In Vitro Modeling of Fatty Acid Synthesis under Conditions Simulating the Zonation of Lipogenic [13C]Acetyl-CoA Enrichment in the Liver
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m403837200
ISSN1083-351X
AutoresIlya Bederman, Takhar Kasumov, Aneta E. Reszko, France David, Henri Brunengraber, Joanne K. Kelleher,
Tópico(s)Lipid metabolism and biosynthesis
ResumoIn the companion report (Bederman, I. R., Reszko, A. E., Kasumov, T., David, F., Wasserman, D. H., Kelleher, J. K., and Brunengraber, H. (2004) J. Biol. Chem. 279, 43207-43216), we demonstrated that, when the hepatic pool of lipogenic acetyl-CoA is labeled from [13C]acetate, the enrichment of this pool decreases across the liver lobule. In addition, estimates of fractional synthesis calculated by isotopomer spectral analysis (ISA), a nonlinear regression method, did not agree with a simpler algebraic two-isotopomer method. To evaluate differences between these methods, we simulated in vitro the synthesis of fatty acids under known gradients of precursor enrichment, and known values of fractional synthesis. First, we synthesized pentadecanoate from [U-13C3]propionyl-CoA and four gradients of [U-13C3]malonyl-CoA enrichment. Second, we pooled the fractions of each gradient. Third, we diluted each pool with pentadecanoate prepared from unlabeled malonyl-CoA to simulate the dilution of the newly synthesized compound by pre-existing fatty acids. This yielded a series of samples of pentadecanoate with known values of (i) lower and upper limits for the precursor enrichment, (ii) the shape of the gradient, and (iii) the fractional synthesis. At each step, the mass isotopomer distributions of the samples were analyzed by ISA and the two-isotopomer method to determine whether each method could correctly (i) detect gradients of precursor enrichment, (ii) estimate the gradient limits, and (iii) estimate the fractional synthesis. The two-isotopomer method did not identify gradients of precursor enrichment and underestimated fractional synthesis by up to 2-fold in the presence of gradients. ISA uses all mass isotopomers, correctly identified imposed gradients of precursor enrichment, and estimated the expected values of fractional synthesis within the constraints of the data. In the companion report (Bederman, I. R., Reszko, A. E., Kasumov, T., David, F., Wasserman, D. H., Kelleher, J. K., and Brunengraber, H. (2004) J. Biol. Chem. 279, 43207-43216), we demonstrated that, when the hepatic pool of lipogenic acetyl-CoA is labeled from [13C]acetate, the enrichment of this pool decreases across the liver lobule. In addition, estimates of fractional synthesis calculated by isotopomer spectral analysis (ISA), a nonlinear regression method, did not agree with a simpler algebraic two-isotopomer method. To evaluate differences between these methods, we simulated in vitro the synthesis of fatty acids under known gradients of precursor enrichment, and known values of fractional synthesis. First, we synthesized pentadecanoate from [U-13C3]propionyl-CoA and four gradients of [U-13C3]malonyl-CoA enrichment. Second, we pooled the fractions of each gradient. Third, we diluted each pool with pentadecanoate prepared from unlabeled malonyl-CoA to simulate the dilution of the newly synthesized compound by pre-existing fatty acids. This yielded a series of samples of pentadecanoate with known values of (i) lower and upper limits for the precursor enrichment, (ii) the shape of the gradient, and (iii) the fractional synthesis. At each step, the mass isotopomer distributions of the samples were analyzed by ISA and the two-isotopomer method to determine whether each method could correctly (i) detect gradients of precursor enrichment, (ii) estimate the gradient limits, and (iii) estimate the fractional synthesis. The two-isotopomer method did not identify gradients of precursor enrichment and underestimated fractional synthesis by up to 2-fold in the presence of gradients. ISA uses all mass isotopomers, correctly identified imposed gradients of precursor enrichment, and estimated the expected values of fractional synthesis within the constraints of the data. In the companion report (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we demonstrated that the mass isotopomer distributions (MID), 1The abbreviations used are: MID, mass isotopomer distribution; GC-MS, gas chromatography-mass spectrometry; ISA, isotopomer spectral analysis. 