A plasma metabolomic signature of Leber hereditary optic neuropathy showing taurine and nicotinamide deficiencies.

Leber's hereditary optic neuropathy (LHON) is the most common disorder due to mitochondrial DNA mutations and complex I deficiency. It is characterized by an acute vision loss, generally in young adults, with a higher penetrance in males. How complex I dysfunction induces the peculiar LHON clinical presentation remains an unanswered question. To gain an insight into this question, we carried out a non-targeted metabolomic investigation using the plasma of 18 LHON patients, during the chronic phase of the disease, comparing them to 18 healthy controls. A total of 500 metabolites were screened of which 156 were accurately detected. A supervised Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) highlighted a robust model for disease prediction with a Q2 (cum) of 55.5%, with a reliable performance during the permutation test (cross-validation analysis of variance, P-value = 5.02284e-05) and a good prediction of a test set (P = 0.05). This model highlighted 10 metabolites with variable importance in the projection (VIP) > 0.8. Univariate analyses revealed nine discriminating metabolites, six of which were the same as those found in the Orthogonal Projections to Latent Structures Discriminant Analysis model. In total, the 13 discriminating metabolites identified underlining dietary metabolites (nicotinamide, taurine, choline, 1-methylhistidine and hippurate), mitochondrial energetic substrates (acetoacetate, glutamate and fumarate) and purine metabolism (inosine). The decreased concentration of taurine and nicotinamide (vitamin B3) suggest interesting therapeutic targets, given their neuroprotective roles that have already been demonstrated for retinal ganglion cells. Our results show a reliable predictive metabolomic signature in the plasma of LHON patients and highlighted taurine and nicotinamide deficiencies.

Metabolomics hallmarks OPA1 variants correlating with their in vitro phenotype and predicting clinical severity.

Interpretation of variants of uncertain significance is an actual major challenge. We addressed this question on a set of OPA1 missense variants responsible for variable severity of neurological impairments. We used targeted metabolomics to explore the different signatures of OPA1 variants expressed in Opa1 deleted mouse embryonic fibroblasts (Opa1-/- MEFs), grown under selective conditions. Multivariate analyses of data discriminated Opa1+/+ from Opa1-/- MEFs metabolic signatures and classified OPA1 variants according to their in vitro severity. Indeed, the mild p.I382M hypomorphic variant was segregating close to the wild-type allele, while the most severe p.R445H variant was close to Opa1-/- MEFs, and the p.D603H and p.G439V alleles, responsible for isolated and syndromic presentations, respectively, were intermediary between the p.I382M and the p.R445H variants. The most discriminant metabolic features were hydroxyproline, the spermine/spermidine ratio, amino acid pool and several phospholipids, emphasizing proteostasis, endoplasmic reticulum (ER) stress and phospholipid remodeling as the main mechanisms ranking OPA1 allele impacts on metabolism. These results demonstrate the high resolving power of metabolomics in hierarchizing OPA1 missense mutations by their in vitro severity, fitting clinical expressivity. This suggests that our methodological approach can be used to discriminate the pathological significance of variants in genes responsible for other rare metabolic diseases and may be instrumental to select possible compounds eligible for supplementation treatment.

Large Extracellular Vesicle-Associated Rap1 Accumulates in Atherosclerotic Plaques, Correlates With Vascular Risks and Is Involved in Atherosclerosis.

