1Department of Pediatrics, The Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania
Falk, M. J., Rao, M., Ostrovsky, J., Daikhin, E., Nissim, I., Yudkoff, M. Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans. J. Vis. Exp. (48), e2288, doi:10.3791/2288 (2011).
Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.
Protocol A: Stable isotopic profiling of intermediary metabolism during C. elegans development on NGM plates.
*Note: Stable isotope enrichment can be successfully measured in young adult nematode populations following isotope exposure (with either universally-labeled 13C glucose or 1,6-13C2-glucose) beginning either in early development or in early adulthood. This protocol facilitates concurrent monitoring of animal health, development, and growth on standard nematode growth media (NGM) plates, which is not as easily achieved in liquid culture.
Protocol B. Stable isotopic profiling of intermediary metabolism in C. elegans young adults in liquid culture.
*NOTE: Pharmacologic effects on intermediary metabolic flux in adult nematodes can be studied following isotopic exposure beginning on the first day of egg laying either on NGM plates (see Protocol A, above) or in liquid culture. We prefer the latter approach to maximize cost efficiency of stable isotope and pharmacologic agent utilization.
|S. Basal with cholesterol||3020 μL||3020 uL - "Drug A" volume|
|Stable Isotope (1,6-13C2-glucose)||80 μL||80 μL|
|K12 E. coli (OD660 2.5 to 3)||400 μL||400 μL|
|Young Adult Worms (2,000)||500 μL||500 μL|
|Drug A (desired concentration)||-||As desired|
PROTOCOL C-1. Monitoring isotopic utilization in adult worms on plates by tracing labeled carbon in atmospheric and dissolved carbon dioxide.
*NOTE: Whereas PROTOCOLS A and B detail methods to monitor isotope incorporation into free metabolites within worms over 2-3 days of development or 1 day of adult life, respectively, gross confirmation of the success and kinetics of isotopic incorporation over a short time course (i.e., minutes to hours) can be achieved by monitoring label in atmospheric carbon dioxide (CO2) released from worms or contained in dissolved carbon dioxide within worm extract. Live or killed bacteria can be fed to worms when grown on plates (PROTOCOL C-1). Bacteria is not necessary for short-term isotope exposure of worms in liquid culture (PROTOCOL C-2).
Alternative Protocol (C-2): Monitoring isotopic utilization in adult worms in liquid culture by tracing labeled carbon in atmospheric and dissolved carbon dioxide.
Protocol D. Processing neutralized samples from Protocols A and B for amino acid and organic acid analysis by gas chromatography/mass spectrometry.
E. Sample Derivitization and Machine Settings for Mass Spectrometry
Add 50 μL of acetonitrile and 50 μL of n-methyl-n-t-butyldimethylsilyl trifluoroacetamide (MTBSTFA) to each sample vial. Cover, mix and incubate for 30 minutes at 60°C. Inject 1-2 μl per sample into Gas Chromatography/Mass Spectrometer (GC/MS).
F. Analysis of Results
Stable isotope is well-incorporated into young adult worms, as evidenced by labeled carbon measured in atmospheric CO2 and dissolved worm CO 2. In worms fed either living or UV-irradiated killed OP50 E. coli, the ratio of 13CO2 to 12CO2 was greater in the dissolved worm CO2 fraction relative to the released atmospheric CO2 (Figure 1). Feeding worms live or killed OP50 E. coli did not significantly alter the measured 13CO2 to 12CO2 ratio in washed worm extract (see PROTOCOL C1).
Prolonged exposure to stable isotope from the early larval period increased isotopic enrichment in intermediary metabolites. Corrected Atoms Percent Excess of labeled carbon determined in intermediary metabolites of young adult wild-type worms was greater across all glutamate species when worms were fed universally-labeled 13C-glucose and live bacteria throughout development relative to similarly treated animals fed labeled bacteria for 48 hours only after reaching the egg-laying adult stage (Figure 2).
Effect of clearing times on isotopic enrichment. Regardless of exposure timecourse, all animals grown on plates were 'cleared' of isotopic label prior to downstream preparation for GC/MS analyses. Comparison of clearing protocols was performed as shown in Figure 2, including feeding worms for 2 hours on NGM agar plates spread with live fresh bacteria without isotope or feeding on NGM agar plates without bacteria for 2 hours, and feeding on NGM agar plates without bacteria for either 2 or 6 hours. Regardless of isotopic exposure course, no significant difference in isotopic enrichment in intermediary metabolites from young adult whole worm extract was observed when animals were cleared on plates without bacteria for either 2 or 6 hours. By contrast, less isotopic enrichment was observed in intermediary metabolites when animals were subsequently 'cleared' of excess isotope by feeding on unlabeled bacteria for two hours. However, increased enrichment in +3, +4, and +5 species was evident when worms were exposed to isotope from the early larval period. Therefore, the optimal clearing protocol was determined to be clearing worms following their incubation on NGM plates spread with stable isotope and bacteria by subsequent incubation for 2 hours on unspread NGM plates. Of note, worms grown in liquid culture were washed clear of isotopic label and bacteria in three volumes of S. basal (see Protocol B); GC/MS analysis of washings showed no significant isotopic enrichment by the third wash.