1The abbreviations used are: MID, mass isotopomer distribution; GC-MS, gas chromatography-mass spectrometry; ISA, isotopomer spectral analysis. of fatty acids and sterols isolated from (i) livers of conscious dogs infused with tracer [1,2-13C2]acetate in the portal vein, and (ii) rat livers perfused with 10 mm [1,2-13C2]acetate, are not compatible with a constant enrichment of lipogenic acetyl-CoA across the liver lobule. We concluded that gradients of precursor enrichment occur even in the presence of flooding [1, 2-13C2]acetate concentrations. This probably results from the inverse zonations (2Jungermann K. Kietzmann T. Annu. Rev. Nutr. 1996; 16: 179-203Crossref PubMed Scopus (410) Google Scholar) of the activities of glycolytic (2Jungermann K. Kietzmann T. Annu. Rev. Nutr. 1996; 16: 179-203Crossref PubMed Scopus (410) Google Scholar, 3Fischer W. Ick M. Katz N.R. Hoppe Seylers. Z. Physiol. Chem. 1982; 363: 375-380Crossref PubMed Scopus (51) Google Scholar, 4Katz N. Teutsch H.F. Jungermann K. Sasse D. FEBS Lett. 1977; 83: 272-276Crossref PubMed Scopus (105) Google Scholar, 5Miethke H. Wittig B. Nath A. Zierz S. Jungermann K. Biol. Chem. Hoppe-Seyler. 1985; 366: 493-501Crossref PubMed Scopus (46) Google Scholar, 6Trus M. Zawalich K. Gaynor D. Matschinsky F. J. Histochem. Cytochem. 1980; 28: 579-581Crossref PubMed Scopus (47) Google Scholar) and lipogenic enzymes (7Katz N. Thiele J. Giffhorn-Katz S. Eur. J. Biochem. 1989; 180: 185-189Crossref PubMed Scopus (12) Google Scholar, 8Katz N.R. Fischer W. Giffhorn S. Eur. J. Biochem. 1983; 135: 103-107Crossref PubMed Scopus (40) Google Scholar, 9Katz N.R. Fischer W. Ick M. Eur. J. Biochem. 1983; 130: 297-301Crossref PubMed Scopus (17) Google Scholar) (perivenous > periportal) versus the activity of cytosolic acetyl-CoA synthetase (periportal > perivenous) (10Knudsen C.T. Immerdal L. Grunnet N. Quistorff B. Eur. J. Biochem. 1992; 204: 359-362Crossref PubMed Scopus (11) Google Scholar). Gradients of precursor enrichment were detected using isotopomer spectral analysis (ISA) (11Kelleher J.K. Masterson T.M. Am. J. Physiol. 1992; 262: E118-E125PubMed Google Scholar). In addition, we found that fractional lipogenesis calculated by the two-isotopomer method (an algebraic method similar to that described by Chinkes et al. (12Chinkes D.L. Aarsland A. Rosenblatt J. Wolfe R.R. Am. J. Physiol. 1996; 271: E373-E383Crossref PubMed Google Scholar)) produces lower estimates of fractional synthesis than those produced by the best fit estimates of ISA. Although the “linear gradient” ISA model (see companion report (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) for model definitions) yielded a better fit than the “Single pool” ISA model, it was not possible to evaluate the effect of gradients on estimates of fractional synthesis. Thus, we could not quantitatively evaluate the performance of the ISA in comparison to the two-isotopomer method, because the true rates of fractional synthesis in the liver dog and rat liver perfusion study were unknown.The goal of the present study was to evaluate the differences in estimates of precursor enrichment and fractional synthesis calculated by the two-isotopomer method and ISA. We used an experimental model where both the gradient in precursor enrichment and the fractional synthesis are known. This was accomplished by in vitro preparations that simulated the zonation of acetyl-CoA enrichment. Lipogenesis from sub-populations of hepatocytes across the liver lobule was simulated, in parallel incubations, by synthesizing a fatty acid using purified fatty acid synthase (13Linn T.C. Arch. Biochem. Biophys. 1981; 209: 613-619Crossref PubMed Scopus (88) Google Scholar, 14Weeks G. Shapiro M. Burns R.O. Wakil S.J. J. Bacteriol. 