RATIONALE: Metabolic syndrome (MetS) is a cluster of interrelated risk factors for cardiovascular diseases and atherosclerosis. Circulating levels of large extracellular vesicles (lEVs), submicrometer-sized vesicles released from plasma membrane, from MetS patients were shown to induce endothelial dysfunction, but their role in early stage of atherosclerosis and on vascular smooth muscle cells (SMC) remain to be fully elucidated. OBJECTIVE: To determine the mechanisms by which lEVs lead to the progression of atherosclerosis in the setting of MetS. METHODS AND RESULTS: Proteomic analysis revealed that the small GTPase, Rap1 was overexpressed in lEVs from MetS patients compared with those from non-MetS subjects. Rap1 was in GTP-associated active state in both types of lEVs, and Rap1-lEVs levels correlated with increased cardiovascular risks, including stenosis. MetS-lEVs, but not non-MetS-lEVs, increased Rap1-dependent endothelial cell permeability. MetS-lEVs significantly promoted migration and proliferation of human aortic SMC and increased expression of proinflammatory molecules and activation of ERK (extracellular signal-regulated kinase) 5/p38 pathways. Neutralization of Rap1 by specific antibody or pharmacological inhibition of Rap1 completely prevented the effects of lEVs from MetS patients. High-fat diet-fed ApoE-/- mice displayed an increased expression of Rap1 both in aortas and circulating lEVs. lEVs accumulated in plaque atherosclerotic lesions depending on the progression of atherosclerosis. lEVs from high-fat diet-fed ApoE-/- mice, but not those from mice fed with a standard diet, enhanced SMC proliferation. Human atherosclerotic lesions were enriched in lEVs expressing Rap1. CONCLUSIONS: These data demonstrate that Rap1 carried by MetS-lEVs participates in the enhanced SMC proliferation, migration, proinflammatory profile, and activation of ERK5/p38 pathways leading to vascular inflammation and remodeling, and atherosclerosis. These results highlight that Rap1 carried by MetS-lEVs may be a novel determinant of diagnostic value for cardiometabolic risk factors and suggest Rap1 as a promising therapeutic target against the development of atherosclerosis. Graphical Abstract: A graphical abstract is available for this article.

Preliminary Metabolomic Profiling of the Vitreous Humor from Hypothermia Fatalities.

The postmortem diagnosis of hypothermia fatalities is often complex due to the absence of pathognomonic lesions and biomarkers. In this study, potential novel biomarkers of hypothermia fatalities were searched in the vitreous humor of known cases of hypothermia fatalities (n = 20) compared to control cases (n = 16), using a targeted metabolomics approach allowing quantitative detection of 188 metabolites. A robust discriminant model with good predictivity was obtained with the supervised OPLS-DA multivariate analysis, showing a distinct separation between the hypothermia and control groups. This signature was characterized by the decreased concentrations of five metabolites (methionine sulfoxide, tryptophan, phenylalanine, alanine, and ornithine) and the increased concentration of 28 metabolites (21 phosphatidylcholines, 3 sphingomyelins, spermine, citrulline, acetylcarnitine, and hydroxybutyrylcarnitine) in hypothermia fatalities compared to controls. The signature shows similarities with already identified features in serum such as the altered concentrations of tryptophan, acylcarnitines, and unsaturated phosphatidylcholines, revealing a highly significant increased activity of methionine sulfoxide reductase, attested by a low methionine sulfoxide-to-methionine ratio. Our results show a preliminary metabolomics signature of hypothermia fatalities in the vitreous humor, highlighting an increased methionine sulfoxide reductase activity.

Tryptophane-kynurenine pathway in the remote ischemic conditioning mechanism.

The actual protective mechanisms underlying cardioprotection with remote ischemic conditioning (RIC) remain unclear. Recent data suggest that RIC induces kynurenine (KYN) and kynurenic acid synthesis, two metabolites derived from tryptophan (TRP), yet a causal relation between TRP pathway and RIC remains to be established. We sought to study the impact of RIC on the levels of TRP and its main metabolites within tissues, and to assess whether blocking kynurenine (KYN) synthesis from TRP would inhibit RIC-induced cardioprotection. In rats exposed to 40-min coronary occlusion and 2-h reperfusion, infarct size was significantly smaller in RIC-treated animals (35.7 ± 3.0% vs. 46.5 ± 2.2%, p = 0.01). This protection was lost in rats that received 1-methyl-tryptophan (1-MT) pretreatment, an inhibitor of KYN synthesis from TRP (infarct size = 46.2 ± 5.0%). Levels of TRP and nine compounds spanning its metabolism through the serotonin and KYN pathways were measured by reversed-phase liquid chromatography-tandem mass spectrometry in the liver, heart, and limb skeletal muscle, either exposed or not to RIC. In the liver, RIC induced a significant increase in xanthurenic acid, nicotinic acid, and TRP. Likewise, RIC increased NAD-dependent deacetylase sirtuin activity in the liver. Pretreatment with 1-MT suppressed the RIC-induced increases in NAD-dependent deacetylase sirtuin activity. Altogether, these findings indicate that RIC mechanism is dependent on TRP-KYN pathway activation.

