3-methylcrotonyl-CoA carboxylase deficiency: Clinical, biochemical, enzymatic and molecular studies in 88 individuals

ABSTRACT: BACKGROUND: Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine metabolism caused by mutations in MCCC1 or MCCC2 encoding the alpha and beta subunit of MCC, respectively. The phenotype is highly variable ranging from acute neonatal onset with fatal outcome to asymptomatic adults. METHODS: We report clinical, biochemical, enzymatic and mutation data of 88 MCC deficient individuals, 53 identified by newborn screening, 26 diagnosed due to clinical symptoms or positive family history and 9 mothers, identified following the positive newborn screening result of their baby. RESULTS: Fifty-seven percent of patients were asymptomatic while 43% showed clinical symptoms, many of which were probably not related to MCC deficiency but due to ascertainment bias. However, 12 patients (5 of 53 identified by newborn screening) presented with acute metabolic decompensations. We identified 15 novel MCCC1 and 16 novel MCCC2 mutant alleles. Additionally, we report expression studies on 3 MCCC1 and 8 MCCC2 mutations and show an overview of all 132 MCCC1 and MCCC2 variants known to date. CONCLUSIONS: Our data confirm that MCC deficiency, despite low penetrance, may lead to a severe clinical phenotype resembling classical organic acidurias. However, neither the genotype nor the biochemical phenotype is helpful in predicting the clinical course.     

Isolated 3-methylcrotonyl-CoA carboxylase deficiency: evidence for an allele-specific dominant negative effect and responsiveness to biotin therapy

Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCC alpha protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA-wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo.     

Mitochondrial targeting signals and mature peptides of 3-methylcrotonyl-CoA carboxylase

Inherited deficiency of 3-methylcrotonyl-CoA carboxylase (MCC), an enzyme of leucine degradation, is an organic acidemia detectable by expanded newborn screening with a variable phenotype that ranges from asymptomatic to death in infancy. Here, we show that the two subunits of the enzyme (MCCalpha; MCCbeta) are imported into the mitochondrial matrix by the classical pathway involving cleavable amino-terminal targeting presequences. We identified the cleavage sites (Tyr41/Thr42 and Ala22/Tyr23 for MCCalpha and MCCbeta, respectively) of the targeting signals and the amino-termini of the mature polypeptides of MCC and propionyl-CoA carboxylase, a mitochondrial paralog. The amino-termini containing 39 (MCCalpha) or 20 amino acids (MCCbeta) were both necessary and sufficient for targeting. Structural requirements for mitochondrial import were defined by site-directed mutagenesis. Our studies provide the prerequisite to understand the impact of specific mutations on the clinical phenotype of MCC deficiency.     

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.     

Characterization of biotin and 3-methylcrotonyl-coenzyme a carboxylase in higher plant mitochondria

Mitochondria from green pea (Pisum sativum) leaves were purified free of peroxisomes and chlorophyll contamination and examined for their biotin content. The bulk of the bound biotin detected in plant mitochondria was shown to be associated with the matrix space to a concentration of about 13 micromolar, and no free biotin was detected. Western blot analysis of mitochondrial polypeptides using horseradish peroxidase-labeled streptavidin revealed a unique biotin-containing polypeptide with a molecular weight of 76,000. This polypeptide was implicated as being the biotinylated subunit of 3-methylcrotonyl-coenzyme A (CoA) carboxylase. Fractionation of pea leaf protoplasts demonstrated that this enzyme activity was located largely in mitochondria. The 3-methylcrotonyl-CoA carboxylase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of mitochondria. Maximal 3-methylcrotonyl-CoA carboxylase activity was found at pH 8.3 in the presence of Mg²⁺. Kinetic constants (apparent K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 0.05 millimolar; ATP, 0.16 millimolar; HCO₃⁻, 2.2 millimolar. The involvement of 3-methylcrotonyl-CoA carboxylase in the leucine degradation pathway in plant mitochondria is proposed.     