1969; 97: 827-836Crossref PubMed Google Scholar) and [U-13C3]malonyl-CoA of varying enrichment. We used gradients of malonyl-CoA enrichment, because fatty acid synthesis involves the conversion to malonyl-CoA of all acetyl units added to the primer. We used [U-13C3]propionyl-CoA as a primer to avoid the possibility of contamination of our newly synthesized pentadecanoate with unlabeled pentadecanoate. In the presence of unlabeled malonyl-CoA, the process yields M3 2Mass isotopomers are designated as M, M1, M2... Mi, where i is the number of mass units above that of the unlabeled isotopomer M. The subscripted notations M1,M2,... Mi are the intensities of the mass spectrometric signals of the corresponding isotopomers. 2Mass isotopomers are designated as M, M1, M2... Mi, where i is the number of mass units above that of the unlabeled isotopomer M. The subscripted notations M1,M2,... Mi are the intensities of the mass spectrometric signals of the corresponding isotopomers. [13,14,15-13C3]pentadecanoate. By monitoring the distribution of M3 to M15 isotopomers of pentadecanoate, we simulated in vitro the polymerization of six [13C]acetyl units into a C-12 fatty acid, for multiple values of acetyl enrichment. Our goal was to simulate lipogenesis as it occurs in a real liver (i) under gradients of acetyl-CoA 13C enrichment and (ii) in the presence of unlabeled lipids. To achieve this goal, we monitored the MID of pentadecanoate from (i) sets of incubations with progressively decreasing malonyl-CoA enrichments, (ii) pools of incubations from each set, and (iii) pools of incubations spiked with increasing amounts of “unlabeled” [13,14,15-13C3]pentadecanoate. The data were analyzed by the two-isotopomer method and by ISA (11Kelleher J.K. Masterson T.M. Am. J. Physiol. 1992; 262: E118-E125PubMed Google Scholar, 15Kelleher J.K. Ann. Biomed. Eng. 1995; 23: S72Google Scholar).EXPERIMENTAL PROCEDURESMaterialsChemicals, biochemicals, and enzymes were purchased from Sigma-Aldrich. Pentafluorobenzyl bromide was from Pierce. Bovine serum albumin (fraction V, fatty acid poor) was purchased from Miles Biochemicals, and dialyzed as a 15% solution against Krebs-Ringer bicarbonate buffer for 48 h. [U-13C3]Propionate and [U-13C3]malonic acid (99%) were from Isotec. [ω-2H3]Myristic acid was from Cambridge Isotopes Laboratories. [U-13C3]Malonyl-CoA and [U-13C3]propionyl-CoA were prepared from the corresponding acids and purified as reported previously (16Kasumov T.K. Martini W.Z. Bian F. Pierce B.A. David F. Roe C.R. Brunengraber H. Anal. Biochem. 2002; 305: 90-96Crossref PubMed Scopus (22) Google Scholar, 17Reszko A.E. Kasumov T. Comte B. Pierce B.A. David F. Bederman I.R. Deutsch J. Des R.C. Brunengraber H. Anal. Biochem. 2001; 298: 69-75Crossref PubMed Scopus (42) Google Scholar). Fatty acid synthase was isolated from livers from rats that were first starved for 2 days then re-fed with a high glucose diet for 2 days (13Linn T.C. Arch. Biochem. Biophys. 1981; 209: 613-619Crossref PubMed Scopus (88) Google Scholar). The enzyme was precipitated with ammonium sulfate from the effluent of an Ultragel AcA-34 column, and the suspension was kept frozen in small aliquots at -80 °C. The enzyme was used as an ammonium sulfate suspension (1 unit/ml).In Vitro Synthesis of PentadecanoateTheory—The protocol was conceived to simulate decreasing gradients of 13C enrichment of lipogenic acetyl-CoA across the liver lobule. Fatty acid synthesis involves the addition to a primer molecule (usually acetyl-CoA) of malonyl-CoA molecules formed by carboxylation of acetyl-CoA. Thus, gradients of acetyl-CoA enrichment can be reflected by gradients of malonyl-CoA enrichment. Because we wanted the acetyl units added to the primer to be labeled on both carbons, we created gradients of [U-13C3]malonyl-CoA enrichment. In the process of fatty acid synthesis, carbon 3 of [U-13C3]malonyl-CoA is lost as 13CO2. Four protocols were followed to generate gradients of malonyl-CoA enrichment within four series of incubations. For three series of 15 incubations each, the gradients of M3 enrichment of malonyl-CoA decreased from 65% to 10% with the three profiles shown below in Fig. 1 (continuous lines). Note that the