Metabolomic Profiling of Aqueous Humor in Glaucoma Points to Taurine and Spermine Deficiency: Findings from the Eye-D Study.

We compared the metabolomic profile of aqueous humor from patients with primary open-angle glaucoma (POAG; n = 26) with that of a group of age- and sex-matched non-POAG controls (n = 26), all participants undergoing cataract surgery. Supervised paired partial least-squares discriminant analysis showed good predictive performance for test sets with a median area under the receiver operating characteristic of 0.89 and a p-value of 0.0087. Twenty-three metabolites allowed discrimination between the two groups. Univariate analysis after the Benjamini-Hochberg correction showed significant differences for 13 of these metabolites. The POAG metabolomic signature indicated reduced concentrations of taurine and spermine and increased concentrations of creatinine, carnitine, three short-chain acylcarnitines, 7 amino acids (glutamine, glycine, alanine, leucine, isoleucine, hydroxyl-proline, and acetyl-ornithine), 7 phosphatidylcholines, one lysophosphatidylcholine, and one sphingomyelin. This suggests an alteration of metabolites involved in osmoprotection (taurine and creatinine), neuroprotection (spermine, taurine, and carnitine), amino acid metabolism (7 amino acids and three acylcarnitines), and the remodeling of cell membranes drained by the aqueous humor (hydroxyproline and phospholipids). Five of these metabolic alterations, already reported in POAG plasma, concern spermine, C3 and C4 acylcarnitines, PC aa 34:2, and PC aa 36:4, thus highlighting their importance in the pathogenesis of glaucoma.

Lipidomics Reveals Triacylglycerol Accumulation Due to Impaired Fatty Acid Flux in Opa1-Disrupted Fibroblasts.

OPA1 is a dynamin GTPase implicated in mitochondrial membrane fusion. Despite its involvement in lipid remodeling, the function of OPA1 has never been analyzed by whole-cell lipidomics. We used a nontargeted, reversed-phase lipidomics approach, validated for cell cultures, to investigate OPA1-inactivated mouse embryonic fibroblasts ( Opa1 -/- MEFs). This led to the identification of a wide range of 14 different lipid subclasses comprising 212 accurately detected lipids. Multivariate and univariate statistical analyses were then carried out to assess the differences between the Opa1 -/- and Opa1 +/+ genotypes. Of the 212 lipids identified, 69 were found to discriminate between Opa1 -/- MEFs and Opa1 +/+ MEFs. Among these lipids, 34 were triglycerides, all of which were at higher levels in Opa1 -/- MEFs with fold changes ranging from 3.60 to 17.93. Cell imaging with labeled fatty acids revealed a sharp alteration of the fatty acid flux with a reduced mitochondrial uptake. The other 35 discriminating lipids included phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamine, and sphingomyelins, mainly involved in membrane remodeling, and ceramides, gangliosides, and phosphatidylinositols, mainly involved in apoptotic cell signaling. Our results show that the inactivation of OPA1 severely affects the mitochondrial uptake of fatty acids and lipids through membrane remodeling and apoptotic cell signaling.

A serum metabolomics signature of hypothermia fatalities involving arginase activity, tryptophan content, and phosphatidylcholine saturation.

