High-Resolution Structural Proteomics of Mitochondria Using the 'Build and Retrieve' Methodology.

The application of integrated systems biology to the field of structural biology is a promising new direction, although it is still in the infant stages of development. Here we report the use of single particle cryo-EM to identify multiple proteins from three enriched heterogeneous fractions prepared from human liver mitochondrial lysate. We simultaneously identify and solve high-resolution structures of nine essential mitochondrial enzymes with key metabolic functions, including fatty acid catabolism, reactive oxidative species clearance, and amino acid metabolism. Our methodology also identified multiple distinct members of the acyl-CoA dehydrogenase family. This work highlights the potential of cryo-EM to explore tissue proteomics at the atomic level.

Dynamin-related protein 1 regulates substrate oxidation in skeletal muscle by stabilizing cellular and mitochondrial calcium dynamics.

Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, which was coupled to loss of ATP-stimulated calcium flux and impaired substrate oxidation stimulated by exogenous calcium. The insights obtained herein suggest that DRP1 regulates substrate oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases.

Time to harmonize mitochondrial syndrome nomenclature and classification: A consensus from the North American Mitochondrial Disease Consortium (NAMDC).

OBJECTIVE: To harmonize terminology in mitochondrial medicine, we propose revised clinical criteria for primary mitochondrial syndromes. METHODS: The North American Mitochondrial Disease Consortium (NAMDC) established a Diagnostic Criteria Committee comprised of members with diverse expertise. It included clinicians, researchers, diagnostic laboratory directors, statisticians, and data managers. The Committee conducted a comprehensive literature review, an evaluation of current clinical practices and diagnostic modalities, surveys, and teleconferences to reach consensus on syndrome definitions for mitochondrial diseases. The criteria were refined after manual application to patients enrolled in the NAMDC Registry. RESULTS: By building upon published diagnostic criteria and integrating recent advances, NAMDC has generated updated consensus criteria for the clinical definition of classical mitochondrial syndromes. CONCLUSIONS: Mitochondrial diseases are clinically, biochemically, and genetically heterogeneous and therefore challenging to classify and diagnose. To harmonize terminology, we propose revised criteria for the clinical definition of mitochondrial disorders. These criteria are expected to standardize the diagnosis and categorization of mitochondrial diseases, which will facilitate future natural history studies and clinical trials.

Plasma Citrate and Succinate Are Associated With Neurocognitive Impairment in Older People With HIV.

BACKGROUND: Neurocognitive impairment (NCI) is associated with monocyte activation in people with HIV (PWH). Activated monocytes increase glycolysis, reduce oxidative phosphorylation, and accumulate citrate and succinate, tricarboxylic acid (TCA) cycle metabolites that promote inflammation-this metabolic shift may contribute to NCI and slowed gait speed in PWH. METHODS: Plasma citrate and succinate were assayed by liquid chromatography-mass spectrometry from 957 participants upon entry to a multicenter, prospective cohort of older PWH. Logistic, linear, and mixed-effects linear regression models were used to examine associations between entry/baseline TCA cycle metabolites and cross-sectional and longitudinal NCI, neuropsychological test scores (NPZ-4), and gait speed. RESULTS: Median age was 51 (range 40-78) years. Each 1 standard deviation (SD) citrate increment was associated with 1.18 higher odds of prevalent NCI at baseline (P = .03), 0.07 SD lower time-updated NPZ-4 score (P = .01), and 0.02 m/s slower time-updated gait speed (P < .0001). Age accentuated these effects. In the oldest age-quartile, higher citrate was associated with 1.64 higher odds of prevalent NCI, 0.17 SD lower NPZ-4, and 0.04 m/s slower gait speed (P ≤ .01 for each). Similar associations were apparent with succinate in the oldest age-quintile, but not with gait speed. In participants without NCI at entry, higher citrate predicted a faster rate of neurocognitive decline. CONCLUSIONS: Higher plasma citrate and succinate are associated with worse cross-sectional and longitudinal measures of neurocognitive function and gait speed that are age-dependent, supporting the importance of altered bioenergetic metabolism in the pathogenesis of NCI in older PWH.