range of values for in vitro gradients from 65% to 10% was not chosen randomly. We observed a similar range of precursor enrichments in our in vivo models (see companion report (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)). In a fourth series of seven incubations, the M3 enrichment of malonyl-CoA decreased linearly from 10% to 1.0%. Control incubations were conducted with unlabeled malonyl-CoA and resulted in the formation of [13,14,15-13C3]pentadecanoate. The latter represents an unlabeled species, because it was prepared from unlabeled malonyl-CoA. In our simulation of liver lipogenesis, [13,14,15-13C3]pentadecanoate also represents the pre-existing, unlabeled fatty acid, which dilutes the MID of the newly synthesized labeled fatty acid. When pentadecanoate synthesis is conducted with 97% enriched [U-13C3]propionyl-CoA and [U-13C3]malonyl-CoA of various enrichments, the MID of pentadecanoate ranged from M3 up to M15 (Fig. 2).Fig. 2Mass isotopomer distributions of pentadecanoate synthesized from 97% enriched [U-13C3]propionyl-CoA and lots of [U-13C3]malonyl-CoA of various M3 enrichments (indicated in the upper right corner of each panel). The M0 to M2 mass isotopomers correspond to traces of unlabeled pentadecanoate present in the fatty acid synthase preparation.View Large Image Figure ViewerDownload (PPT)Incubations—For each set of incubations, we prepared 15 or 7 solutions of malonyl-CoA of decreasing enrichment by mixing high-performance liquid chromatography-standardized stock solutions of unlabeled and M3 malonyl-CoA. To verify the malonyl-CoA enrichments and the shape of the gradients, aliquots of all mixed malonyl-CoA solutions were hydrolyzed in alkali, neutralized, and treated to form the tert-butyldimethylsilyl derivative of malonate, which was assayed by GC-MS (17Reszko A.E. Kasumov T. Comte B. Pierce B.A. David F. Bederman I.R. Deutsch J. Des R.C. Brunengraber H. Anal. Biochem. 2001; 298: 69-75Crossref PubMed Scopus (42) Google Scholar).Each incubation included 35 nmol of [U-13C3]propionyl-CoA, 100 nmol of malonyl-CoA, 600 nmol of NADPH, 0.02 unit of fatty acid synthase, in 2 ml of 0.2 m potassium phosphate buffer, pH 7.0. After 1 h of incubation at 37 °C, each incubation medium in a given series was split evenly between two tubes. To one set of tubes, we added 15 nmol of [ω-2H3]myristate (14:0) internal standard before deproteinization with sulfosalicylic acid, extraction of fatty acids, derivatization with pentafluorobenzyl bromide, and ammonia-negative chemical ionization GC-MS assay (see companion report (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)). The analyses yielded the amount and MID of pentadecanoate synthesized in each “fraction.” The other halves of all incubations of each series were pooled to simulate the extraction of a real liver and the mixing of fatty acids synthesized in all cell sub-populations. Then, the pool was redistributed into a new set of 11 tubes (1 ml/tube) to which we added increasing amounts of unlabeled [13,14,15-13C3]pentadecanoate (0-75-nmol by 7.5-nmol increments) to simulate the dilution of newly synthesized labeled fatty acids by endogenous unlabeled fatty acids. Also, 15 nmol of [ω-2H3]myristate internal standard was added to each tube. Then the samples were treated for GC-MS analysis as above. GC-MS analyses of pentadecanoate, derivatized with pentafluorobenzyl bromide (m/z 244-256), were conducted as described in the companion report (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), except that the amounts of pentadecanoate synthesized in the incubations were calculated using a standard of [ω-2H3]myristate (m/z 230). Derivatization of fatty acids with pentafluorobenzyl bromide (18Ostlund Jr., R.E. Hsu F.F. Bosner M.S. Stenson W.F. Hachey D.L. J. Mass Spectrom. 