INTRODUCTION: Hypothermia is a potentially lethal condition whose postmortem diagnosis is often complex to perform due to the absence of pathognomonic lesions and biomarkers. Our first study of human serum and urinary metabolome in hypothermia fatalities sought novel biomarkers with better diagnostic performances than those already existing. MATERIAL AND METHOD: Thirty-two cases of hypothermia deaths and 16 cases excluding known antemortem exposure to cold or postmortem elements suggesting hypothermia were selected. A targeted metabolomic study allowing the detection and quantitation of 188 metabolites was performed on collected serum and urine using direct flow injection (FIA) and liquid chromatography (LC) separation, both coupled to tandem mass spectrometry (MS/MS). Amino acid quantification was also carried on using an in-house LC-MS/MS method in order to replicate the results obtained with the metabolomic study. RESULTS: A discriminant metabolic signature allowing a clear separation between hypothermia and control groups was obtained in the serum. This signature was characterized by increased arginase activity and fatty acid unsaturation along with decreased levels of tryptophan in hypothermia fatalities compared to controls. By contrast, no discriminant metabolic signature separating hypothermia from control fatalities was found in urines. DISCUSSION: The serum metabolic signature of hypothermia fatalities herein observed pointed toward metabolic adaptations that likely aimed at heat production enhancement, endothelial function, and cell membrane fluidity preservation. Novel biomarkers potentially useful in a hypothermia diagnosis were also identified.

Metabolomics and Lipidomics Profiling of a Combined Mitochondrial Plus Endoplasmic Reticulum Fraction of Human Fibroblasts: A Robust Tool for Clinical Studies.

Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles' functions, dysfunctions, and interactions. We developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with excellent reproducibility. Seventy-two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12%, among which many were specific to the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and "omics" combination will be useful for investigating the pathophysiology of complex diseases.

Mitochondrial energetic defects in muscle and brain of a Hmbs-/- mouse model of acute intermittent porphyria.

Acute intermittent porphyria (AIP), an autosomal dominant metabolic disease (MIM #176000), is due to a deficiency of hydroxymethylbilane synthase (HMBS), which catalyzes the third step of the heme biosynthetic pathway. The clinical expression of the disease is mainly neurological, involving the autonomous, central and peripheral nervous systems. We explored mitochondrial oxidative phosphorylation (OXPHOS) in the brain and skeletal muscle of the Hmbs(-/-) mouse model first in the basal state (BS), and then after induction of the disease with phenobarbital and treatment with heme arginate (HA). The modification of the respiratory parameters, determined in mice in the BS, reflected a spontaneous metabolic energetic adaptation to HMBS deficiency. Phenobarbital induced a sharp alteration of the oxidative metabolism with a significant decrease of ATP production in skeletal muscle that was restored by treatment with HA. This OXPHOS defect was due to deficiencies in complexes I and II in the skeletal muscle whereas all four respiratory chain complexes were affected in the brain. To date, the pathogenesis of AIP has been mainly attributed to the neurotoxicity of aminolevulinic acid and heme deficiency. Our results show that mitochondrial energetic failure also plays an important role in the expression of the disease.

A Nontargeted UHPLC-HRMS Metabolomics Pipeline for Metabolite Identification: Application to Cardiac Remote Ischemic Preconditioning.

In recent years, the number of investigations based on nontargeted metabolomics has increased, although often without a thorough assessment of analytical strategies applied to acquire data. Following published guidelines for metabolomics experiments, we report a validated nontargeted metabolomics strategy with pipeline for unequivocal identification of metabolites using the MSMLS molecule library. We achieved an in-house database containing accurate m/z values, retention times, isotopic patterns, full MS, and MS/MS spectra. A UHPLC-HRMS Q-Exactive method was developed, and experimental variations were determined within and between 3 experimental days. The extraction efficiency as well as the accuracy, precision, repeatability, and linearity of the method were assessed, the method demonstrating good performances. The methodology was further blindly applied to plasma from remote ischemic pre-conditioning (RIPC) rats. Samples, previously analyzed by targeted metabolomics using completely different protocol, analytical strategy, and platform, were submitted to our analytical pipeline. A combination of multivariate and univariate statistical analyses was employed. Selection of putative biomarkers from OPLS-DA model and S-plot was combined to jack-knife confidence intervals, metabolites' VIP values, and univariate statistics. Only variables with strong model contribution and highly statistical reliability were selected as discriminated metabolites. Three biomarkers identified by the previous targeted metabolomics study were found in the current work, in addition to three novel metabolites, emphasizing the efficiency of the current methodology and its ability to identify new biomarkers of clinical interest, in a single sequence. The biomarkers were identified to level 1 according to the metabolomics standard initiative and confirmed by both RPLC and HILIC-HRMS.