Exercise training and diet-induced weight loss increase markers of hepatic bile acid (BA) synthesis and reduce serum total BA concentrations in obese women.

Regular exercise has profound metabolic influence on the liver, but effects on bile acid (BA) metabolism are less well known. BAs are synthesized exclusively in the liver from cholesterol via the rate-limiting enzyme cholesterol 7 alpha-hydroxylase (CYP7A1). BAs contribute to the solubilization and absorption of lipids and serve as important signaling molecules, capable of systemic endocrine function. Circulating BAs increase with obesity and insulin resistance, but effects following exercise and diet-induced weight loss are unknown. To test if improvements in fitness and weight loss as a result of exercise training enhance BA metabolism, we measured serum concentrations of total BAs (conjugated and unconjugated primary and secondary BAs) in sedentary, obese, insulin-resistant women (N = 11) before (PRE) and after (POST) a ∼14-wk exercise and diet-induced weight loss intervention. BAs were measured in serum collected after an overnight fast and during an oral glucose tolerance test (OGTT). Serum fibroblast growth factor 19 (FGF19; a regulator of BA synthesis) and 7-alpha-hydroxy-cholesten-3-one (C4, a marker of CYP7A1 enzymatic activity) also were measured. Using linear mixed-model analyses and the change in V̇O2peak (mL/min/kg) as a covariate, we observed that exercise and weight loss intervention decreased total fasting serum BA by ∼30% (P = 0.001) and increased fasting serum C4 concentrations by 55% (P = 0.004). C4 was significantly correlated with serum total BAs only in the POST condition, whereas serum FGF19 was unchanged. These data indicate that a fitness and weight loss intervention modifies BA metabolism in obese women and suggest that improved metabolic health associates with higher postabsorptive (fasting) BA synthesis. Furthermore, pre- vs. postintervention patterns of serum C4 following an OGTT support the hypothesis that responsiveness of BA synthesis to postprandial inhibition is improved after exercise and weight loss.NEW & NOTEWORTHY Exercise and weight loss in previously sedentary, insulin-resistant women facilitates a significant improvement in insulin sensitivity and fitness that may be linked to changes in bile acid metabolism. Diet-induced weight loss plus exercise-induced increases in fitness promote greater postabsorptive bile acid synthesis while also sensitizing the bile acid metabolic system to feedback inhibition during a glucose challenge when glucose and insulin are elevated.

Anesthetic Hypersensitivity in a Case-Controlled Series of Patients With Mitochondrial Disease.

BACKGROUND: Children with mitochondrial disease undergo anesthesia for a wide array of surgical procedures. However, multiple medications used for their perioperative care can affect mitochondrial function. Defects in function of the mitochondrial electron transport chain (ETC) can lead to a profound hypersensitivity to sevoflurane in children. We studied the sensitivities to sevoflurane, during mask induction and maintenance of general anesthesia, in children presenting for muscle biopsies for diagnosis of mitochondrial disease. METHODS: In this multicenter study, 91 children, aged 6 months to 16 years, presented to the operating room for diagnostic muscle biopsy for presumptive mitochondrial disease. General anesthesia was induced by a slow increase of inhaled sevoflurane concentration. The primary end point, end-tidal (ET) sevoflurane necessary to achieve a bispectral index (BIS) of 60, was recorded. Secondary end points were maximal sevoflurane used to maintain a BIS between 40 and 60 during the case, and maximum and minimum heart rate and blood pressures. After induction, general anesthesia was maintained according to the preferences of the providers directing the cases. Primary data were analyzed comparing data from patients with complex I deficiencies to other groups using nonparametric statistics in SPSS v.27. RESULTS: The median sevoflurane concentration to reach BIS of 60 during inductions (ET sevoflurane % [BIS = 60]) was significantly lower for patients with complex I defects (0.98%; 95% confidence interval [CI], 0.5-1.4) compared to complex II (1.95%; 95% CI, 1.2-2.7; P < .001), complex III (2.0%; 95% CI, 0.7-3.5; P < .001), complex IV (2.0%; 95% CI, 1.7-3.2; P < .001), and normal groups (2.2%; 95% CI, 1.8-3.0; P < .001). The sevoflurane sensitivities of complex I patients did not reach significance when compared to patients diagnosed with mitochondrial disease but without an identifiable ETC abnormality (P = .172). Correlation of complex I activity with ET sevoflurane % (BIS = 60) gave a Spearman's coefficient of 0.505 (P < .001). The differences in sensitivities between groups were less during the maintenance of the anesthetic than during induction. CONCLUSIONS: The data indicate that patients with complex I dysfunction are hypersensitive to sevoflurane compared to normal patients. Hypersensitivity was less common in patients presenting with other mitochondrial defects or without a mitochondrial diagnosis.

