High resolution oxymetry study (HROS) of skeletal muscle usually requires 90–120 min preparative phase (dissection, permeabilization and washing). This work reports on the suitability of a rapid ...muscle preparation which by-passes this long preparation. For a few seconds only, muscle biopsy from pigs is submitted to gentle homogenization at 8000 rotations per minute using an ultra-dispersor apparatus. Subsequent HROS is performed using FCCP instead of ADP, compounds crossing and not plasma membrane, respectively. This simplified procedure compares favorably with classical (permeabilized fibers) HROS in terms of respiratory chain complex activities. Mitochondria from cells undergoing ultradispersion were functionally preserved as attested by relative inefficacy of added cytochrome C (not crossing intact mitochondrial outer membrane) to stimulate mitochondrial respiration. Responsiveness of respiration to ADP (in the absence of FCCP) suggested that these intact mitochondria were outside cells disrupted by ultradispersion or within cells permeated by this procedure.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Abstract The ketogenic diet (KD) and the modified Atkins diet are effective therapies for intractable epilepsy. We compared retrospectively the KD and modified Atkins diet in 27 children and also ...assessed serum long chain fatty acid profiles. After 3 months, using an intent-to-treat analysis, the KD was more successful, with >50% seizure reduction in 11/17 (65%) vs. 2/10 (20%) with the modified Atkins diet, p = 0.03. After 6 months, however, the difference was no longer significant: 7/17 (41%) vs. 2/10 (20%) ( p = 0.24). We observed a preventive effect of both diets on the occurrence of status epilepticus. After 1 and 3 months of either diet, responders experienced a significant decrease in serum arachidonic acid concentration compared to non-responders. The KD and modified Atkins diet led to seizure reduction in this small pilot series, with slightly better results after 3 months with the KD, but not after 6 months. The decrease of serum arachidonic acid levels might be involved in the anticonvulsive effects of KD or modified Atkins diet.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Acylcarnitine ...profile allows the identification and quantification of more than thirty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit acylcarnitine profile. Validation parameters and recommendations are discussed in this specific framework.
Abstract
Context
Pheochromocytomas and paragangliomas (PPGLs) with SDHx pathogenic variants (PVs) are characterized by a higher intratissular succinate/fumarate ratio (RS/F) than non-SDHx–mutated ...ones. Also, an increase in serum succinate levels has been reported in patients with germline SDHB or SDHD PV.
Objective
To assess whether measurement of serum succinate, fumarate levels, and RS/F might aid identification of an SDHx germline PV/likely pathogenic variant (LPV) in patients with PPGL or in asymptomatic relatives; and to guide identification of a PV/LPV among the variants of unknown significance (VUS) identified in SDHx by next-generation sequencing.
Methods
This prospective monocentric study included 93 patients attending an endocrine oncogenetic unit for genetic testing. Succinate and fumarate were measured in serum by gas chromatography coupled to mass spectrometry. The RS/F was calculated to assess SDH enzymatic function. Diagnostic performance was assessed by receiver operating characteristic analysis.
Results
RS/F had a higher discriminant power than succinate alone to identify an SDHx PV/LPV in patients with PPGL. However, SDHD PVs/LPVs are frequently missed. Only RS/F differed between asymptomatic SDHB/SDHD PV/LPV carriers and SDHB/SDHD-linked patients with PPGL. Finally RS/F could be helpful to easily evaluate the functional impact of VUS in SDHx.
Conclusion
Measurement of serum RS/F in patients with PPGL and in asymptomatic relatives is a valuable initial workup tool to detect those carrying a germline PV/LPV in SDHx. Its discriminative power is equal or superior to those of succinate measured alone. SDHD PVs/LPVs are less frequently identified by these biochemical tools. Use of RS/F for SDHx VUS reclassification needs to be evaluated further.
