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Quantifying cholesterol synthesis in vivo using 2H2O: enabling back-to-back studies in the same subject

2011; Elsevier BV; Volume: 52; Issue: 7 Linguagem: Inglês

10.1194/jlr.d014993

1539-7262

Stephen F. Previs, Ablatt Mahsut, Alison Kulick, Keiana Dunn, Genevieve Andrews-Kelly, Christopher Johnson, Gowri Bhat, Kithsiri Herath, Paul L. Miller, Shengping Wang, Karim Azer, Jing Xu, Douglas G. Johns, Brian K. Hubbard, Thomas P. Roddy,

Fatty Acid Research and Health

The advantages of using 2H2O to quantify cholesterol synthesis include i) homogeneous precursor labeling, ii) incorporation of 2H via multiple pathways, and iii) the ability to perform long-term studies in free-living subjects. However, there are two concerns. First, the t1/2 of tracer in body water presents a challenge when there is a need to acutely replicate measurements in the same subject. Second, assumptions are made regarding the number of hydrogens (n) that are incorporated during de novo synthesis. Our primary objective was to determine whether a step-based approach could be used to repeatedly study cholesterol synthesis a subject. We observed comparable changes in the 2H-labeling of plasma water and total plasma cholesterol in African-Green monkeys that received five oral doses of 2H2O, each dose separated by one week. Similar rates of cholesterol synthesis were estimated when comparing data in the group over the different weeks, but better reproducibility was observed when comparing replicate determinations of cholesterol synthesis in the same nonhuman primate during the respective dosing periods. Our secondary objective was to determine whether n depends on nutritional status in vivo; we observed n of ∼25 and ∼27 in mice fed a high-carbohydrate (HC) versus carbohydrate-free (CF) diet, respectively. We conclude that it is possible to acutely repeat studies of cholesterol synthesis using 2H2O and that n is relatively constant. The advantages of using 2H2O to quantify cholesterol synthesis include i) homogeneous precursor labeling, ii) incorporation of 2H via multiple pathways, and iii) the ability to perform long-term studies in free-living subjects. However, there are two concerns. First, the t1/2 of tracer in body water presents a challenge when there is a need to acutely replicate measurements in the same subject. Second, assumptions are made regarding the number of hydrogens (n) that are incorporated during de novo synthesis. Our primary objective was to determine whether a step-based approach could be used to repeatedly study cholesterol synthesis a subject. We observed comparable changes in the 2H-labeling of plasma water and total plasma cholesterol in African-Green monkeys that received five oral doses of 2H2O, each dose separated by one week. Similar rates of cholesterol synthesis were estimated when comparing data in the group over the different weeks, but better reproducibility was observed when comparing replicate determinations of cholesterol synthesis in the same nonhuman primate during the respective dosing periods. Our secondary objective was to determine whether n depends on nutritional status in vivo; we observed n of ∼25 and ∼27 in mice fed a high-carbohydrate (HC) versus carbohydrate-free (CF) diet, respectively. We conclude that it is possible to acutely repeat studies of cholesterol synthesis using 2H2O and that n is relatively constant. There are two general tracer approaches for quantifying lipid synthesis in vivo: either administer a carbon-labeled isotope (e.g., [14C] or [13C]acetate) or give labeled water (1Casazza J.P. Veech R.L. Quantitation of the rate of fatty acid synthesis.Lab. Res. Methods Biol. Med. 1984; 10: 231-240PubMed Google Scholar, 2Andersen J.M. Dietschy J.M. Absolute rates of cholesterol synthesis in extrahepatic tissues measured with 3H-labeled water and 14C-labeled substrates.J. Lipid Res. 1979; 20: 740-752Abstract Full Text PDF PubMed Google Scholar). Concerns generally surround the use of carbon-labeled tracers since one typically estimates the flux by determining