1996; 31: 1291-1296Crossref PubMed Scopus (29) Google Scholar) was selected, (i) because of the sensitivity of the negative chemical ionization assay and (ii) because the pentafluorobenzyl group splits off the fatty acyl group in the ion source and, thus, does not contribute to the MID of pentadecanoate. All analyses were run with double injection.Calculations—Calculations of the parameters of fatty acid synthesis (precursor enrichment and fractional synthesis) were conducted using the two-isotopomer method and two variants of isotopomer spectral analysis (“Single pool” and “Gradient”) that assume that the precursor enrichment is either constant, or follows a gradient of the shape defined by the model's equations (11Kelleher J.K. Masterson T.M. Am. J. Physiol. 1992; 262: E118-E125PubMed Google Scholar, 15Kelleher J.K. Ann. Biomed. Eng. 1995; 23: S72Google Scholar).The following four gradients were set up for in vitro simulation: Linear, Convex, Concave, and Low linear. For the large linear gradient, each synthesized sample was prepared with the precursor fractional abundance D(c) given by the following relationship, Linear D(c)=(Dmin−Dmax)(c/14)+Dmax (Eq. 1) where c is an integer ranging from 0 to 14, and Dmax and Dmin are the upper and lower limits for the variable D(c). By solving for linear D(c) at each value of c, the mix of labeled D(c) and natural malonyl-CoA [1 - D(c)] is specified for each of the 15 samples comprising the gradient. The equation is constructed so that as c increases from 0 to 14, the value of D(c) decreases. Dmax and Dmin were set at 0.65 and 0.1, respectively. The low linear gradient was constructed similarly with c ranging from 0 to 5, and Dmax and Dmin were set at 0.1 and 0.01, respectively.To compare the effect of gradient shape on the fit of model to data, two additional equations were used to generate concave and convex gradients with 15 distinct values of D(c), Concave D(c)=(Dmin−Dmax)[1−e(−kc14)]+Dmax (Eq. 2) Convex D(c)=(Dmin−Dmax)[e(−k(14−c)14)]+Dmax (Eq. 3) where k specifies the degree of nonlinearity of the concave and convex gradients. k was set to 5.ISA Models for Gradients—A key feature of ISA is that it uses all measurable isotopomer data to find the best fit of model to data. As originally designed (11Kelleher J.K. Masterson T.M. Am. J. Physiol. 1992; 262: E118-E125PubMed Google Scholar), ISA solves for two unknown parameters, the precursor enrichment, D, and the fraction of new synthesis at the time of sampling g(t). However, the nonlinear regression feature of ISA allows for models with additional parameters. First, gradients in precursor enrichment are modeled via ISA in discrete steps. We use 15 steps to model the gradients for ISA computed exactly as for the in vitro synthesis procedure described above. For each step of the gradient a different value is used for the precursor enrichment, D(c), as indicated by the equations above. The gradient is created by combining the values for all isotopomers for the 15 steps of the gradient and computing the fractional abundances for the combined gradient. Second, as with the conventional form of ISA, the program compares the fractional abundance values for isotopomers between data and model by calculating the weighted sum of square errors. The program searches for the best fit values of the three parameters, Dmin, Dmax, and g(t) yielding the smallest error using the Levenberg-Marquardt algorithm (11Kelleher J.K. Masterson T.M. Am. J. Physiol. 1992; 262: E118-E125PubMed Google Scholar). ISA requires no correction for natural 13C abundance, which is included in the model. A spreadsheet is included in the Supplementary Materials to demonstrate how the gradient ISA fractional abundances are created. The algebraic equations describing the steps of the gradient were developed with the assistance of the symbolic algebra facility of Mathcad (Maple) (Mathsoft, Cambridge, MA). Although the spreadsheet provides sample calculations, it does not have the capacity to perform the complete ISA calculations. The ISA program requires additional modeling that is not available in Excel; this allows finding the best-fit solution for all isotopomer equations simultaneously. For additional details about the ISA program, contact one of us (J. K. K.).The Two-isotopomer Method—We used the following two-isotopomer equations to compute precursor enrichment, p, and fractional synthesis, f, using the notation of Hellerstein (19Hellerstein M.K. Neese R.A. Am. J. Physiol. 