The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q 2cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as the greater expression of C/EBP homologous protein and the increased XBP1 splicing, in fibroblasts from affected patients, all these changes being reversed by the endoplasmic reticulum stress inhibitor, TUDCA (tauroursodeoxycholic acid). Thus, our metabolomic analysis reveals a pharmacologically-reversible endoplasmic reticulum stress in complex I-related Leber's hereditary optic neuropathy fibroblasts, a finding that may open up new therapeutic perspectives for the treatment of Leber's hereditary optic neuropathy with endoplasmic reticulum-targeting drugs.

Time window-dependent effect of perinatal maternal protein restriction on insulin sensitivity and energy substrate oxidation in adult male offspring.

Epidemiological and experimental evidence suggests that a suboptimal environment during perinatal life programs offspring susceptibility to the development of metabolic syndrome and Type 2 diabetes. We hypothesized that the lasting impact of perinatal protein deprivation on mitochondrial fuel oxidation and insulin sensitivity would depend on the time window of exposure. To improve our understanding of underlying mechanisms, an integrative approach was used, combining the assessment of insulin sensitivity and untargeted mass spectrometry-based metabolomics in the offspring. A hyperinsulinemic-euglycemic clamp was performed in adult male rats born from dams fed a low-protein diet during gestation and/or lactation, and subsequently exposed to a Western diet (WD) for 10 wk. Metabolomics was combined with targeted acylcarnitine profiling and analysis of liver gene expression to identify markers of adaptation to WD that influence the phenotype outcome evaluated by body composition analysis. At adulthood, offspring of protein-restricted dams had impaired insulin secretion when fed a standard diet. Moreover, rats who demonstrated catch-up growth at weaning displayed higher gluconeogenesis and branched-chain amino acid catabolism, and lower fatty acid ß-oxidation compared with control rats. Postweaning exposure of intrauterine growth restriction-born rats to a WD exacerbated incomplete fatty acid ß-oxidation and excess fat deposition. Control offspring nursed by protein-restricted mothers showed peculiar low-fat accretion through adulthood and preserved insulin sensitivity even after WD-exposure. Altogether, our findings suggest a testable hypothesis about how maternal diet might influence metabolic outcomes (insulin sensitivity) in the next generation such as mitochondrial overload and/or substrate oxidation inflexibility dependent on the time window of perinatal dietary manipulation.

Creatine and guanidinoacetate reference values in a French population.

Creatine and guanidinoacetate are biomarkers of creatine metabolism. Their assays in body fluids may be used for detecting patients with primary creatine deficiency disorders (PCDD), a class of inherited diseases. Their laboratory values in blood and urine may vary with age, requiring that reference normal values are given within the age range. Despite the long known role of creatine for muscle physiology, muscle signs are not necessarily the major complaint expressed by PCDD patients. These disorders drastically affect brain function inducing, in patients, intellectual disability, autistic behavior and other neurological signs (delays in speech and language, epilepsy, ataxia, dystonia and choreoathetosis), being a common feature the drop in brain creatine content. For this reason, screening of PCDD patients has been repeatedly carried out in populations with neurological signs. This report is aimed at providing reference laboratory values and related age ranges found for a large scale population of patients with neurological signs (more than 6 thousand patients) previously serving as a background population for screening French patients with PCDD. These reference laboratory values and age ranges compare rather favorably with literature values for healthy populations. Some differences are also observed, and female participants are discriminated from male participants as regards to urine but not blood values including creatine on creatinine ratio and guanidinoacetate on creatinine ratio values. Such gender differences were previously observed in healthy populations; they might be explained by literature differential effects of testosterone and estrogen in adolescents and adults, and by estrogen effects in prepubertal age on SLC6A8 function. Finally, though they were acquired on a population with neurological signs, the present data might reasonably serve as reference laboratory values in any future medical study exploring abnormalities of creatine metabolism and transport.

An immuno-lipidomic signature revealed by metabolomic and machine-learning approaches in labial salivary gland to diagnose primary Sjögren's syndrome.