a precursor/product labeling ratio. Questions arise regarding the identity of the true precursor labeling and the ability to correctly measure that value. In a clas­sical study, Andersen and Dietschy demonstrated that different 14C-tracers yielded unique rates of hepatic cholesterol synthesis (2Andersen J.M. Dietschy J.M. Absolute rates of cholesterol synthesis in extrahepatic tissues measured with 3H-labeled water and 14C-labeled substrates.J. Lipid Res. 1979; 20: 740-752Abstract Full Text PDF PubMed Google Scholar). Such discrepancies typically arise from heterogeneity of the underlying biochemistry. The administered tracer may not enter all cells or in the same proportion with other cholesterogenic substrates. This situation may result from various factors, including membrane permeability and activation/compartmentalization of substrates within a cell/organ, which can influence the dilution of the label and lead to uncertainty regarding the true precursor labeling.Dietschy et al. demonstrated that labeled water is a preferred tracer for studying cholesterol synthesis (2Andersen J.M. Dietschy J.M. Absolute rates of cholesterol synthesis in extrahepatic tissues measured with 3H-labeled water and 14C-labeled substrates.J. Lipid Res. 1979; 20: 740-752Abstract Full Text PDF PubMed Google Scholar, 3Dietschy J.M. Spady D.K. Measurement of rates of cholesterol synthesis using tritiated water.J. Lipid Res. 1984; 25: 1469-1476Abstract Full Text PDF PubMed Google Scholar, 4Lowenstein J.M. Brunengraber H. Wadke M. Measurement of rates of lipogenesis with deuterated and tritiated water.Methods Enzymol. 1975; 35: 279-287Crossref PubMed Scopus (78) Google Scholar). In addition to the fact that it enters all cells equally, labeled water allows investigators to perform integrative studies. For example, Taylor et al. administered 2H2O and quantified cholesterol synthesis in free-living humans over approximately 40 days, while rodent studies have been run for several months (5Peng S.K. Ho K.J. Mikkelson B. Taylor C.B. Studies on cholesterol metabolism in rats by application of D2O and mass spectrometry.Atherosclerosis. 1973; 18: 197-213Abstract Full Text PDF PubMed Scopus (9) Google Scholar, 6Taylor C.B. Mikkelson B. Anderson J.A. Forman D.T. Human serum cholesterol synthesis measured with the deuterium label.Arch Path. 1966; 81: 213-231Google Scholar). Although the last point represents a potentially important and novel advantage for labeled water, one of the perceived limitations concerns the t1/2 of the tracer in vivo. Because the t1/2 of 2H in body water may approach two weeks in humans, investigators may feel the need to allow approximately two months (or about four half-lives of the precursor labeling) to elapse before repeating a study in the same subjects. In addition, investigators typically assume that there is a constant number of exchangeable sites in a given product molecule. Namely, although 2H2O readily distributes in body water, 2H is incorporated into cholesterol via three distinct sources, including water, NADPH, and acetyl-CoA (7Lakshmanan M.R. Veech R.L. Measurement of rate of rat-liver sterol synthesis invivo using tritiated-water.J. Biol. Chem. 1977; 252: 4667-4673Abstract Full Text PDF PubMed Google Scholar). When using 2H2O to quantify rates of synthesis, one assumes that the relative labeling of each hydrogen source is constant, which may be problematic in cases where building blocks are generated via multiple pathways (e.g., NADPH is made following glucose oxidation in the pentose pathway and/or by malic enzyme, whereas acetyl-CoA can be derived from glucose and/or fatty acid oxidation).We previously demonstrated that one could administer 2H2O using a two-step strategy to study gluconeogenesis in rodents (8Katanik J. Mccabe B.J. Brunengraber D.Z. Chandramouli V. Nishiyama F.J. Anderson V.E. Previs S.F. Measuring gluconeogenesis using a low dose of 2H2O: advantage of isotope fractionation during gas chromatography.Am. J. Physiol. Endocrinol. Metab. 