Possible Bioenergetic Biomarker for Chronic Cancer-Related Fatigue.

BACKGROUND: Cancer-related fatigue (CRF) is a highly prevalent, debilitating, and persistent symptom experienced by patients receiving cancer treatments. Up to 71% of men with prostate cancer receiving radiation therapy experience acute and persistent CRF. There is neither an effective therapy nor a diagnostic biomarker for CRF. This pilot study aimed to discover potential biomarkers associated with chronic CRF in men with prostate cancer receiving radiation therapy. METHODS: We used a longitudinal repeated-measures research design. Twenty men with prostate cancer undergoing radiation therapy completed all study visits. CRF was evaluated by a well-established and validated questionnaire, the Patient-Reported Outcomes Measurement Information System for Fatigue (PROMIS-F) Short Form. In addition, peripheral blood mononuclear cells were harvested to quantify ribonucleic acid (RNA) gene expression of mitochondria-related genes. Data were collected before, during, on completion, and 24 months postradiation therapy and analyzed using paired t-tests and repeated-measures analysis of variance. RESULTS: The mean of the PROMIS-F T score was significantly increased over time in patients with prostate cancer, remaining elevated at 24 months postradiation therapy compared to baseline. A significant downregulated BC1 ubiquinol-cytochrome c reductase synthesis-like (BCS1L) was observed over time during radiation therapy and at 24 months postradiation therapy. An increased PROMIS-F score was trended with downregulated BCS1L in patients 24 months after completing radiation therapy. DISCUSSION: This is the first evidence to describe altered messenger RNA for BCS1L in chronic CRF using the PROMIS-F measure with men receiving radiation therapy for prostate cancer. CONCLUSION: Our results suggest that peripheral blood mononuclear cell messenger RNA for BCS1L is a potential biomarker and therapeutic target for radiation therapy-induced chronic CRF in this clinical population.

Identification of unusual phospholipids from bovine heart mitochondria by HPLC-MS/MS.

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.

Mutations in the mitochondrial ribosomal protein MRPS22 lead to primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is characterized by amenorrhea and loss or dysfunction of ovarian follicles prior to the age of 40. POI has been associated with autosomal recessive mutations in genes involving hormonal signaling and folliculogenesis, however, the genetic etiology of POI most often remains unknown. Here we report MRPS22 homozygous missense variants c.404G>A (p.R135Q) and c.605G>A (p.R202H) identified in four females from two independent consanguineous families as a novel genetic cause of POI in adolescents. Both missense mutations identified in MRPS22 are rare, occurred in highly evolutionarily conserved residues, and are predicted to be deleterious to protein function. In contrast to prior reports of mutations in MRPS22 associated with severe mitochondrial disease, the POI phenotype is far less severe. Consistent with this genotype-phenotype correlation, mitochondrial defects in oxidative phosphorylation or rRNA levels were not detected in fibroblasts derived from the POI patients, suggesting a non-bioenergetic or tissue-specific mitochondrial defect. Furthermore, we demonstrate in a Drosophila model that mRpS22 deficiency specifically in somatic cells of the ovary had no effect on fertility, whereas flies with mRpS22 deficiency specifically in germ cells were infertile and agametic, demonstrating a cell autonomous requirement for mRpS22 in germ cell development. These findings collectively identify that MRPS22, a component of the small mitochondrial ribosome subunit, is critical for ovarian development and may therefore provide insight into the pathophysiology and treatment of ovarian dysfunction.

