TITLE: Case - Citric Acid Cycle and Related Intermediates
AUTHORS: Henri Brunengraber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>Analysis of the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) using gas chromatography mass spectrometry (GC-MS) is an analytical approach to identifying unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their absolute concentrations or relative ratios by using known amounts of added reference standards to the sample. GC-MS can also distinguish between heavy labelled molecules (eg. </span><span style = "vertical-align:super;">2</span><span>H- or </span><span style = "vertical-align:super;">13</span><span>C-labelled) and the naturally occurring most abundant molecules. The use of metabolic tracers (eg. </span><span style = "vertical-align:super;">2</span><span>H- or </span><span style = "vertical-align:super;">13</span><span>C-labelled) can offer additional information that can lead to the understanding of interrelationships between the pools of intermediary metabolism, such as with the CAC, and specific metabolic pathways. Applications using tracers can also assess the turnover of specific metabolic pools under various physiological and pathological conditions.  </span></div><div class = "text-block">The following SOP presents a relatively simple method that is sensitive for simultaneously measuring concentrations and heavy labelled molecules of CAC intermediates (relative and absolute) and other related intermediates of energy metabolism using GC-MS technology. Note: the following extraction procedure can also be used parallel analysis for acyl-CoA’s and lipids (for details on isolating and mass spectrometry assay procedures see, refs 4-6).</div><div class = "text-block"><span style = "font-weight:bold;">References:  </span></div><div class = "text-block">1. Yang, L., Kasumov, T., Kombu, R. S., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern. J. Biol. Chem. (2008) 283, 21988-21996 </div><div class = "text-block">2. Yang, L., Kombu, R. S., Kasumov, T., Zhu, S. H., Cendrowski, A. V., David, F., Anderson, V. E., Kelleher, J. K., and Brunengraber, H. Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. I. Interrelation between gluconeogenesis and cataplerosis; formation of methoxamates from aminooxyacetate and ketoacids. J. Biol. Chem. (2008) 283, 21978-21987  </div><div class = "text-block">3. Kombu RS, Brunengraber H, and Puchowicz MA. Analysis of the Citric Acid Cycle Intermediates using Gas Chromatography-Mass Spectrometry In: Methods Molec Biol, “Metabolic Profiling: Methods and Protocols” (ed. Metz,TO), Humana Press, (2011) 708:14757  </div><div class = "text-block">4. Zhang Y, Zhang S, Marin-Valencia I, Puchowicz MA. Decreased Carbon Shunting From Glucose Towards Oxidative Metabolism In Diet-Induced Ketotic Rat Brain. J Neurochem. (2015) 132(3): 301-12 </div><div class = "text-block">5. Harris SR, Zhang GF, Sadhukhan S, Wang H, Shi C, Puchowicz MA, Anderson VE, Salomon RG, Tochtrop GP, Brunengraber H. Metabolomics and Mass Isotopomer Analysis as a Strategy for Pathway Discovery: Pyrrolyl and Cyclopentenyl Derivatives of the Pro-Drug of Abuse, Levulinate. Chem Res Toxicol. (2012) 26(2): 213-20  </div><div class = "text-block">6. Zhang, G. F., Kombu, R. S., Kasumov, T., Han, Y., Sadhukhan, S., Zhang, J., Sayre, L. M., Ray, D., Gibson, K. M., Anderson, V. A., Tochtrop, G. P., and Brunengraber, H. Catabolism of 4-hydroxyacids and 4-hydroxynonenal via 4-hydroxy-4-phosphoacyl-CoAs. J. Biol. Chem. (2009) 284, 33521-33534</div></div>

[STEPS]
?. 5% acetic acid in methanol/water (1:1) tissue extraction procedure 1. Weigh about 0.1-0.5 g of powdered frozen (-80 °C) tissue; eg. use a pre-weighed/tarred 15 mL conical tube chilled over dry ice; record the tissue weight (note: when using smaller sample sizes, may use less amounts of solvents and internal standards) 2. Then spike the powdered frozen tissue with the selected internal standards (~50 nM of [13C6]citrate, ~30 nM and [13C4]succinate (may also use ~30 nM of (RS)-3-hydroxy[2H5]glutarate; see refs 1-4) 3. Homogenize the tissue with 3-5 mL of 5% acetic acid in methanol/water (1:1) extraction buffer (chilled on ice) for 2 min on ice bath (note: for acyl-CoA extraction, see modifications, refs 3-6) 4. Centrifuge the homogenate at 670 x g for 30 min at 4°C.  Decant the supernatant into a glass test tube and save on ice and process immediately (note: may freeze the supernatant at -80°C until assaying for GC-MS (note: for acyl-CoA extraction, see modifications, refs 3-6) 5. TMSC/TBDMS derivatization procedure: pipette 100-200 µL of the supernatant collected in step 3-4 into test tube. Notes: (i) if performing parallel analysis for lipids/cholesterol or acyl-CoA assays use 80% of the supernatant collected in step 3, refs 3-6 , (ii) addition of methoxylamine-HCL is necessary if measuring analytes that contain keto groups; follow the details outlined in references (1-3)6. Dry completely under nitrogen gas 7. Then react by adding 70 µL of either derivatizing TMSC or TBDMS reagents (in pyridine based solvent) and heat on a heating  block at 80°C for 1 hour (notes: may react at room temperature overnight) 8. transfer to GC insert and cap; follow GC parameters and the monitored SIM ions (m/z) for each analyte, as outlined in refs 1-4GC-MS Analysis: TMSC or TBDMS derivatives are analyzed using an Agilent 5973N-MSD equipped with an Agilent 6890 GC system, and a DB-17MS capillary column (30m x 0.25 mm x 0.25 um; may use 60m). The mass spectrometer is operated under electron impact mode (EI) or ammonia chemical ionization mode (CI) and selective ion monitoring (SIM) m/z for each analyte; When stable isotopes are applied, the SIM for M0-M+ m/z are monitored, see refs1-4