In vivo metabolic flux profiling with stable isotopes discriminates sites and quantifies effects of mitochondrial dysfunction in C. elegans.

[pyruvate dehydrogenase deficiency]

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 .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 )C 2 -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 N 2 adult worms .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 .S table 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 .

Diseases
Validation

Diseases presenting "mitochondrial respiratory chain" symptom

inclusion body myositis

pyruvate dehydrogenase deficiency

zellweger syndrome

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