2003; 284: E1043-E1048Crossref PubMed Scopus (33) Google Scholar). For example, we quantified the contribution gluconeogenesis to glucose production by giving a low dose of 2H2O on one day and then repeated the study the following day by giving a higher dose. This approach allowed us to overcome the apparent limitation of the 2H2O method for measuring the source(s) of blood glucose in vivo (8Katanik J. Mccabe B.J. Brunengraber D.Z. Chandramouli V. Nishiyama F.J. Anderson V.E. Previs S.F. Measuring gluconeogenesis using a low dose of 2H2O: advantage of isotope fractionation during gas chromatography.Am. J. Physiol. Endocrinol. Metab. 2003; 284: E1043-E1048Crossref PubMed Scopus (33) Google Scholar).The current study had two objectives. The first objective was to determine whether it was possible to use a step-based protocol to estimate cholesterol synthesis in vivo by using a nonhuman primate model in which cholesterol synthesis was estimated over five intervals. Our second objective was to examine a major assumption regarding the number of hydrogens (n) that are incorporated during cholesterol synthesis in vivo (9Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 10Lee W.N. Bassilian S. Ajie H.O. Schoeller D.A. Edmond J. Bergner E.A. Byerley L.O. In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E699-E708Crossref PubMed Google Scholar, 11Lee W.N. Bassilian S. Guo Z. Schoeller D. Edmond J. Bergner E.A. Byerley L.O. Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E372-E383Crossref PubMed Google Scholar). As noted above, the hydrogen bound to cholesterol is derived from unique sources, two of which may be labeled to a different degree compared with body water. In this study, attention was directed toward determining whether n would remain constant under different nutritional states. These studies required the use of a rodent model, which has a faster metabolic rate and in which we could increase the precursor labeling to quantify the singly- and doubly-labeled mass isotopomers. Uncertainty concerning n is problematic in some settings (e.g., human and nonhuman primates) since one will typically only observe an increase in the abundance of singly-labeled product molecules (i.e., the M1 isotopomer); therefore, investigators are obligated to assume a value for n, unlike some experimental models (e.g., rodents and isolated organs or cells) in which multiple mass isotopomers can be quantified, and therefore, n can be calculated (9Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 10Lee W.N. Bassilian S. Ajie H.O. Schoeller D.A. Edmond J. Bergner E.A. Byerley L.O. In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E699-E708Crossref PubMed Google Scholar, 11Lee W.N. Bassilian S. Guo Z. Schoeller D. Edmond J. Bergner E.A. Byerley L.O. Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E372-E383Crossref PubMed Google Scholar, 12Ajie H.O. Connor M.J. Lee W.N. Bassilian S. Bergner E.A. Byerley L.O. In vivo study of the biosynthesis of long-chain fatty acids using deuterated water.Am. J. Physiol. 1995; 269: E247-E252PubMed Google Scholar).MATERIALS AND METHODSUnless noted, chemicals and reagents, including 99.8% 2H2O, were purchased from Sigma-Aldrich. [3-2H1]Cholesterol was purchased from Cambridge Isotopes (Andover, MA). African-Green monkeys were fed a commercial diet (5050 Purina Laboratory Fiber-Plus Monkey Diet). Rodent diets were purchased from Research Diets, Inc. (New Brunswick, NJ). All animal studies were approved by our Institutional Animal Care and Use Committee.Biological studiesAcutely replicating measurements of cholesterol synthesis in vivo.African-Green monkeys (4.1 ± 0.3 kg, females) were individually housed and trained to drink from 30 ml syringes; ∼2 g of Prang (Bio-Serv, Frenchtown, NJ) was added to the drinking water to encourage their training. Cholesterol synthesis was quantified over five weeks using the following protocol. On day 0 of a given week, a baseline blood sample (∼1 ml) was drawn, animals were then given an oral bolus of 2H2O (1.35 ml 2H2O × kg−1, i.e., ∼5 ml 2H2O) mixed in ∼18 ml of reverse osmosis water to which ∼2 g of Prang was added (to maintain the normal routine of