Worm grinding yielded maximal enrichment in intermediary metabolites. To optimize percent enrichment in intermediary metabolites following similar treatment conditions, comparison was made of worm samples disrupted by sonication alone or sonication plus grinding. Figure 3 shows that worms disrupted following isotope exposure by sonication plus grinding had greater isotopic enrichment than samples disrupted only by sonication. These data suggest that more sub-organismal fractions were disrupted by grinding than by sonication. Subsequent studies revealed grinding alone without sonication was sufficient to achieve maximal isotopic enrichment (data not shown).
Whole worm isotopic exposure permits analysis of intermediary metabolic pathway flux. Feeding living worms with stable isotopic precursor either during development (PROTOCOL A, Figure 4A) or beginning in adult stage animals (PROTOCOL B, Figure 4B) permits sensitive analysis of isotopic enrichment among metabolites indicative of flux through glycolysis (lactate, alanine), pyruvate metabolism (alanine), and the tricarboxylic acid cycle (citrate, malate, succinate, aspartate, glutamate, and glutamine). Universally-labeled 13C-glucose provides robust labeling of all carbon species, whereas utilization of 1,6-13C2-glucose permits robust labeling only in +1 species of each metabolite. This technique can sensitively discern differences from wild-type worm intermediary metabolic flux among mitochondrial respiratory chain mutant strains, such as the complex III mitochondrial respiratory chain subunit mutant isp-1(qm150). Figure 5 illustrates the magnitude of differences observed in absolute label between isp-1(qm150) and N2 worms each exposed for 24 hours to 1,6-13C2-glucose in liquid culture with K12 E. coli bacteria from the first day of egg-laying young adult stage (Protocol B). Additional stable isotopic studies in a range of mitochondrial mutant worms are underway.
Figure 1. Universally-labeled 13C-glucose stable isotope was well-incorporated into young adult worms. 13CO2:12CO2 ratio (atoms percent excess (APE), corrected) in wild-type (N2) worms following two hours feeding universally-labeled 13C-glucose and either UV-killed or live OP50 bacteria on plates (PROTOCOL C1). 13C incorporation into worm metabolites was robust after two hours of isotope exposure. Blue and red bars indicate relative isotopic enrichment in gas phase ("atmospheric CO2") and liquid phase ("worm CO2"), respectively.
Figure 2. Prolonged isotope exposure from the early larval period and subsequently 'clearing' worms on NGM agar plates without bacteria increased isotopic enrichment in free glutamate of young adult worms. Isotopic enrichment in glutamate species is shown for worms fed on NGM agar plates with universally-labeled-13C-glucose and OP50 bacteria either throughout development from the L1 larval stage through the 959 cell young adult stage ("L1", see PROTOCOL A), or for forty-eight hours beginning when worms reached the first day of egg-laying young adult stage ("YA"). X-axis indicates the total number of labeled carbon atoms in each glutamate species. Y-axis indicates the percent enrichment (corrected atoms per excess (APE)). Green bars indicate worms cleared following isotope exposure by feeding OP50 E. coli without isotope on NGM plates for two hours prior to PCA extraction. Blue and gray bars indicate worms cleared following isotope exposure on NGM plates without bacteria or isotope for two or six hours, respectively, prior to PCA extraction.
Figure 3. Disrupting worms by grinding yields maximal isotopic enrichment in intermediary worm metabolites. Percent of carbon enrichment measured in +1 citrate species in wild-type worms treated for 24 hours in liquid culture with 1,6-13C2-glucose without bacteria (PROTOCOL B). Black and gray bars indicate worms that were disrupted by sonication only or by sonication and grinding, respectively. Isotopic enrichment following exposure to 1,6-13C2-glucose is best assessed in metabolites labeled on one carbon. In contrast, label is enriched in all species of each metabolite when universally-labeled-13C-glucose is utilized.
Figure 4. Representative results illustrate the extent of isotopic enrichment in worm intermediary metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Corrected atoms percent excess (APE) was determined in young adult wild-type (N2 Bristol) worms following 1,6-13C2-glucose exposure either (A) during development from L1 stage on plates (PROTOCOL A), or (B) beginning on the first day of egg-laying as young adults for 24 hours in liquid culture (PROTOCOL B). Error bars indicate standard deviation where reliable isotopic data from three biological replicates was available.