In amyotrophic lateral sclerosis (ALS), motor neuron degeneration occurs simultaneously with systemic metabolic impairment and neuroinflammation. Playing an important role in the regulation of both ...phenomena, interleukin (IL)-6, a major cytokine of the inflammatory response has been proposed as a target for management of ALS. Although a pilot clinical trial provided promising results in humans, another recent preclinical study showed that knocking out the IL-6 gene in mice carrying ALS did not improve clinical outcome. In this study, we aimed to determine the relevance of the IL-6 pathway blockade in a mouse model of ALS by using a pharmacological antagonist of IL-6, a murine surrogate of tocilizumab, namely MR16-1. We analyzed the immunological and metabolic effects of IL-6 blockade by cytokine measurement, blood cell immunophenotyping, targeted metabolomics, and transcriptomics. A deleterious clinical effect of MR16-1 was revealed, with a speeding up of weight loss (
p
= 0.0041) and decreasing body weight (
p
< 0.05). A significant increase in regulatory T-cell count (
p
= 0.0268) and a decrease in C-X-C ligand-1 concentrations in plasma (
p
= 0.0479) were observed. Metabolomic and transcriptomic analyses revealed that MR16-1 mainly affected branched-chain amino acid, lipid, arginine, and proline metabolism. IL-6 blockade negatively affected body weight, despite a moderated anti-inflammatory effect. Metabolic effects of IL-6 were mild compared with metabolic disturbances observed in ALS, but a modification of lipid metabolism by therapy was identified. These results indicate that IL-6 blockade did not improve clinical outcome of a mutant superoxide dismutase 1 mouse model of ALS.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UL, UM, UPCLJ, UPUK, VKSCE, VSZLJ, ZAGLJ, ZRSKP
Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Amino acid ...chromatography allows the identification and quantification of more than forty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit amino acid chromatography. Validation parameters and recommendations are discussed in this specific framework.
The biochemical diagnosis of mitochondrial fatty acid oxidation defects (FAOD) currently rests on enzyme assays. A dynamic
ex vivo exploration consisting of incubations of whole-blood samples with ...stable-labeled palmitate and determining leukocyte capacities to produce deuterated acylcarnitines was developed on healthy controls (
n
=
52) and patients with very-long- (VLCADD) (
n
=
2), medium- (MCADD) (
n
=
6), or short- (SCADD) (
n
=
1) chain acyl-CoA dehydrogenase deficiencies.
Incubations were optimized with
l-carnitine and 16-
2H
3, 15-
2H
2-palmitate at 37 °C for various time periods on MCADD and control whole-blood samples. Labeled acylcarnitines were quantified by electrospray-ionization tandem mass spectrometry after thawing, extraction and derivatization to their butyl esters and the method was applied to patients with defects mentioned above.
The production of acylcarnitines was linear until 6 h of incubation and optimal on 50 to 200 nmol deuterated substrate. A good discrimination between MCADD patient and control data was found, with median C8/C4 acylcarnitine production rate ratios of 81.0 (5th–95th percentile range: 16.6–209.9) and 0.21 (5th–95th percentile range: 0.06–0.79), respectively. The method also discriminated from controls the VLCADD and SCADD patients. Preliminary studies on a healthy control indicated that the storage at 4 °C does little or not alter capacities of whole-blood samples to generate labeled acylcarnitines over a period of 48 h.
The rapid management afforded by the method, its abilities to characterize patients and to work on whole-blood samples after a stay of 24–48 h at 4 °C make it promising for the diagnostic exploration of FAOD.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Lipin-1 deficiency is associated with massive rhabdomyolysis episodes in humans, precipitated by febrile illnesses. Despite well-known roles of lipin-1 in lipid biosynthesis and transcriptional ...regulation, the pathogenic mechanisms leading to rhabdomyolysis remain unknown. Here we show that primary myoblasts from lipin-1-deficient patients exhibit a dramatic decrease in LPIN1 expression and phosphatidic acid phosphatase 1 activity, and a significant accumulation of lipid droplets (LD). The expression levels of LPIN1-target genes peroxisome proliferator-activated receptors delta and alpha (PPARδ, PPARα), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), acyl-coenzyme A dehydrogenase, very long (ACADVL), carnitine palmitoyltransferase IB and 2 (CPT1B and CPT2) were not affected while lipin-2 protein level, a closely related member of the family, was increased. Microarray analysis of patients' myotubes identified 19 down-regulated and 51 up-regulated genes, indicating pleiotropic effects of lipin-1 deficiency. Special attention was paid to the up-regulated ACACB (acetyl-CoA carboxylase beta), a key enzyme in the fatty acid synthesis/oxidation balance. We demonstrated that overexpression of ACACB was associated with free fatty acid accumulation in patients' myoblasts whereas malonyl-carnitine (as a measure of malonyl-CoA) and CPT1 activity were in the normal range in basal conditions accordingly to the normal daily activity reported by the patients. Remarkably ACACB invalidation in patients' myoblasts decreased LD number and size while LPIN1 invalidation in controls induced LD accumulation. Further, pro-inflammatory treatments tumor necrosis factor alpha+Interleukin-1beta(TNF1α+IL-1ß) designed to mimic febrile illness, resulted in increased malonyl-carnitine levels, reduced CPT1 activity and enhanced LD accumulation, a phenomenon reversed by dexamethasone and TNFα or IL-1ß inhibitors. Our data suggest that the pathogenic mechanism of rhabdomyolysis in lipin-1-deficient patients combines the predisposing constitutive impairment of lipid metabolism and its exacerbation by pro-inflammatory cytokines.