1992; 263: E988-E1001PubMed Google Scholar), p=(2M7)(5M5)+(2M7) (Eq. 4) f=M5/ΣM6p(1−p)5 (Eq. 5) where Mi is the intensity of the signal for various isotopomers corrected for natural abundance. 2Mass isotopomers are designated as M, M1, M2... Mi, where i is the number of mass units above that of the unlabeled isotopomer M. The subscripted notations M1,M2,... Mi are the intensities of the mass spectrometric signals of the corresponding isotopomers. These equations are identical to those described in the companion report (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) except that they are adjusted for pentadecanoate synthesized from unlabeled malonyl-CoA and six labeled acetyl-CoA molecules. Note that the equation for p is a function of the relative intensities of M5 and M7. There is no need to divide each isotopomer intensity by that of the unlabeled M or by the sum of all isotopomers as these factors cancel out. However, the equation for f requires that the intensity at M5 be divided by the sum of all isotopomers, ∑M. These equations are based on probabilities and differ slightly from those proposed by Chinkes et al. (12Chinkes D.L. Aarsland A. Rosenblatt J. Wolfe R.R. Am. J. Physiol. 1996; 271: E373-E383Crossref PubMed Google Scholar). Note that the two-isotopomer approach requires that the data are first corrected for the natural abundance of carbon and other atoms in the mass ion analyzed. These corrections have proved to be nontrivial (21Rosenblatt J. Chinkes D. Wolfe M. Wolfe R.R. Am. J. Physiol. 1992; 263: E584-E596Crossref PubMed Google Scholar). A spreadsheet included in the Supplementary Material demonstrates the validity of the two-isotopomer method for ideal, error-free, data using any adjoining two isotopomer pair. The spreadsheet includes algebraic equations developed with Mathcad and demonstrates the agreement between the algebraic equations and idealized data.Comparison of the Methods—To compare the two methodologies (gradient form of ISA and two-isotopomer method), we created error-free mass isotopomer spectra of pentadecanoate from given precursor enrichment and fractional synthesis parameters. Then we computed the latter parameters based on the ideal MID using ISA and the twoisotopomer method.RESULTS AND DISCUSSIONThe computations of the parameters of the synthesis of biopolymers from the analysis of mass isotopomer patterns are based on solid considerations of combinatorial analysis. This has been demonstrated in test tube experiments where polymeric compounds were synthesized from monomers labeled with heavy atoms (13C, 2H, 15N, and 18O). Such polymers include glucose penta[13C]acetate (22Lee W.N. Bergner E.A. Guo Z.K. Biol. Mass Spectrom. 1992; 21: 114-122Crossref PubMed Scopus (77) Google Scholar), hexamethylenetetramine labeled from 15NH4+ (23Yang D. Puchowicz M.A. David F. Powers L. Halperin M.L. Brunengraber H. J. Mass Spectrom. 1999; 34: 1130-1136Crossref PubMed Scopus (8) Google Scholar) or from [2H2]formaldehyde (24Brunengraber H. Kelleher J.K. Des Rosiers C. Annu. Rev. Nutr. 1997; 17: 559-596Crossref PubMed Scopus (94) Google Scholar), acetone labeled from deuterated water by keto-enol tautomerism (25Yang D. Diraison F. Beylot M. Brunengraber D.Z. Samols M.A. Anderson V.E. Brunengraber H. Anal. Biochem. 1998; 258: 315-321Crossref PubMed Scopus (93) Google Scholar), and trimethylphosphate labeled from 18O-labeled water (26Brunengraber D.Z. McCabe B.J. Katanik J. Previs S.F. Anal. Biochem. 2002; 306: 278-282Crossref PubMed Scopus (20) Google Scholar). In all these cases, precise values of precursor enrichment were calculated. As a result, an important application of mass isotopomer analysis is the assay of the low isotopic enrichment of compounds that can be polymerized into a compound assayable by GC-MS (23Yang D. Puchowicz M.A. David F. Powers L. Halperin M.L. Brunengraber H. J. Mass Spectrom. 1999; 34: 1130-1136Crossref PubMed Scopus (8) Google Scholar, 25Yang D. Diraison F. Beylot M. Brunengraber D.Z. Samols M.A. Anderson V.E. Brunengraber H. Anal. Biochem. 