the animal), although the animals were allowed to drink as they desired the tracer was typically ingested within 20 to 40 min. Blood samples (∼1 ml) were collected 24 and 48 h after the ingestion of 2H2O. This paradigm was repeated for five consecutive weeks, i.e., each week a baseline blood sample was drawn prior to the administration of 2H2O (1.35 ml 2H2O × kg−1, to which ∼2 g Prang was added) which was then followed by the collection of blood samples 24 and 48 h posttracer administration. Samples were collected in EDTA tubes and plasma was frozen until analyses. Note that throughout this study all animals were maintained on their normal feeding schedule and allowed access to ad libitum unlabeled water.Determining the number of hydrogens incorporated during the synthesis of cholesterol in vivo.Male C57BL/6J mice (11 weeks old) were randomized to either a high-carbohydrate, low-fat (HC) diet (D12450B, kcal distribution equal to 10% fat, 70% carbohydrate, and 20% protein) or a carbohydrate-free, high-fat (CF) diet (D12369B, 90% fat, 0% carbohydrate, and 10% protein). Mice in each group were fed the respective diets ad libitum for 13 days, all mice were then given an intraperitoneal injection of 99% 2H2O (20 μl × g−1 of body weight). After injection, mice were returned to their cages and maintained on 5% 2H-labeled drinking water. As previously demonstrated, this design will maintain a steady-state 2H-labeling of body water (9Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 12Ajie H.O. Connor M.J. Lee W.N. Bassilian S. Bergner E.A. Byerley L.O. In vivo study of the biosynthesis of long-chain fatty acids using deuterated water.Am. J. Physiol. 1995; 269: E247-E252PubMed Google Scholar, 13Brunengraber D.Z. Mccabe B.J. Kasumov T. Alexander J.C. Chandramouli V. Previs S.F. Influence of diet on the modeling of adipose tissue triglycerides during growth.Am. J. Physiol. Endocrinol. Metab. 2003; 285: E917-E925Crossref PubMed Scopus (78) Google Scholar). Mice were euthanized after four days of 2H2O labeling, and plasma samples were frozen until analyses were performed.AnalyticalWater labeling.The 2H-labeling of plasma water was determined as described by Shah et al. (14Shah V. Herath K. Previs S.F. Hubbard B.K. Roddy T.P. Headspace analyses of acetone: a rapid method for measuring the 2H-labeling of water.Anal. Biochem. 2010; 404: 235-237Crossref PubMed Scopus (47) Google Scholar). Briefly, 2H present in water was exchanged with hydrogen bound to acetone by incubating 10 μl of plasma or known standards in a 2 ml glass screw-top GC vial at room temperature for 4 h with 2 μl 10N NaOH (Fisher Scientific) and 5 μl of acetone. The instrument was programmed to inject 5 μl of headspace gas from the GC vial in a splitless mode. Samples were analyzed using a 0.8 min isothermal run (Agilent 5973 MS coupled to a 6890 GC oven fitted with an Agilent DB-17 MS column, 15 m × 250 µm × 0.15 µm, the oven was set at 170°C, and the helium carrier flow was set at 1.0 ml × min−1), acetone elutes at ∼0.4 min; the mass spectrometer was set to perform selected ion monitoring of m/z 58 and 59 (10 ms dwell time per ion) in the electron impact ionization mode.Cholesterol labeling: gas chromatography-pyrolysis-isotope ratio mass spectrometry.5-α-Cholestane and 25-hydoxy-cholesterol (Steraloids, Newport, RI) were used as internal standards for cholesterol, and 25 μl 5-α-cholestane (2.8 mg × ml−1) and 35 μl (3.3 mg × ml−1) 25-hydoxy-cholesterol were added to the plasma samples prior to processing. Total (free plus bound) cholesterol was extracted as follows: 80 μl of plasma was saponified in 1N KOH at 60°C for 2 h, samples were cooled to room temperature and then extracted into 150 μl chloroform (JT Baker, Cambridge, MA) upon addition of 25 μl 6N HCl. The samples were then centrifuged for 5 min, and then the bottom layer (chloroform) was collected, evaporated to dryness, and reacted with 65 μl acetic anhydride/pyridine (2:1, v:v) at 75°C for 30 min before drying under a stream of N2. The dried residue was reconstituted in 90 μl ethyl acetate and subjected to gas