Figure: Working hypothesis for an experimental mechanistic link between lipin-1 deficiency and muscle lipid droplet formation along with an attempt for a clinical transposition in terms of predisposition and triggering of rhabdomyolysis episodes. Depicted events are either explicitly or implicitly supported by the present work. They are thought to confer muscle predisposition to lysis. Their experimental exacerbation by inflammation is put forward as a coherent trigger of muscle lysis. Display omitted
•Lipin-1-deficient myoblasts have increased LPIN2 expression and lipid droplet content.•Microarray study shows an up-regulation of ACACB in lipin-1-deficient myotubes.•ACACB up-regulation induced malonyl-carnitine increase and secondary CPT1 inhibition.•ACACB siRNA on lipin-1-deficient myoblasts decreases lipid droplets number and size.•Inflammatory cytokines mimicking triggering factors of rhabdomyolysis enhance lipid anomalies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Despite ACADS (acyl-CoA dehydrogenase, short-chain) gene susceptibility variants (c.511C>T and c.625G>A) are considered to be non-pathogenic, encoded proteins are known to exhibit altered kinetics. ...Whether or not, they might affect overall fatty acid β-oxidation still remains, however, unclear.
De novo biosynthesis of acylcarnitines by whole blood samples incubated with deuterated palmitate (16-2H3,15-2H2-palmitate) is suitable as a fluxomic exploration to distinguish between normal and disrupted β-oxidation, abnormal profiles and ratios of acylcarnitines with different chain-lengths being indicative of the site for enzymatic blockade. Determinations in 301 control subjects of ratios between deuterated butyrylcarnitine and sum of deuterated C2 to C14 acylcarnitines served here as reference values to state specifically functional SCAD impairment in patients addressed for clinical and/or biological suspicion of a β-oxidation disorder.
Functional SCAD impairment was found in 39 patients. The 27 patients accepting subsequent gene studies were all positive for ACADS mutations. Twenty-six of 27 patients were positive for c.625G>A variant. Twenty-three of 27 patients harbored susceptibility variants as sole ACADS alterations (18 homozygous and 3 heterozygous for c.625G>A, 2 compound heterozygous for c.625G>A/c.511C>T).
Our present fluxomic assessment of SCAD suggests a link between ACADS susceptibility variants and abnormal β-oxidation consistent with known altered kinetics of these variants.
•ACADS susceptibility variants (c.511C>T, c.625G>A) are considered to be non-pathogenic.•Fluxomic acylcarnitine profiling documents the site(s) for β-oxidation enzyme blockade.•It also detected symptomatic patients with ACADS susceptibility variants.•In these patients, SCAD contribution to mitochondrial palmitate oxidation was impaired.•This finding is consistent with known altered kinetics of encoded proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
This review is aimed at illustrating that mitochondrial dysfunction and altered lipid homeostasis may concur in a variety of pathogenesis states, being either contributive or consecutive to primary ...disease events. Underlying mechanisms for this concurrence are far from being the exhaustive elements taking place in disease development. They may however complicate, contribute or cause the disease. In the first part of the review, physiological roles of mitochondria in coordinating lipid metabolism and in controlling reactive oxygen species (ROS), ATP and calcium levels are briefly presented. In a second part, clues for how mitochondria-driven alterations in lipid metabolism may induce toxicity are discussed. In the third part, it is illustrated how mitochondrial dysfunction and lipid homeostasis disruption may be associated (i) to complicate type 1 diabetes (pancreatic β-cell mitochondrial dysfunction in ATP yield induces reduced insulin secretion and hence disruption of glucose and lipid metabolism), (ii) to contribute to type 2 diabetes and other insulin resistant states (mitochondrial impairment may induce adipocyte dysfunction with subsequent increase in circulating free fatty acids and their abnormal deposit in non adipose tissues (pancreatic β-cells, skeletal muscle and liver) which results in lipotoxicity and mitochondrial dysfunction), (iii) to offer new clues in our understanding of how the brain controls feeding supply and energy expenditure, (iv) to promote cancer development notably via fatty acid oxidation/synthesis imbalance (in favor of synthesis) further strengthened in some cancers by a lipogenetic benefit induced by a HER2/fatty acid synthase cross-talk, and (v) to favor cardiovascular disorders by impacting heart function and arterial wall integrity.
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