Introduction: Assessing labial salivary gland exocrinopathy is a cornerstone in primary Sjögren's syndrome. Currently this relies on the histopathologic diagnosis of focal lymphocytic sialadenitis and computing a focus score by counting lym=phocyte foci. However, those lesions represent advanced stages of primary Sjögren's syndrome, although earlier recognition of primary Sjögren's syndrome and its effective treatment could prevent irreversible damage to labial salivary gland. This study aimed at finding early biomarkers of primary Sjögren's syndrome in labial salivary gland combining metabolomics and machine-learning approaches. Methods: We used a standardized targeted metabolomic approach involving high performance liquid chromatography coupled with mass spectrometry among newly diagnosed primary Sjögren's syndrome (n=40) and non- primary Sjögren's syndrome sicca (n=40) participants in a prospective cohort. A metabolic signature predictive of primary Sjögren's syndrome status was explored using linear (logistic regression with elastic-net regularization) and non-linear (random forests) machine learning architectures, after splitting the data set into training, validation, and test sets. Results: Among 126 metabolites accurately measured, we identified a discriminant signature composed of six metabolites with robust performances (ROC-AUC = 0.86) for predicting primary Sjögren's syndrome status. This signature included the well-known immune-metabolite kynurenine and five phospholipids (LysoPC C28:0; PCaa C26:0; PCaaC30:2; PCae C30:1, and PCaeC30:2). It was split into two main components: the first including the phospholipids was related to the intensity of lymphocytic infiltrates in salivary glands, while the second represented by kynurenine was independently associated with the presence of anti-SSA antibodies in participant serum. Conclusion: Our results reveal an immuno-lipidomic signature in labial salivary gland that accurately distinguishes early primary Sjögren's syndrome from other causes of sicca symptoms.

A Metabolomic Signature of Ischemic Stroke Showing Acute Oxidative and Energetic Stress.

Metabolomics is a powerful data-driven tool for in-depth biological phenotyping that could help identify the specific metabolic profile of cryptogenic strokes, for which no precise cause has been identified. We performed a targeted quantitative metabolomics study in West African patients who had recently suffered an ischemic stroke, which was either cryptogenic (n = 40) or had a clearly identified cause (n = 39), compared to a healthy control group (n = 40). Four hundred fifty-six metabolites were accurately measured. Multivariate analyses failed to reveal any metabolic profile discriminating between cryptogenic ischemic strokes and those with an identified cause but did show superimposable metabolic profiles in both groups, which were clearly distinct from those of healthy controls. The blood concentrations of 234 metabolites were significantly affected in stroke patients compared to controls after the Benjamini-Hochberg correction. Increased methionine sulfoxide and homocysteine concentrations, as well as an overall increase in saturation of fatty acids, were indicative of acute oxidative stress. This signature also showed alterations in energetic metabolism, cell membrane integrity, monocarbon metabolism, and neurotransmission, with reduced concentrations of several metabolites known to be neuroprotective. Overall, our results show that cryptogenic strokes are not pathophysiologically distinct from ischemic strokes of established origin, and that stroke leads to intense metabolic remodeling with marked oxidative and energetic stresses.

Barth syndrome in a female patient.

BACKGROUND: Barth syndrome (BTHS) is an X-linked recessive disorder characterized by cardiomyopathy, skeletal myopathy and cyclic neutropenia in male patients. It is caused by mutations in the TAZ gene coding for the tafazzin, a protein involved in the remodeling of cardiolipin. Loss of cardiolipin in the inner mitochondrial membrane results in respiratory chain dysfunction. No specific symptom has been identified in female carriers. CASE REPORT: We report the first case of BTHS confirmed by TAZ gene analysis in a female patient. This girl experienced severe heart failure at 1-month of age. Echocardiography diagnosed dilated-hypokinetic and hypertrophic cardiomyopathy with noncompaction of the left ventricle. Initial metabolic screening was normal, except for a cyclic neutropenia. Respiratory chain analysis performed on skin fibroblasts revealed a decreased activity of complexes I, III and IV. Screening on a bloodspot showed abnormal monolysocardiolipin:cardiolipin ratio, later confirmed on cultured fibroblasts, indicative of BTHS. Genetic analyses finally confirmed the diagnosis of BTHS, by showing a large intragenic deletion of exons 1 through 5 in the TAZ gene. Cytogenetic analysis showed mosaicism for monosomy X and for a ring X chromosome with a large deletion of the long arm including the Xq28 region. The girl presented recurrent episodes of severe acute heart failure, progressive muscle weakness, and had a fatal septic shock at 3 years. CONCLUSION: This case highlights that the diagnosis of BTHS should also be suspected in female patients presenting a phenotype similar to affected boys. In these cases, analysis of the monolysocardiolipin:cardiolipin ratio in bloodspots is a rapid and sensitive screening tool for BTHS. However clinical expression in a carrier female requires hemizygosity for the mutated allele of the TAZ gene, which supposes a rearrangement of the TAZ gene region on the other X chromosome.

