TITLE: Case - Heavy Water Assays by GC-mass spectrometry
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>Heavy water can be used as a tracer for estimating metabolic rates (</span><span style = "font-style:italic;">in vivo, in vitro</span><span>) such as total energy expenditure (TEE) and fractional synthesis rates (FSR; eg. protein, lipids, triglycerides). When using both ²H</span><span style = "vertical-align:sub;">²</span><span>O and H</span><span style = "vertical-align:sub;vertical-align:super;">²</span><span style = "vertical-align:super;">18</span><span>O (DLW), TEE is estimated from the total production of CO</span><span style = "vertical-align:sub;">² </span><span>as measured by the differences in decay rates of labeled the </span><span style = "vertical-align:super;">18</span><span>O and ²H in body water over time following a single bolus of DLW (1).  </span><span style = "vertical-align:super;"> </span><span>²H</span><span style = "vertical-align:sub;">²</span><span>O can be used to estimate fractional synthesis rates of metabolic reactions such as those associated with proteins, lipids, triglycerides, and cholesterol (2,4).</span></div><div class = "text-block"><span style = "font-weight:bold;">References: </span></div><div class = "text-block">1. Gas chromatography-mass spectrometry assay of the (18) O enrichment of water as trimethyl phosphate. Brunengraber DZ, McCabe BJ, Katanik J, and Previs SF. Anal Biochem 306: 278– 282 (2002). </div><div class = "text-block">2. Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis. Fang D, West RH, Manson ME, Ruddy J, Jiang D, Previs SF, Sonawane ND, Burgess JD, Kelley TJ.Respir Res.; 11:61 (2010). </div><div class = "text-block">3. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem.; 284(10):6101-8 (2009). </div><div class = "text-block">4. Novel application of the "doubly labeled" water method: measuring CO2 production and the tissue-specific dynamics of lipid and protein in vivo. Bederman IR, Dufner DA, Alexander JC, Previs SF. Am J Physiol Endocrinol Metab.; 290(5):E1048-56 (2006).</div></div>

[STEPS]
?. 1. Quantitation by ²H²O  Standard Curve:2. Standards are made from deuterium oxide (Aldrich 617385) 3. Pipette 10μl of plasma or standard into Eppendorf4. ²H²O standards     a. Blank (MQ H²O)     b. 0.1% D²O in (MQ H²O)     c. 0.5% D²O in (MQ H²O)     d. 0.10% D²O in (MQ H²O)     e. 1.5% D²O in (MQ H²O)     f.  2.0% D²O in (MQ H²O)     g. 2.5% D²O in (MQ H²O)     h. 3.0% D²O in (MQ H²O)     i.  3.5% D²O in (MQ H²O)     j.  4.0% D²O in (MQ H²O)5. Add 2μl of a 10N NaOH solution to each sample or standard 6. Add 4μl of acetone/acetonitrile solution (10μl of acetone +200μl of acetonitrile) to each sample 7. Be careful when taking samples out of the centrifuge to make sure that all of the drops are at the bottom of the tube 8. Cap and briefly centrifuge samples (~5sec) to ensure NaOH and acetonitrile react with sample  9. Let samples sit overnight (at least 10hrs) 10. Add 500μl of chloroform to samples 11. Add ~ 50mg Na2SO4 salt to each sample   12. Centrifuge sample for 2 minutes13. Pipette 100μl of chloroform layer into glass insert, place inserts in GC vials and cap, then assay on a GC-MS system, EI mode (see below for parameters and references).
?. For doubly labelled water studies, such as total energy expenditure; TEE (1,4): 1. H²18O standards:      a. Blank (MQ H²O)     b. 0.01% H²18O in (MQ H²O)     c. 0.05% H²18O in (MQ H²O)     d. 0.10% H²18O in (MQ H²O)     e. 0.15% H²18O in (MQ H²O)     f. 0.20% H²18O in (MQ H²O)     g. 0.25% H²18O in (MQ H²O)     h. 0.30% H²18O in (MQ H²O)     i. 0.35% H²18O in (MQ H²O)     j. 0.40% H²18O in (MQ H²O)2. H²18O assay: 5 ul of blood or plasma sample or standard into a 12 x 75-mm glass tube   3. Then add ~3 mg of PCl5 to samples or standards to generate phosphoric acid; let stand for 20 min 4. To generate TMP, then react samples or standards by adding 300 µl of freshly prepared etheral-diazomethane (to derivatize samples) and allow to stand at room temperature during reaction, until the ether evaporated; may use an additional 120µl of diethyl ether in hexane solution 5. TMP is extracted by addition of 150 ul of water and 600 µl of chloroform (1:4 ratio) followed by addition of 0.5 g Na2 SO4 6. Samples are then vigorously mixed, and a small aliquot of the chloroform is transferred to a GC-MS vial and assayed on GC-MS system, EI mode Gas Chormatography Mass Spectrometry, GC-MS (EI mode):  Acetone and the TMP derivatives are analyzed using an Agilent 5973N-MSD equipped with an Agilent 6890 GC system, and a DB-17MS capillary column (30 m x 0.25 mm x 0.25 um). The mass spectrometer is operated in the electron impact mode (EI; 70 eV), (1,4).7. Selective ion monitoring of mass-to-charge ratios (m/z)      a. For ²H enrichments monitor acetone (58, 59) and for 18O enrichments monitor TMP (140,142)     b. The ²H enrichments are calculated from the²H²O standard curve and the 18O enrichments are calculated from the signal ratio (142)/(142 + 140).