Mitochondrial Dysfunction and Myocardial Ischemia-Reperfusion: Implications for Novel Therapies.

Mitochondria have emerged as key participants in and regulators of myocardial injury during ischemia and reperfusion. This review examines the sites of damage to cardiac mitochondria during ischemia and focuses on the impact of these defects. The concept that mitochondrial damage during ischemia leads to cardiac injury during reperfusion is addressed. The mechanisms that translate ischemic mitochondrial injury into cellular damage, during both ischemia and early reperfusion, are examined. Next, we discuss strategies that modulate and counteract these mechanisms of mitochondrial-driven injury. The new concept that mitochondria are not merely stochastic sites of oxidative and calcium-mediated injury but that they activate cellular responses of mitochondrial remodeling and cellular reactions that modulate the balance between cell death and recovery is reviewed, and the therapeutic implications of this concept are discussed.

Alterations of skeletal muscle bioenergetics in a mouse with F508del mutation leading to a cystic fibrosis-like condition.

High energy expenditure is reported in cystic fibrosis (CF) animal models and patients. Alterations in skeletal muscle oxidative capacity, fuel utilization, and the creatine kinase-phosphocreatine system suggest mitochondrial dysfunction. Studies were performed on congenic C57BL/6J and F508del (Cftrtm1kth) mice. Indirect calorimetry was used to measure gas exchange to evaluate aerobic capacity during treadmill exercise. The bioenergetic function of skeletal muscle subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) was evaluated using an integrated approach combining measurement of the rate of oxidative phosphorylation by polarography and of electron transport chain activities by spectrophotometry. CF mice have reduced maximal aerobic capacity. In SSM of these mice, oxidative phosphorylation was impaired in the presence of complex I, II, III, and IV substrates except when glutamate was used as substrate. This impairment appeared to be caused by a defect in complex V activity, whereas the oxidative system of the electron transport chain was unchanged. In IFM, oxidative phosphorylation and electron transport chain activities were preserved, whereas complex V activity was reduced, in CF. Furthermore, creatine kinase activity was reduced in both SSM and IFM of CF skeletal muscle. The decreased complex V activity in SSM resulted in reduced oxidative phosphorylation, which could explain the reduced skeletal muscle response to exercise in CF mice. The decrease in mitochondrial creatine kinase activity also contributed to this poor exercise response.

Exercise plasma metabolomics and xenometabolomics in obese, sedentary, insulin-resistant women: impact of a fitness and weight loss intervention.

Insulin resistance has wide-ranging effects on metabolism, but there are knowledge gaps regarding the tissue origins of systemic metabolite patterns and how patterns are altered by fitness and metabolic health. To address these questions, plasma metabolite patterns were determined every 5 min during exercise (30 min, ∼45% of V̇o2peak, ∼63 W) and recovery in overnight-fasted sedentary, obese, insulin-resistant women under controlled conditions of diet and physical activity. We hypothesized that improved fitness and insulin sensitivity following a ∼14-wk training and weight loss intervention would lead to fixed workload plasma metabolomics signatures reflective of metabolic health and muscle metabolism. Pattern analysis over the first 15 min of exercise, regardless of pre- versus postintervention status, highlighted anticipated increases in fatty acid tissue uptake and oxidation (e.g., reduced long-chain fatty acids), diminution of nonoxidative fates of glucose [e.g., lowered sorbitol-pathway metabolites and glycerol-3-galactoside (possible glycerolipid synthesis metabolite)], and enhanced tissue amino acid use (e.g., drops in amino acids; modest increase in urea). A novel observation was that exercise significantly increased several xenometabolites ("non-self" molecules, from microbes or foods), including benzoic acid-salicylic acid-salicylaldehyde, hexadecanol-octadecanol-dodecanol, and chlorogenic acid. In addition, many nonannotated metabolites changed with exercise. Although exercise itself strongly impacted the global metabolome, there were surprisingly few intervention-associated differences despite marked improvements in insulin sensitivity, fitness, and adiposity. These results and previously reported plasma acylcarnitine profiles support the principle that most metabolic changes during submaximal aerobic exercise are closely tethered to absolute ATP turnover rate (workload), regardless of fitness or metabolic health status.

