In vivo metabolic flux profiling with stable isotopes discriminates sites and quantifies effects of mitochondrial dysfunction in C. elegans.
Mitochondrial respiratory chain (RC) disease diagnosis is complicated both by an absence of biomarkers that sufficiently divulge all cases and limited capacity to quantify adverse effects across intermediary metabolism. We applied high performance liquid chromatography (HPLC) and mass spectrometry (MS) studies of stable-isotope based precursor-product relationships in the nematode, C. elegans, to interrogate in vivo differences in metabolic flux among distinct genetic models of primary RC defects and closely related metabolic disorders.


METHODS
C. elegans strains studied harbor single nuclear gene defects in complex I, II, or III RC subunits (gas-1, mev-1, isp-1); enzymes involved in coenzyme Q biosynthesis (clk-1), the tricarboxylic acid cycle (TCA, idh-1), or pyruvate metabolism (pdha-1); and central nodes of the nutrient-sensing signaling network that involve insulin response (daf-2) or the sirtuin homologue (sir-2.1). Synchronous populations of 2000 early larval stage worms were fed standard Escherichia coli on nematode growth media plates containing 1,6-(13)C2-glucose throughout their developmental period, with samples extracted on the first day of adult life in 4% perchloric acid with an internal standard. Quantitation of whole animal free amino acid concentrations and isotopic incorporation into amino and organic acids throughout development was performed in all strains by HPLC and isotope ratio MS, respectively. GC/MS analysis was also performed to quantify absolute isotopic incorporation in all molecular species of key TCA cycle intermediates in gas-1 and N2 adult worms.


RESULTS
Genetic mutations within different metabolic pathways displayed distinct metabolic profiles. RC complex I (gas-1) and III (isp-1) subunit mutants, together with the coenzyme Q biosynthetic mutant (clk-1), shared a similar amino acid profile of elevated alanine and decreased glutamate. The metabolic signature of the complex II mutant (mev-1) was distinct from that of the other RC mutants but resembled that of the TCA cycle mutant (idh-1) and both signaling mutants (daf-2 and sir-2.1). All branched chain amino acid levels were significantly increased in the complex I and III mutants but decreased in the PDH mutant (pdha-1). The RC complex I, coenzyme Q, TCA cycle, and PDH mutants shared significantly increased relative enrichment of lactate+1 and absolute concentration of alanine+1, while glutamate+1 enrichment was significantly decreased uniquely in the RC mutants. Relative intermediary flux analyses were suggestive of proximal TCA cycle disruption in idh-1, completely reduced TCA cycle flux in sir-2.1, and apparent distal TCA cycle alteration in daf-2. GC/MS analysis with universally-labeled (13)C-glucose in adult worms further showed significantly increased isotopic enrichment in lactate, citrate, and malate species in the complex I (gas-1) mutant.


CONCLUSIONS
Stable isotopic/mass spectrometric analysis can sensitively discriminate primary RC dysfunction from genetic deficiencies affecting either the TCA cycle or pyruvate metabolism. These data are further suggestive that metabolic flux analysis using stable isotopes may offer a robust means to discriminate and quantify the secondary effects of primary RC dysfunction across intermediary metabolism.
Division of Human Genetics, The Children's Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.|Division of Metabolic Disease, The Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.|Children's Hospital of The King's Daughters, Division of Medical Genetics and Metabolism, Norfolk, VA, USA.
Samantha Schrier
SS
Vergano
Division of Human Genetics, The Children's Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Meera
M
Rao
Division of Endocrinology, Department of Pediatrics, The Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Shana
S
McCormack
Division of Human Genetics, The Children's Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Julian
J
Ostrovsky
Division of Human Genetics, The Children's Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.|Division of Metabolic Disease, The Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Colleen
C
Clarke
Division of Human Genetics, The Children's Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Judith
J
Preston
Department of Pathology & Laboratory Medicine, The Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Michael J
MJ
Bennett
Division of Metabolic Disease, The Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Marc
M
Yudkoff
Department of Biostatistics and Epidemiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Rui
R
Xiao
Division of Human Genetics, The Children's Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.|Division of Metabolic Disease, The Children's Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
Marni J
MJ
Falk
D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D042967:Electron Transport Complex I / Q000235:genetics* / Q000378:metabolism; D042963:Electron Transport Complex II / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology*; D028361:Mitochondrial Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D009154:Mutation
D029742:Caenorhabditis elegans Proteins; C478189:respiratory complex II; D042963:Electron Transport Complex II; D042967:Electron Transport Complex I
High performance liquid chromatography; Mass spectrometry; Pyruvate dehydrogenase deficiency; Respiratory chain mutants; Signaling pathway mutant; TCA cycle mutant