1998; 258: 315-321Crossref PubMed Scopus (93) Google Scholar, 26Brunengraber D.Z. McCabe B.J. Katanik J. Previs S.F. Anal. Biochem. 2002; 306: 278-282Crossref PubMed Scopus (20) Google Scholar, 27Landau B.R. Wahren J. Chandramouli V. Schumann W.C. Ekberg K. Kalhan S.C. J. Clin. Invest. 1995; 95: 172-178Crossref PubMed Scopus (177) Google Scholar, 28Landau B.R. Wahren J. Chandramouli V. Schumann W.C. Ekberg K. Kalhan S.C. J. Clin. Invest. 1996; 98: 378-385Crossref PubMed Scopus (389) Google Scholar).The usual application of mass isotopomer analysis to the synthesis of polymers in live cells assumes that the precursor enrichment is identical in all cells and does not change with time. The metabolic zonation of the liver (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), resulting from the organ's lobular architecture that functions as a plug-flow reactor (29Tisseyre B. Coquille J.C. Gervais P. Bioprocess Eng. 1995; 13: 113-118Crossref Scopus (0) Google Scholar), poses particular challenges to the measurement of fractional synthesis of biopolymers by MID analysis using this assumption. This is because each cell along the liver lobule is in contact with blood of continuously changing composition in terms of substrate concentrations and isotopic enrichment of tracers. Several studies reported that the concentration and enrichment of glycerol (30Landau B.R. Wahren J. Previs S.F. Ekberg K. Chandramouli V. Brunengraber H. Am. J. Physiol. 1996; 271: E1110-E1117PubMed Google Scholar, 31Previs S.F. Martin S.K. Hazey J.W. Soloviev M.V. Keating A.P. Lucas D. David F. Koshy J. Kirschenbaum D.W. Tserng K.Y. Brunengraber H. Am. J. Physiol. 1996; 271: E1118-E1124PubMed Google Scholar), NH4+ (32Yang D. Hazey J.W. David F. Singh J. Rivchum R. Streem J.M. Halperin M.L. Brunengraber H. Am. J. Physiol. 2000; 278: E469-E476Crossref PubMed Google Scholar), and acetate (20Puchowicz M.A. Bederman I.R. Comte B. Yang D. David F. Stone E. Jabbour K. Wasserman D.H. Brunengraber H. Am. J. Physiol. 1999; 277: E1022-E1027PubMed Google Scholar) markedly decrease across the liver. In addition, the activities of enzymes involved in the synthesis of biopolymers also vary across the lobule. For example, there is an inverse zonation of the enzymes, which fuels lipogenesis (glucokinase (3Fischer W. Ick M. Katz N.R. Hoppe Seylers. Z. Physiol. Chem. 1982; 363: 375-380Crossref PubMed Scopus (51) Google Scholar) and ATP-citrate lyase and fatty acid synthase (7Katz N. Thiele J. Giffhorn-Katz S. Eur. J. Biochem. 1989; 180: 185-189Crossref PubMed Scopus (12) Google Scholar, 8Katz N.R. Fischer W. Giffhorn S. Eur. J. Biochem. 1983; 135: 103-107Crossref PubMed Scopus (40) Google Scholar, 9Katz N.R. Fischer W. Ick M. Eur. J. Biochem. 1983; 130: 297-301Crossref PubMed Scopus (17) Google Scholar)) and cytosolic acetyl-CoA synthetase (which introduces label from [13C]acetate in the lipogenic pathway (10Knudsen C.T. Immerdal L. Grunnet N. Quistorff B. Eur. J. Biochem. 1992; 204: 359-362Crossref PubMed Scopus (11) Google Scholar)).In the companion report (1Bederman I.R. Reszko A.E. Kasumov T. David F. Wasserman D.H. Kelleher J.K. Brunengraber H. J. Biol. Chem. 2004; 279: 43207-43216Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), we demonstrated the existence of translobular gradients of enrichment of lipogenic acetyl-CoA (labeled from [1,2-13C2]acetate) in the livers of live dogs and in perfused rat livers. In the latter animal preparations, ISA indicated the presence of a gradient of acetyl-CoA enrichment even in the absence of gradients of acetate concentration and enrichment across the liver. The MIDs of fatty acids isolated from the various livers were analyzed by the two state-of-theart computation techniques, i.e. the two-isotopomer method (modified from Chinkes (12Chinkes D.L. Aarsland A. Rosenblatt J. Wolfe R.R. Am. J. Physiol. 1996; 271: E373-E383Crossref PubMed Google Scholar)) and by ISA (11Kelleher J.K. Masterson T.M. Am. J. Physiol. 1992; 262: E118-E125PubMed Google Scholar). The two-isotopomer method, like the more widely applied MID analysis method (19Hellerstein M.K. Neese R.A. Am. J. 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