Analyzing mitochondrial function in human peripheral blood mononuclear cells.

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for producing most of the adenosine triphosphate required by eukaryotic cells. Lymphocytes make up the majority of the peripheral blood mononuclear cells. Peripheral blood mononuclear cells are readily obtainable, providing an ideal sample to monitor systemic changes and understand molecular signaling mechanisms in disease processes. Mitochondrial energy metabolism of lymphocyte has been used to screen for OXPHOS disorders. While there are increasing studies of lymphocyte OXPHOS, few studies examined activity of electron transport chain of lymphocyte mitochondria. We present an optimal protocol to harvest fresh peripheral blood mononuclear cells from human whole blood, determine integrated mitochondrial function, and analyze electron transport chain complex activity. Analyzing integrated mitochondrial function using OXPHOS provides data to uncover defects in the transport of substrates into the mitochondria, generation of reducing equivalents, the electron transport chain, and coupling to the production of adenosine triphosphate. The optimal conditions to harvest peripheral blood mononuclear cells were using blood anticoagulated with ethylenediaminetetraacetic acid, processed utilizing Lymphoprep™, and washed in phosphate buffered saline, all at room temperature. Using isolated peripheral blood mononuclear cells, integrated mitochondrial function and the activities of electron transport chain were determined.

Mitochondrial Metabolism in Aging Heart.

Altered mitochondrial metabolism is the underlying basis for the increased sensitivity in the aged heart to stress. The aged heart exhibits impaired metabolic flexibility, with a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism. Aging impairs mitochondrial oxidative phosphorylation, with a greater role played by the mitochondria located between the myofibrils, the interfibrillar mitochondria. With aging, there is a decrease in activity of complexes III and IV, which account for the decrease in respiration. Furthermore, aging decreases mitochondrial content among the myofibrils. The end result is that in the interfibrillar area, there is ≈50% decrease in mitochondrial function, affecting all substrates. The defective mitochondria persist in the aged heart, leading to enhanced oxidant production and oxidative injury and the activation of oxidant signaling for cell death. Aging defects in mitochondria represent new therapeutic targets, whether by manipulation of the mitochondrial proteome, modulation of electron transport, activation of biogenesis or mitophagy, or the regulation of mitochondrial fission and fusion. These mechanisms provide new ways to attenuate cardiac disease in elders by preemptive treatment of age-related defects, in contrast to the treatment of disease-induced dysfunction.

Investigating the link of ACAD10 deficiency to type 2 diabetes mellitus.

The Native American Pima population has the highest incidence of insulin resistance (IR) and type 2 diabetes mellitus (T2DM) of any reported population, but the pathophysiologic mechanism is unknown. Genetic studies in Pima Indians have linked acyl-CoA dehydrogenase 10 (ACAD10) gene polymorphisms, among others, to this predisposition. The gene codes for a protein with a C-terminus region that is structurally similar to members of a family of flavoenzymes-the acyl-CoA dehydrogenases (ACADs)-that catalyze α,ß-dehydrogenation reactions, including the first step in mitochondrial FAO (FAO), and intermediary reactions in amino acids catabolism. Dysregulation of FAO and an increase in plasma acylcarnitines are recognized as important in the pathophysiology of IR and T2DM. To investigate the deficiency of ACAD10 as a monogenic risk factor for T2DM in human, an Acad-deficient mouse was generated and characterized. The deficient mice exhibit an abnormal glucose tolerance test and elevated insulin levels. Blood acylcarnitine analysis shows an increase in long-chain species in the older mice. Nonspecific variable pattern of elevated short-terminal branch-chain acylcarnitines in a variety of tissues was also observed. Acad10 mice accumulate excess abdominal adipose tissue, develop an early inflammatory liver process, exhibit fasting rhabdomyolysis, and have abnormal skeletal muscle mitochondria. Our results identify Acad10 as a genetic determinant of T2DM in mice and provide a model to further investigate genetic determinants for insulin resistance in humans.

The Failing Heart Relies on Ketone Bodies as a Fuel.