Immunocytochemical localization of 3-methylcrotonyl-CoA carboxylase in cultured ependymal, microglial and oligodendroglial cells

To evaluate the ability of ependymal, microglial and oligodendroglial cells to degrade leucine, the presence of 3-methylcrotonyl-CoA carboxylase (MCC) was investigated in cultures of these cells. MCC is a biotin-containing heterodimeric enzyme that is specific for the irreversible part of the leucine catabolic pathway. It has been reported previously that in cell culture MCC is expressed in astrocytes and a subpopulation of neurones. In the present study ependymal, microglial and oligodendroglial cell cultures, derived from the brains of newborn rats, were examined for the expression of MCC by RT-PCR, western blotting and immunocytochemistry. The results of RT-PCR and western blotting showed the presence of mRNA as well as protein of both subunits of MCC in ependymal, microglial and oligodendroglial cell cultures. Immunocytochemical investigation of the cellular and subcellular distribution of MCC demonstrated a mitochondrial location of MCC in all neuroglial cell types investigated. The ubiquitous expression of MCC in glial cells demonstrates the ability of the cells to engage in the catabolism of leucine transported into the brain, mainly for the generation of energy.     

Expression, purification, characterization of human 3-methylcrotonyl-CoA carboxylase (MCCC)

The current study reports the use of baculovirus system to express functionally active human recombinant 3-methylcrotonyl-CoA carboxylase (MCCC), a heteromultimeric complex that is composed of alpha and beta subunits which are encoded by distinct genes. Using immuno-affinity purification, an efficient protocol has been developed to purify the active MCCC which appears to reside in a approximately 500-800kDa complex in Superpose-6 gel-filtration chromatography. Consistent with the native enzyme, in the recombinant human MCCC, the stoichiometry of alpha and beta subunits are at a one:one ratio. The k(cat) value of the recombinant enzyme is determined to be approximately 4.0s(-1). It also possesses K(m) values (ATP: 45+/-11microM; 3-methylcrotonyl-CoA: 74+/-7microM) similar to those reported for the native enzyme. The recombinant human MCCC described here may provide a counter-screen enzyme source for testing cross reactivity for inhibitors against acetyl-CoA carboxylases which are designed to treat obesity, type 2 diabetes and other metabolic disorders.     

Evaluation of biotin responsiveness in cultured fibroblasts from patients with propionic acidemia: Absence of response by structurally altered carboxylases

We have demonstrated that, although propionyl CoA carboxylase (PCC) activity is deficient in fibroblast extracts from PCC-deficient patients belonging to the two major and two minor genetic complementation groups, the activity of another biotin-dependent carboxylase, -methylcrotonyl CoA carboxylase (MCC), is normal. Moreover, MCC activity is stimulated when the fibroblasts are cultured in high concentrations of biotin, in the same way that it is in normal fibroblasts, whereas the depressed PCC activity remains essentially unchanged. Because these results are parallel with the in vivo failure of high-dose biotin to stimulate PCC activity in peripheral blood leukocytes, we conclude that the biotin responsiveness of PCC in cultured fibroblasts from patients with PCC deficiency may be used to predict or confirm biotin responsiveness in vivo.     

Differential Accumulation of Biotin Enzymes during Carrot Somatic Embryogenesis

The activities of four biotin enzymes, acetyl-coenzyme A (CoA) carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase, and the accumulation of six biotin-containing polypeptides were determined during development of somatic embryos of carrot (Daucus carota). Acetyl-CoA carboxylase activity increased more than sevenfold, whereas the activities of 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase were relatively unaltered. An increase also occurred in the accumulation of three of the biotin-containing polypeptides (molecular masses of 220, 62, and 34 kilodaltons). Of these, the most dramatic change was in the accumulation of the 62-kilodalton biotin-containing polypeptide, which increased by at least 50-fold as embryogenic cell clusters developed into torpedo embryos.     

3-Methylcrotonylglycine Disrupts Mitochondrial Energy Homeostasis and Inhibits Synaptic Na+,K+-ATPase Activity in Brain of Young Rats

Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO₂ production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I–IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na⁺,K⁺-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO₂ production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na⁺,K⁺-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na⁺,K⁺-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na⁺,K⁺-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.     

Human biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase gene (MCCA): cDNA sequence, genomic organization, localization to chromosomal band 3q27, and expression

3-Methylcrotonyl-CoA carboxylase (MCCase; EC 6.4.1.4) is a mitochondrial biotin enzyme and plays an essential role in the catabolism of leucine and isovalerate in animals, bacterial species, and plants. MCCase consists of two subunits, those that are biotin-containing and non-biotin-containing. The genes responsible for these subunits have been isolated in soybean, Arabidopsis thaliana, and tomatoes, but not in mammals. In humans, MCCase deficiency has been thought to be a rare metabolic disease, but the number of patients with MCCase deficiency appears to be increasing with a wide range of clinical presentations, some that result in a lethal condition and others that are asymptomatic. In this report, we have isolated and carried out chromosomal mapping of the gene for the biotin-containing subunit (A subunit) of the human MCCase gene, MCCA. The cDNA predicts an open reading frame coding for a 725-amino-acid protein with mitochondrial signal peptide, biotin carboxylase, and biotin-carrier domains. The gene is composed of at least 19 exons and covers more than 70 kb of sequence on band q27 of chromosome 3. MCCA was abundantly expressed in mitochondria-rich organs, such as the heart, skeletal muscles, kidney, and liver. In exon 13, we observed a His/Pro polymorphism at codon 464 (an A to C transition at nucleotide position 1391 in the cDNA sequence). Then, we determined the DNA sequences of the 5' untranslated region and entire coding regions in two patients with MCCase deficiency, but no sequence substitution was detected, suggesting that the gene mutations might be in the non-biotin-containing subunit (B subunit) gene, MCCB, in these patients.     

