of statin treatment. Currently, there is insufficient evidence from human studies to link statin therapy unequivocally to pathologically significantly decreased tissue CoQ(10) levels. Although statin treatment has been reported to lower plasma/serum CoQ(10) status, few human studies have assessed tissue CoQ(10) status. The plasma/serum CoQ(10) level is influenced by a number of physiological factors and, therefore, has limited value as a means of assessing intracellular CoQ(10) status. In those limited studies that have assessed the effect of statin treatment upon tissue CoQ(10) levels, none have shown evidence of a fall in CoQ(10) levels. This may reflect the doses of statins used, since many appear to have been used at doses below those recommended for their maximum therapeutic effects. Moreover, the poor bioavailability in those peripheral tissues tested may not reflect the effects the agents are having in liver and muscle, the tissues commonly affected in those patients who do not tolerate statins. This article reviews the biochemistry of CoQ(10), its role in cellular metabolism and the available evidence linking possible CoQ(10) deficiency to statin therapy.

Biochemical diagnosis of coenzyme q10 deficiency.

Coenzyme Q10 (CoQ10) deficiency appears to have a particularly heterogeneous clinical presentation. However, there appear to be 5 recognisable clinical phenotypes: encephalomyopathy, severe infantile multisystemic disease, nephropathy, cerebellar ataxia, and isolated myopathy. However, although useful, clinical symptoms alone are insufficient for the definitive diagnosis of CoQ10 deficiency which relies upon biochemical assessment of tissue CoQ10 status. In this article, we review the biochemical methods used in the diagnosis of human CoQ10 deficiency and indicate the most appropriate tissues for this evaluation.

Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes.

We evaluated coenzyme Q10 (CoQ) levels in patients studied under suspicion of mitochondrial DNA depletion syndromes (MDS) (n=39). CoQ levels were quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A high percentage of MDS patients presented with CoQ deficiency as compared to other mitochondrial patients (Mann-Whitney-U test: p=0.001). Our findings suggest that MDS are frequently associated with CoQ deficiency, as a possible secondary consequence of disease pathophysiology. Assessment of muscle CoQ status seems advisable in MDS patients since the possibility of CoQ supplementation may then be considered as a candidate therapy.

Levels of 5-methyltetrahydrofolate and ascorbic acid in cerebrospinal fluid are correlated: implications for the accelerated degradation of folate by reactive oxygen species.

Deficiency of 5-methyltetrahydrofolate (5-MTHF) in cerebrospinal fluid (CSF) is associated with a number of neurometabolic conditions including mitochondrial electron transport chain defects. Whilst failure of the active transport of 5-methyltetrahydrofolate (5-MTHF) into the CSF compartment has been proposed as a potential mechanism responsible for the 5-MTHF deficiency seen in mitochondrial disorders, it is becoming increasingly clear that other mechanisms are involved. Here, we have considered the role of oxidative stress as a contributing mechanism. Concerning, ascorbic acid (AA), we have established a CSF reference range (103-303µM) and demonstrated a significant positive correlation between 5-MTHF and AA. Furthermore, CSF itself was also shown to convey antioxidant properties towards 5-MTHF. However, this protection could be overcome by the introduction of a hydroxyl radical generating system. Using a neuronal model system, inhibition of mitochondrial complex I, by 58%, was associated with a 23% increase in superoxide generation and a significantly increased loss of 5-MTHF from the extracellular medium. Addition of AA (150µM) was able to prevent this increased 5-MTHF catabolism. We conclude that increased generation of reactive oxygen species and/or loss of CSF antioxidants are also factors to consider with regard to the development of a central 5-MTHF deficiency. Co-supplementation of AA together with appropriate folate replacement may be of therapeutic benefit.

Dopamine but not l-dopa stimulates neural glutathione metabolism. Potential implications for Parkinson's and other dopamine deficiency states.

Dopamine is produced first by hydroxylalation of l-tyrosine to l-dihydroxyphenylalanine (l-dopa) and subsequently by the decarboxylation of l-dopa to dopamine catalysed by the enzymes tyrosine hydroxylase and aromatic l-amino acid decarboxylase (AADC) respectively. Reduced glutathione (GSH) acts as a major cellular antioxidant. We have investigated the role of dopamine in the control of GSH homeostasis in brain cells. The SH-SY5Y human neuroblastoma cell line was found to increase intracellular GSH levels in response to 50µM dopamine treatment. Similarly the 1321N1 human astrocytoma cell line was found to increase GSH release in response to 50µM dopamine. The same concentration of l-dopa was also found to increase intracellular GSH in SH-SY5Y cells, however when AADC was inhibited this affect was abolished. Furthermore 1321N1 cells which were found to have almost undetectable levels of AADC activity did not increase GSH release in response to 50µM l-dopa. These results suggest that at these concentrations dopamine has the potential to act as a signal for the upregulation of GSH synthesis within neuronal-like cells and for the increased trafficking of GSH from astrocytes to neurons. This effect could potentially relate to the activation of antioxidant response elements leading to the induction of phase II detoxifying enzymes including those involved in GSH synthesis and release. The inability of l-dopa to produce a similar effect when AADC was inhibited or when AADC activity was absent indicates that these effects are relatively specific to dopamine. Additionally dopamine but not l-dopa treatment led in an increase in complex I activity of the respiratory chain in SH-SY5Y cells which may be related to the effect of dopamine on GSH levels.