BACKGROUND: Significant evidence indicates that the failing heart is energy starved. During the development of heart failure, the capacity of the heart to utilize fatty acids, the chief fuel, is diminished. Identification of alternate pathways for myocardial fuel oxidation could unveil novel strategies to treat heart failure. METHODS AND RESULTS: Quantitative mitochondrial proteomics was used to identify energy metabolic derangements that occur during the development of cardiac hypertrophy and heart failure in well-defined mouse models. As expected, the amounts of proteins involved in fatty acid utilization were downregulated in myocardial samples from the failing heart. Conversely, expression of ß-hydroxybutyrate dehydrogenase 1, a key enzyme in the ketone oxidation pathway, was increased in the heart failure samples. Studies of relative oxidation in an isolated heart preparation using ex vivo nuclear magnetic resonance combined with targeted quantitative myocardial metabolomic profiling using mass spectrometry revealed that the hypertrophied and failing heart shifts to oxidizing ketone bodies as a fuel source in the context of reduced capacity to oxidize fatty acids. Distinct myocardial metabolomic signatures of ketone oxidation were identified. CONCLUSIONS: These results indicate that the hypertrophied and failing heart shifts to ketone bodies as a significant fuel source for oxidative ATP production. Specific metabolite biosignatures of in vivo cardiac ketone utilization were identified. Future studies aimed at determining whether this fuel shift is adaptive or maladaptive could unveil new therapeutic strategies for heart failure.

Anesthesia and bariatric surgery gut preparation alter plasma acylcarnitines reflective of mitochondrial fat and branched-chain amino acid oxidation.

The period around bariatric surgery offers a unique opportunity to characterize metabolism responses to dynamic shifts in energy, gut function, and anesthesia. We analyzed plasma acylcarnitines in obese women (n = 17) sampled in the overnight fasted/postabsorptive state approximately 1-2 wk before surgery (condition A), the morning of surgery (prior restriction to a 48-h clear liquid diet coupled in some cases a standard polyethylene glycol gut evacuation: condition B), and following induction of anesthesia (condition C). Comparisons tested if 1) plasma acylcarnitine derivatives reflective of fatty acid oxidation (FAO) and xenometabolism would be significantly increased and decreased, respectively, by preoperative gut preparation/negative energy balance (condition A vs. B), and 2) anesthesia would acutely depress markers of FAO. Acylcarnitines associated with fat mobilization and FAO were significantly increased in condition B: long-chain acylcarnitines (i.e., C18:1, ~70%), metabolites from active but incomplete FAO [i.e., C14:1 (161%) and C14:2 (102%)] and medium- to short-chain acylcarnitines [i.e., C2 (91%), R-3-hydroxybutyryl-(245%), C6 (45%), and cis-3,4-methylene-heptanoyl-(17%), etc.]. Branched-chain amino acid markers displayed disparate patterns [i.e., isobutyryl-(40% decreased) vs. isovaleryl carnitine (51% increased)]. Anesthesia reduced virtually every acylcarnitine. These results are consistent with a fasting-type metabolic phenotype coincident with the presurgical "gut preparation" phase of bariatric surgery, and a major and rapid alteration of both fat and amino acid metabolism with onset of anesthesia. Whether presurgical or anesthesia-associated metabolic shifts in carnitine and fuel metabolism impact patient outcomes or surgical risks remains to be evaluated experimentally.

Parkinson's disease-associated pathogenic VPS35 mutation causes complex I deficits.

Defect in the complex I of the mitochondrial electron-transport chain is a characteristic of Parkinson's disease (PD) which is thought to play a critical role in the disease pathogenesis. Mutations in vacuolar protein sorting 35 (VPS35) cause autosomal dominant PD and we recently demonstrated that pathogenic VPS35 mutations cause mitochondrial damage through enhanced mitochondrial fragmentation. In this study, we aimed to determine whether pathogenic VPS35 mutation impacts the activity of complex I and its underlying mechanism. Indeed, VPS35 D620N mutation led to decreased enzymatic activity and respiratory defects in complex I and II in patient fibroblasts. While no changes in the expression of the complex I and II subunits were noted, the level of assembled complex I and II as well as the supercomplex was significantly reduced in D620N fibroblasts. Importantly, inhibition of mitochondrial fission rescued the contents of assembled complexes as well as the functional defects in complex I and II. Overall, these results suggest that VPS35 D620N mutation-induced excessive mitochondrial fission leads to the defects in the assembled complex I and supercomplex and causes bioenergetics deficits.