chromatography-pyrolysis-isotope ratio mass spectrometry (GCpIRMS) analysis.The 2H-labeling of cholesterol was determined using a Thermo Electron Isotope Ratio MS Delta V Plus (Bremen, Germany) coupled to Agilent 7890 Trace GC (San Jose, CA). Samples were analyzed using a splitless injection (1.5 μl injection volume at an inlet temperature of 230°C), the column (Agilent DB-5MS, 30 m × 250 um × 0.25 um) was programmed using a temperature gradient according to the following conditions: initial column temperature was set to 90°C with a 1 min hold followed by a ramp at 15°C per minute to 230°C, then ramped at 5°C per minute to 260°C and then ramped at 50°C per minute again to 330°C and held for 6 min. Compounds eluting off of the chromatographic column were directed into the pyrolysis reactor (heated at 1400°C) and converted to hydrogen gas, cholesterol elutes at ∼21 min.Measured isotope ratios are typically expressed as delta (δ) values using the equation:δ(HD/HH)=(Rsample−RRef)/Rref×1000where δ is the fraction of heavy isotope represented in parts per thousand notation, Rref is the ratio of standard hydrogen gas, and Rsample is the ratio of the unknown sample. Since we aimed to determine cholesterol synthesis, it was necessary to compare the precursor/product labeling (see below). A standard curve was used to convert the delta values to enrichment (to compare with the water labeling, expressed as enrichment) by mixing known quantities of unlabeled cholesterol with known quantities of [3-2H1]cholesterol and analyzing in parallel with unknown samples.Cholesterol labeling: gas chromatography-quadrupole-mass spectrometry.Plasma samples for gas chromatography-quadrupole-mass spectrometry (GCqMS) analysis were processed in 1.5 ml Eppendorf tubes. An amount of 50 μl of plasma and 100 μl 1N KOH in 80% ethanol were heated at 65°C for 1 h. Samples were acidified with 25 μl 6N HCl and extracted in 125 μl chloroform followed by vigorous vortexing for 20 s The samples were centrifuged at 3,000 rpm for 5 min, and then 100 μl of chloroform (lower layer) was collected and evaporated to dryness under N2. Samples were derivatized using bis-trimethylsilyl trifluoroacetamide (BSTFA) plus 10% trimethylchlorosilane (TMCS), 50 μl was added to the sample, and then it was incubated at 75°C for 1 h. Excess BSTFA-TMCS reagent was evaporated to dryness under N2. The trimethylsilyl derivative was reconstituted in 50 μl ethyl acetate for analysis by GC-MS.Samples were analyzed by GCqMS using the Agilent 6890 gas chromatograph linked to an Agilent 5973 mass selective detector (Palo Alto, CA) operated at 70 eV, gas chromatography was performed using an Agilent DB-5MS capillary column (30.0 m × 250 um × 0.25 um), and 2 μl of sample was injected in a 20/1 split. The inlet temperature was set at 250°C and the helium gas carrier flow was set at 1 ml × min−1. The oven temperature was started at 150°C, raised at 20°C × min−1 to 310°C and held for 6 min. The mass spectrometer was set for selected ion monitoring of m/z 368, 369, and 370 for the trimethylsilyl cholesterol derivative with 10 ms dwell time per ion (15Kasturi S. Bederman I.R. Christopher B. Previs S.F. Ismail-Beigi F. Exposure to azide markedly decreases the abundance of mRNAs encoding cholesterol synthetic enzymes and inhibits cholesterol synthesis.J. Cell. Biochem. 2007; 100: 1034-1044Crossref PubMed Scopus (2) Google Scholar).Mathematical modeling and calculationsIn our studies, the water (or precursor) labeling was maintained at a pseudo steady-state during the initial 48 h immediately following the administration of each dose of 2H2O, while a fairly linear change in cholesterol (or product) labeling was observed over the same period. Therefore, the contribution of cholesterol synthesis was determined using the equation:%newlymadecholesterol=change in cholesterollabeling0 to 48hours, week x/(water labeling0 to 48hours, week x×n)×100where the subscript refers to the time over which measurements were integrated each week and n is the number of exchangeable hydrogens (assumed to equal 26, see below). The absolute rate of