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and -methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10–24 hr after fusion. This profile is similar to those observed for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinical findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.This work was supported by an NIH research grant (AM 25675) and an A. D. Williams research grant (6-48360). B. Wolf is the recipient of an NIH Research Career Development Award (AM 00677) and is aided by a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (5-263). G. Feldman is the recipient of an NIH predoctoral training grant (GM 07492). This article is No. 100 from the Department of Human Genetics at the Medical College of Virginia.     

Iron deficiency and the structure and physiology of maize chloroplasts

The ultrastructure of mesophyll chloroplasts of maize (Zea mays L.) was more severely affected by iron deficiency that induced mild chlorosis than was the ultrastructure of bundle sheath plastids. Ferredoxin and ribulose diphosphate carboxylase levels were severely decreased by iron deficiency. Malic enzyme was less affected, and phosphoenolpyruvate carboxylase activity remained high even under severe iron deficiency. Iron deficient leaves fixed carbon into malic and aspartic acids but the rate of entrance of carbon into the sugar phosphates and sucrose was greatly reduced compared to the control. Chlorophyll a/b ratios ranged from low values of less than 2 in severely iron deficient leaves to high values exceeding 4 in leaves showing little iron deficiency.     

Genetic complementation analysis of 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency in cultured fibroblasts.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase deficiency is an inherited metabolic disorder of leucine catabolism showing variability in clinical expression. We have examined the possibility of a biochemical and genetic basis for this heterogeneity by measuring the residual enzyme activities in fibroblast cultured from seven patients. The mean activity of HMG-CoA lyase was 1.1% +/- 0.3% of normal with no significant differences between the patients. Genetic complementation was studied in heterokaryons obtained by fusion with polyethylene glycol using the incorporation of 1-[14C]isovaleric acid into trichloroacetic acid precipitable material to determine the activity of the leucine catabolic pathway. Unfused cells from the patients with a deficiency of HMG-CoA lyase had incorporations of less than 5% of normal. Unfused cells from patients with isovaleric acidemia or a deficiency of 3-methylcrotonyl-CoA carboxylase also had incorporations of less than 5% of normal, and when fused with cells of patients with a deficiency of HMG-CoA lyase, gave positive complementation with an incorporation of 30% of normal. None of the fusions between the seven different lines deficient in HMG-CoA lyase resulted in increased incorporation. Thus, no evidence was obtained for biochemical or genetic heterogeneity in fibroblasts of these seven patients with a deficiency of HMG-CoA lyase that would account for their different clinical presentations.     

Biochemical characterization of biotin-responsive multiple carboxylase deficiency: heterogeneity within the bio genetic complementation group

Three biotin-dependent enzymes, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC), and beta-methylcrotonyl CoA carboxylase (beta MCC), were biochemically characterized in fibroblasts from two patients with neonatal multiple carboxylase deficiency. Genetic complementation analyses indicated that both cell lines, designated lines 1 and 2, were deficient in the various carboxylase activities and belonged to the bio complementation group. The activities of the three carboxylases became normal when line 2 cells were incubated in medium supplemented with biotin (1 mg/l) for 24 hrs, whereas 4-6 days were required to achieve maximum activities of PC, PCC, and beta MCC (57%, 46%, and 29% of mean normal enzyme activity, respectively) in line 1 cells incubated in medium containing up to 10 mg/1 biotin. Furthermore, PC activity in line 2 continued to increase under apparent gluconeogenic conditions in culture, but not in line 1. Thermostability studies suggested that biotin stabilizes PC and beta MCC in both cell lines. PC in line 1 cells incubated with or without biotin was less stable than that in normal or line 2 cells, and the less than normal increase of enzyme activities in line 1, especially that of PC, may represent incomplete biotination. These results indicate that there is biochemical heterogeneity within the bio complementation group. Immunotitration with antibodies prepared against purified pig heart PCC demonstrated normal quantities of cross-reacting material in both lines and no differences in the amount of this material after incubation with supplemental biotin, despite the seven- to 20-fold increase in PCC activity. Thus, the increase in carboxylase activity in both bio lines appears to represent activation of rpe-existing apocarboxylase rather than de novo enzyme synthesis. The primary defect in this form of multiple carboxylase deficiency may be in a common holocarboxylase synthetase or in biotin transport. If the defect is in the synthetase, the differences noted between the two bio lines could be explained by a difference in the enzyme's Km for biotin.     