Systems biology of antioxidants.

Understanding the role of oxidative injury will allow for therapy with agents that scavenge ROS (reactive oxygen species) and antioxidants in the management of several diseases related to free radical damage. The majority of free radicals are generated by mitochondria as a consequence of the mitochondrial cycle, whereas free radical accumulation is limited by the action of a variety of antioxidant processes that reside in every cell. In the present review, we provide an overview of the mitochondrial generation of ROS and discuss the role of ROS in the regulation of endothelial and adipocyte function. Moreover, we also discuss recent findings on the role of ROS in sepsis, cerebral ataxia and stroke. These results provide avenues for the therapeutic potential of antioxidants in a variety of diseases.

A Metabolomic Profiling of Intra-Uterine Growth Restriction in Placenta and Cord Blood Points to an Impairment of Lipid and Energetic Metabolism.

(1) Background: Intrauterine growth restriction (IUGR) involves metabolic changes that may be responsible for an increased risk of metabolic and cardiovascular diseases in adulthood. Several metabolomic profiles have been reported in maternal blood and urine, amniotic fluid, cord blood and newborn urine, but the placenta has been poorly studied so far. (2) Methods: To decipher the origin of this metabolic reprogramming, we conducted a targeted metabolomics study replicated in two cohorts of placenta and one cohort of cord blood by measuring 188 metabolites by mass spectrometry. (3) Results: OPLS-DA multivariate analyses enabled clear discriminations between IUGR and controls, with good predictive capabilities and low overfitting in the two placental cohorts and in cord blood. A signature of 25 discriminating metabolites shared by both placental cohorts was identified. This signature points to sharp impairment of lipid and mitochondrial metabolism with an increased reliance on the creatine-phosphocreatine system by IUGR placentas. Increased placental insulin resistance and significant alteration of fatty acids oxidation, together with relatively higher phospholipase activity in IUGR placentas, were also highlighted. (4) Conclusions: Our results show a deep lipid and energetic remodeling in IUGR placentas that may have a lasting effect on the fetal metabolism.

A Tear Metabolomic Profile Showing Increased Ornithine Decarboxylase Activity and Spermine Synthesis in Thyroid-Associated Orbitopathy.

About half of patients with Graves' disease develop an orbitopathy related to an inflammatory expansion of the periorbital adipose tissue and muscles. We used a targeted metabolomic approach measuring 188 metabolites by mass spectrometry to compare the metabolic composition of tears in patients with active (n = 21) versus inactive (n = 24) thyroid-associated orbitopathy. Among the 44 metabolites accurately measured, 8 showed a significant alteration of their concentrations between the two groups. Two short-chain acylcarnitines, propionylcarnitine and butyrylcarnitine, and spermine showed increased concentrations in the tears of patients with active orbitopathy, whereas ornithine, glycine, serine, citrulline and histidine showed decreased concentrations in this group. In addition, the ratio putrescine/ornithine, representing the activity of ornithine decarboxylase, was significantly increased in patients with active compared to inactive orbitopathy (p = 0.0011, fold change 3.75). The specificity of this candidate biomarker was maintained when compared to a control group with unclassified dry eye disease. Our results suggest that the stimulation of ornithine decarboxylase by TSH receptor autoantibodies in orbital fibroblasts could lead to increased synthesis of spermine, through the increased activity of ornithine decarboxylase, that may contribute to periorbital expansion in Graves' ophthalmopathy.