cholesterol synthesis was determined by multiplying the % of newly made cholesterol by the average concentration of total plasma cholesterol measured during the respective 48 h interval.Mass isotopomer analyses for determining the number of exchangeable hydrogens (n) in cholesterol.The distribution of different mass isotopomers in a product molecule depends on the abundance of the isotopic precursor and the number of times the precursor is incorporated into a given product molecule (9Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 10Lee W.N. Bassilian S. Ajie H.O. Schoeller D.A. Edmond J. Bergner E.A. Byerley L.O. In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E699-E708Crossref PubMed Google Scholar, 16Strong J.M. Upton D.K. Anderson L.W. Monks A. Chisena C.A. Cysyk R.L. A novel approach to the analysis of mass spectrally assayed stable isotope-labeling experiments.J. Biol. Chem. 1985; 260: 4276-4281Abstract Full Text PDF PubMed Google Scholar, 17Hellerstein M.K. Neese R.A. Mass isotopomer distribution analysis at eight years: theoretical, analytic, and experimental considerations.Am. J. Physiol. 1999; 276: E1146-E1170Crossref PubMed Google Scholar, 18Brunengraber H. Kelleher J.K. Des R.C. Applications of mass isotopomer analysis to nutrition research.Annu. Rev. Nutr. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar). When 2H2O is used to quantify cholesterol synthesis, this can be represented by a binomial distribution where the variable is the ratio of hydrogen/deuterium. The ratio of doubly-to-singly-labeled isotopomers (i.e., the M2/M1 ratio) in cholesterol can be expressed as:M2/M1ratio=[(n−1)/2]/[waterlabeling/(1−waterlabeling)] where n refers to the number of exchangeable hydrogens in a cholesterol molecule. Therefore, by measuring the M2/M1 labeling ratio of cholesterol and the labeling of body water, it is possible to solve the equation for n (9Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 10Lee W.N. Bassilian S. Ajie H.O. Schoeller D.A. Edmond J. Bergner E.A. Byerley L.O. In vivo measurement of fatty acids and cholesterol synthesis using D2O and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E699-E708Crossref PubMed Google Scholar, 11Lee W.N. Bassilian S. Guo Z. Schoeller D. Edmond J. Bergner E.A. Byerley L.O. Measurement of fractional lipid synthesis using deuterated water (2H2O) and mass isotopomer analysis.Am. J. Physiol. 1994; 266: E372-E383Crossref PubMed Google Scholar, 12Ajie H.O. Connor M.J. Lee W.N. Bassilian S. Bergner E.A. Byerley L.O. In vivo study of the biosynthesis of long-chain fatty acids using deuterated water.Am. J. Physiol. 1995; 269: E247-E252PubMed Google Scholar). These calculations were performed in mice given 2H2O.Statistics.Statistical tests were run for nonhuman primate data using ANOVA with Tukey's posthoc testing and unpaired t-tests for rodent studies. Unless noted, data are expressed as the mean ± SEM.RESULTSFigure 1 (top panel) demonstrates the time-dependent changes in 2H-labeling of the precursor during the course of the study. Based on the dose of tracer (1.35 ml 2H2O × kg−1), we expected an initial excess labeling of ∼0.23% (assuming ∼60% body water). As expected when giving a bolus of tracer, we observed a decrease in the labeling of body water between weeks 1 and 2. Since the subsequent doses of water were the same, each should have resulted in comparable changes in water labeling. Consistent with that logic, we observed comparable increases in the labeling of body water following the respective doses of 2H2O, with similar eliminations observed between the dosing periods. From the decreases in water labeling, we estimated that the t1/2 of water was equal to 6.2 ± 0.4 days, 6.1 ± 0.3 days, 5.1 ± 0.5 days, and 6.0 ± 0.7 days between weeks 1 and 2, weeks 2 and 3, weeks 3 and 4, and weeks 4 and 5, respectively.Fig. 1 (middle panel) demonstrates the time-dependent changes in 2H-labeling of total plasma cholesterol. As expected, there was a sharp increase in the labeling of cholesterol during the 48 h immediately after each dosing with 2H2O, and the labeling tended