Correcting false positive medium-chain acyl-CoA dehydrogenase deficiency results from newborn screening; synthesis, purification, and standardization of branched-chain C8 acylcarnitines for use in their selective and accurate absolute quantitation by UHPLC-MS/MS.

While selectively quantifying acylcarnitines in thousands of patient samples using UHPLC-MS/MS, we have occasionally observed unidentified branched-chain C8 acylcarnitines. Such observations are not possible using tandem MS methods, which generate pseudo-quantitative acylcarnitine "profiles". Since these "profiles" select for mass alone, they cannot distinguish authentic signal from isobaric and isomeric interferences. For example, some of the samples containing branched-chain C8 acylcarnitines were, in fact, expanded newborn screening false positive "profiles" for medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Using our fast, highly selective, and quantitatively accurate UHPLC-MS/MS acylcarnitine determination method, we corrected the false positive tandem MS results and reported the sample results as normal for octanoylcarnitine (the marker for MCADD). From instances such as these, we decided to further investigate the presence of branched-chain C8 acylcarnitines in patient samples. To accomplish this, we synthesized and chromatographically characterized several branched-chain C8 acylcarnitines (in addition to valproylcarnitine): 2-methylheptanoylcarnitine, 6-methylheptanoylcarnitine, 2,2-dimethylhexanoylcarnitine, 3,3-dimethylhexanoylcarnitine, 3,5-dimethylhexanoylcarnitine, 2-ethylhexanoylcarnitine, and 2,4,4-trimethylpentanoylcarnitine. We then compared their behavior with branched-chain C8 acylcarnitines observed in patient samples and demonstrated our ability to chromographically resolve, and thus distinguish, octanoylcarnitine from branched-chain C8 acylcarnitines, correcting false positive MCADD results from expanded newborn screening.

Lethal neonatal case and review of primary short-chain enoyl-CoA hydratase (SCEH) deficiency associated with secondary lymphocyte pyruvate dehydrogenase complex (PDC) deficiency.

Mutations in ECHS1 result in short-chain enoyl-CoA hydratase (SCEH) deficiency which mainly affects the catabolism of various amino acids, particularly valine. We describe a case compound heterozygous for ECHS1 mutations c.836T>C (novel) and c.8C>A identified by whole exome sequencing of proband and parents. SCEH deficiency was confirmed with very low SCEH activity in fibroblasts and nearly absent immunoreactivity of SCEH. The patient had a severe neonatal course with elevated blood and cerebrospinal fluid lactate and pyruvate concentrations, high plasma alanine and slightly low plasma cystine. 2-Methyl-2,3-dihydroxybutyric acid was markedly elevated as were metabolites of the three branched-chain α-ketoacids on urine organic acids analysis. These urine metabolites notably decreased when lactic acidosis decreased in blood. Lymphocyte pyruvate dehydrogenase complex (PDC) activity was deficient, but PDC and α-ketoglutarate dehydrogenase complex activities in cultured fibroblasts were normal. Oxidative phosphorylation analysis on intact digitonin-permeabilized fibroblasts was suggestive of slightly reduced PDC activity relative to control range in mitochondria. We reviewed 16 other cases with mutations in ECHS1 where PDC activity was also assayed in order to determine how common and generalized secondary PDC deficiency is associated with primary SCEH deficiency. For reasons that remain unexplained, we find that about half of cases with primary SCEH deficiency also exhibit secondary PDC deficiency. The patient died on day-of-life 39, prior to establishing his diagnosis, highlighting the importance of early and rapid neonatal diagnosis because of possible adverse effects of certain therapeutic interventions, such as administration of ketogenic diet, in this disorder. There is a need for better understanding of the pathogenic mechanisms and phenotypic variability in this relatively recently discovered disorder.