The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis

The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-bisphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4].In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively, in the deficient cell, whereas those of aldolase and citrate synthase decreased by 65 and 27%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities.The thermostability of phosphofructokinase (at 53°C) increased, while that of aldolase (at 48°C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52°C) was also unchanged.     

Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.     

Myocardium proteome remodelling after nutritional deprivation of methyl donors

Methyl donor (MD: folate, vitamin B12 and choline) deficiency causes hyperhomocysteinemia, a risk factor for cardiovascular diseases. However, the mechanisms of the association between MD deficiency, hyperhomocysteinemia, and cardiomyopathy remain unclear. Therefore, we performed a proteomic analysis of myocardium of pups from rat dams fed a MD-depleted diet to understand the impact of MD deficiency on heart at the protein level. Two-dimension gel electrophoresis and mass spectrometry-based analyses allowed us to identify 39 proteins with significantly altered abundance in MD-deficient myocardium. Ingenuity Pathway Analysis showed that 87% of them fitted to a single protein network associated with developmental disorder, cellular compromise and lipid metabolism. Concurrently increased protein carbonylation, the major oxidative post-translational protein modification, could contribute to the decreased abundance of many myocardial proteins after MD deficiency. To decipher the effect of MD deficiency on the abundance of specific proteins identified in vivo, we developed an in vitro model using the cardiomyoblast cell line H9c2. After a 4-day exposure to a MD-deprived (vs. complete) medium, cells were deficient of folate and vitamin B12, and released abnormal amounts of homocysteine. Western blot analyses of pup myocardium and H9c2 cells yielded similar findings for several proteins. Of specific interest is the result showing increased and decreased abundances of prohibitin and α-crystallin B, respectively, which underlines mitochondrial injury and endoplasmic reticulum stress within MD deficiency. The in vitro findings validate the MD-deficient H9c2 cells as a relevant model for studying mechanisms of the early metabolic changes occurring in cardiac cells after MD deprivation.     

Treatment of CoQ10 Deficient Fibroblasts with Ubiquinone,CoQ Analogs,and Vitamin C: Time- and Compound-Dependent Effects

Background

Coenzyme Q₁₀ (CoQ₁₀) and its analogs are used therapeutically by virtue of their functions as electron carriers, antioxidant compounds, or both. However, published studies suggest that different ubiquinone analogs may produce divergent effects on oxidative phosphorylation and oxidative stress.

Methodology/Principal Findings

To test these concepts, we have evaluated the effects of CoQ₁₀, coenzyme Q₂ (CoQ₂), idebenone, and vitamin C on bioenergetics and oxidative stress in human skin fibroblasts with primary CoQ₁₀ deficiency. A final concentration of 5 µM of each compound was chosen to approximate the plasma concentration of CoQ₁₀ of patients treated with oral ubiquinone. CoQ₁₀ supplementation for one week but not for 24 hours doubled ATP levels and ATP/ADP ratio in CoQ₁₀ deficient fibroblasts therein normalizing the bioenergetics status of the cells. Other compounds did not affect cellular bioenergetics. In COQ2 mutant fibroblasts, increased superoxide anion production and oxidative stress-induced cell death were normalized by all supplements.

Conclusions/Significance

These results indicate that: 1) pharmacokinetics of CoQ₁₀ in reaching the mitochondrial respiratory chain is delayed; 2) short-tail ubiquinone analogs cannot replace CoQ₁₀ in the mitochondrial respiratory chain under conditions of CoQ₁₀ deficiency; and 3) oxidative stress and cell death can be counteracted by administration of lipophilic or hydrophilic antioxidants. The results of our in vitro experiments suggest that primary CoQ₁₀ deficiencies should be treated with CoQ₁₀ supplementation but not with short-tail ubiquinone analogs, such as idebenone or CoQ₂. Complementary administration of antioxidants with high bioavailability should be considered if oxidative stress is present.