to plateau between weekly intervals as expected, since the water labeling was decreasing (Fig. 1, top panel). However, note that there appears to be substantial 2H-labeling in the initial sample (i.e., ∼0.5% 2H-labeling compared with theoretical 2H-labeling of ∼0.02%), due to the fact that these animals had been used in a pilot tracer study prior to this experiment. We assumed that this seemingly exaggerated baseline cholesterol labeling did not impact the results of the current study. Rather, this observation emphasized the need to obtain baseline samples to account for differences in the measured versus the expected isotope labeling ratios.Fig. 1 (bottom panel) demonstrates the concentration of total plasma cholesterol over the time course of the study. A reasonable steady-state was achieved as expected as animals were maintained in the same environment.Fig. 2 demonstrates cholesterol kinetics calculated from the 2H-labeling and plasma concentrations. The top panel demonstrates the group averages over a given week, while the bottom panel demonstrates the averages in a given animal over the five weeks. In both cases, the data are expressed as the percentage contribution of de novo synthesis (solid bars) and the absolute amount of newly made cholesterol (shaded bars). Although the data are relatively reproducible over the various periods (top panel), the coefficient of variation is reduced by ∼50% when individual animals are studied (bottom panel); i.e., ∼20-25% in the top panel versus ∼12-15% in the bottom panel. The bottom panel demonstrates that although there is variation across individual animals, results in given subjects are fairly reproducible.Fig. 2Calculated rates of cholesterol synthesis. 2H-labeling data were used to estimate cholesterol synthesis. Data are expressed as the percent of newly made cholesterol (solid bars) and the absolute amount of newly made cholesterol (shaded bars). The top panel demonstrates the weekly estimates of cholesterol synthesis showing the collective data from the group, while the bottom panel demonstrates cholesterol synthesis in a given animal measured over five weeks. In all cases, data are mean ± SEM (n = 5); *P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The mass isotopomer distribution of cholesterol obtained from mice fed a high-carbohydrate or a carbohydrate-free diet is presented in Table 1. The baseline samples obtained from control mice demonstrate that quadrupole-based mass spectrometers are capable of measuring isotopic labeling with a relatively high degree of precision (the coefficient of variation is less than 0.5%) and that the experimentally measured values closely agree with theoretical values. Spectra were corrected for natural background labeling, and the excess M1 and M2 isotopomers were used to calculate n. Note that this calculation requires an estimate of the water labeling (i.e., the precursor or p), which was approximately what we expected based on the experimental design (i.e., ∼2.9%). The estimated value for n was comparable in the different diet groups.TABLE 1Estimated Number of Exchangeable Hydrogens in CholesterolCholesterol Isotopomer Distribution (%)GroupM1M2Water Labeling (% Excess)nTheoretical29.94.4——Control30.2 ± 0.14.4 ± 0.1——CF diet48.4 ± 2.417.0 ± 1.72.9 ± 0.127.0 ± 1.0HC diet39.8 ± 2.010.8 ± 1.42.9 ± 0.225.2 ± 1.0The mass isotopomer profile of cholesterol and the labeling of plasma water were determined using GCqMS. The theoretical isotope distribution profile for cholesterol was determined using the isotope calculator reported at http://www.sisweb.com/mstools/isotope.htm. In all cases, the abundance of the M0 isotopomer of cholesterol is 100% (data not shown), and the abundance of the M1 and M2 isotopomers of cholesterol are expressed as a percentage of M0. The number of exchangeable hydrogens (n) was determined from the M2/M1 labeling ratio of cholesterol. Water labeling was determined as described in Materials and Methods. Data are mean ± SEM (five mice per group). Open table in a new tab DISCUSSIONThe use of isotope tracers in metabolic resea