Figure 5. Absolute amino acid quantitation of amino acid metabolite species in wild-type and mitochondrial mutant worm strains. Absolute label in each species of four amino acids was calculated by multiplying HPLC-determined free amino acid concentration (nmol/mg worm protein) by corrected atoms percent excess (APE) for each species. Gray and black bars indicate wild-type (N2 Bristol) and mitochondrial complex III subunit mutant strains (isp-1(qm150)), respectively. Bars indicate average of three biological replicate experiments per strain.
Applying mass spectrometry to measure isotopic abundance in intermediary metabolites affords a wonderfully detailed picture of critical biochemical changes5. Here, we have provided detailed protocols for exploiting this highly sensitive and specific methodology to assess intermediary metabolic flux in the genetically versatile model animal, C. elegans. Indeed, feeding living animals with stable isotope labeled metabolic precursors (such as 13C-glucose) permits novel insight into intermediary metabolic pathway flux when measuring isotopic enrichment in downstream metabolites. We have developed reliable methodology to obtain robust analysis of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. This can be achieved both in animals fed stable isotope from early larval developmental stages or beginning in the post-mitotic adult period. Isotopic enrichment in different metabolite species can be studied in conjunction with whole worm free amino acid profiling by high performance liquid chromatography, as previously described4, to determine absolute isotopic enrichment in different species of free worm alanine, aspartate, glutamate, and glutamine. Indeed, consistent patterns of isotopic enrichment are obtained when measuring amino acid and organic acid analytes in aliquots of 1,000 to 2,000 young adult synchronous worms. Such methodology can sensitively discriminate abnormal intermediary flux in nuclear gene-based mitochondrial mutant strains relative to wild-type worms.
This technique holds value to investigate flux alterations in specific biochemical pathways of relevance to common inborn errors of metabolism. In particular, the utilization of glucose labeled with stable isotope informs carbon flux through central metabolic pathways of relevance to mitochondrial disease. Indeed, our data suggest application of such methodology can sensitively discriminate abnormal intermediary flux in a nuclear gene-based mitochondrial complex III subunit mutant strain (isp-1(qm150)) relative to wild-type worms.
It is important to recognize that since isotopic exposure is completed over a period of one to several days, quantified isotopic enrichment represents a "steady state" enrichment value rather than quantifiable flux over a defined period of time as might be determined in cell-based models exposed to isotope for a period of minutes to hours. Another potential limitation of interrogating pathway flux over the course of nematode development relates to differing lengths of larval development that occur in particular mutant strains. For example, many severe mitochondrial mutations are known to cause arrest at the third (L3) larval stage6. Thus, analysis of isotope incorporation only in animals that survive to adulthood may not be representative of the entire mutant population. However, this methodology could be adapted to assess isotopic incorporation in intermediary metabolites at a specific larval stage (such as L2) rather than waiting for animals to reach the adult stage. Similarly, animals that enter the alternative larval stage known as dauer likely have significantly altered (likely lower) metabolic flux relative to animals who proceed directly through the four larval stages. Future study could also be performed to directly assess isotopic incorporation in dauer stage animals of different genetic backgrounds. Exposing animals to stable isotope for a fixed time period (here, 24 to 48 hours) beginning once animals reached the young adult stage (as detailed in Protocol B) removes the impact of variable developmental periods. While 13C-glucose only interrogates glycolysis, pyruvate metabolism and tricarboxylic acid cycle flux, other isotopic tracers could potentially be assessed to interrogate biochemical pathway flux through other pathways of interest in nematodes. For example, 15N-glycine might be utilized to assess amino acid turnover in nematodes.
Another potential limitation of this approach is the level of sensitivity of metabolite detection. Our experience suggests a minimum of 500 young adult nematodes are required to successfully quantify isotopic incorporation by the HPLC and GC/MS methods employed here. Use of instruments with greater sensitivity, such as ultra performance liquid chromatography (UPLC), may permit further reduction of animal number studied, although we anticipate this is unlikely to permit reliable quantitation of metabolites and isotopic incorporation into metabolite species from single worms. We studied worm populations of 1,000 animals per experiment, which we found to reliably detect low abundance metabolites in each strain. However, some metabolites, such as GABA, can only be reliably quantified in much larger populations of nematodes, on the order of 1x106 animals4.
In summary, stable isotope profiling provides a non-invasive and safe (non-radioactive) approach that has long been utilized to study inborn errors of metabolism. Application of this methodology to C. elegans provides the novel capacity to investigate in vivo, real-time, metabolic alterations that occur at the whole animal level, which may result from individual genetic disorders and/or pharmacologic agent exposures.
No conflicts of interest declared.
This work was funded in part by the National Institutes of Health (K08-DK073545 and NICHD-sponsored Intellectual and Developmental Disabilities Research Center New Investigator Award), The Philadelphia Foundation, and University of Pennsylvania McCabe award (M.J.F.), as well as the Tristan Mullen Fund (M.J.F and M.Y.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National institutes of Health.
|S. basal||1 - page 59||Protocols A and B|
|OP50 E. coli||Caenorhabditis Genetics Center||Protocols A and C|
|K12 E. coli||Caenorhabditis Genetics Center||Protocol B|
|NGM agar||Research Products International Corp.||N81800-1000.0||Protocols A, B, and C|
|13C glucose (universally labeled)||Cambridge Isotope Laboratories||CLM-1396||Protocol A and C|
|13C glucose (1,6 labeled)||Cambridge Isotope Laboratories||CLM-2717||Protocol B|
|60% Perchloric acid (PCA)||Fisher Scientific||MK2766500||Protocols A and B|
|ε-aminocaproic acid (Internal Standard)||Sigma-Aldrich||A-2504||Protocols A and B|
|AG 1-X8, 100-200, Chloride form resin||Bio-Rad||140-1441||Protocols A and B (organic acid extraction)|
|AG 50W-X8, 100-200, Hydrogen form resin||Bio-Rad||142-1441||Protocols A and B (amino acid extraction)|
|3N HCl||Sigma-Aldrich||320331||Protocol D|
|1N HCl||Sigma-Aldrich||320331||Protocol A|
|0.1 N HCl||Sigma-Aldrich||320331||Protocol D|
|4N NH4OH||Sigma-Aldrich||30501||Protocol D|
|4N KOH||Sigma-Aldrich||484016||Protocols A and B|
|Deionized water||Milli Q Biocel||ZMQA60F01||Protocol D|
|SMZ-800 Zoom Stereo-Microscope||Nikon Instruments||NI MNA41000||Protocols A, B, and C|
|pH meter||Fisher Scientific||S68167||Protocols A and B|
|Table top centrifuge - 5804R||Eppendorf||05-400-93||Protocols A and B|
|Incubated platform shaker||New Brunswick Scientific||M1324-0004||Protocol B|
|Mini LabRoller Rotator||Labnet International||H-5500||Protocols A and B|
|Pestles||Kontes Corp||K749520-0090||Protocols A and B|
|Drill||Kontes Corp||K749540-0000||Protocols A and B|
|1 L glass beaker||Fisher Scientific||02-539P|
|1 L Erlenmeyer Flasks||VWR international||89000-368|
|25 mL Erlenmeyer Flasks||VWR international||89000-356||Protocol B|
|7ml round-bottom glass tubes + rubber stopper||VWR international||VT6431||Protocols A, B, and C1,C2|
|10 ml round-bottom glass tubes + rubber stopper||VWR international||VT6430||Protocol C2|
|Blue top tubes||Labco||438B|
|50 ml conical plastic tubes||Falcon BD||14-959-49A||All protocols|
|1.5 ml microfuge tubes||Fisher Scientific||02-681-320||Protocols A and B|
|Syringes (1 mL, 10 mL, 20 mL)||BD Biosciences||301025, 301029, 301031||Protocols C1 and C2|
|25 gauge needles||Fisher Scientific||22-253-131||Protocols C1 and C2|
|Poly-Prep Chromatography Columns||Bio-Rad||731-1550||Protocol D|
|Pasteur pipettes||VWR international||14673-043||Protocol D|
|60mm Petri dishes||VWR international||25373-085||Protocol C|
|Glass chamber (cut bottom of 1 L flask) fitted with grease-sealed 3-way stopcock||Custom Made||Protocol C|
|Optically transparent glass plate||Custom Made||Protocol C|
|Reacti-Vap III Evaporator||Thermo Fisher Scientific, Inc.||18826||Protocol D|
|Cotton||VWR international||14224-516||Protocol D|
|High Vacuum Grease||Dow Corning||1597418||Protocol C1|
|Aluminum Foil||Fisher Scientific||01-213-18||Protocol B|
|Timer||ISC Bioexpress||T-2504-5||Protocol C|
|Gas-Ratio Mass Spectrometer||Thermo Fisher Scientific, Inc.||Protocol D|
|GC-MS||Agilent Technologies||6980N/5973N||Protocol D|
|HPLC||Varian Inc., Agilent||9010||Protocol D|