Rates of gluconeogenesis and citric acid cycle in perfused livers, assessed from the mass...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 6, Issue of February 25, pp, 4170-4180,1993 Printed in U. S. A.

Rates of Gluconeogenesis and Citric Acid Cycle in Perfused Livers, Assessed from the Mass Spectrometric Assay of the 13C Labeling Pattern of Glutamate*

(Received for publication, April 30, 1992)

Lorella Di Donato$§, Christine Des Rosiers$V, Jane A. MontgomeryV, France David[[, Michel GarneauV, and Henri Brunengraberll From the Departments of $Biochemistry and YNutrition, University of Montreal, Montreal Quebec H3C 357, Canada and the 11 Department of Nutrition, Case Western Reserve University, Cleveland Ohio 44106

Absolute rates of gluconeogenesis and of the citric acid cycle were assessed in livers isolated from 24-h starved rats, perfused with physiological concentrations of [3-’3C]lactate and [3-13C]pyruvate 2 0.2 mM octanoate. Calculations are based on (i) the 13C-labeling pattern of glutamate determined by gas chromatography-mass spectrometry combined with isotopomer analysis, (ii) substrate balance, and (iii) equations developed by Magnusson et al. (Magnusson, I., Schu- mann, W. C., Bartsch, G . E., Chandramouli, V., Ku- maran, K., Wahren, J., and Landau, B. R. (1991) J. Biol. Chem. 266, 6975-6984) based on a citric acid cycle model proposed by Katz (Katz, J. (1985) Am. J. Physiol. 248, R391-R399). Glutamate, isolated from liver extracts, is enzymatically or chemically converted to y-aminobutyrate, cY-hydroxyglutarate, isocitrate, and glutamine before mass spectrometric analysis. General equations have been developed (“Appendix I”) to determine the isotopic enrichment of each carbon of glutamate from the isotopic enrichment of fragments obtained from the mass spectra of trimethylsilyl or t-butyldimethylsilyl derivatives of glutamate and of derived compounds (“Appendix 11”). In the presence of octanoate, (i) the rate of the citric acid cycle decreases from 0.25 to 0.13 pmol/min % g wet weight which are one-third and one-sixth of the rate of pyruvate carboxylation, and (ii) the rate of gluconeogenesis increases from 0.65 to 0.83 pmol/min % g wet weight. The rate of pyruvate carboxylation is 13 and 34-fold faster than that of pyruvate dehydrogenation in the absence or presence of octanoate, respectively. The rate of oxaloacetate to fumarate interconversion is at least six times greater than that of the citric acid cycle. Our data closely agree with those obtained by Magnusson et al. who used a non-invasive “chemical biopsy” of the human liver and support the use of labeled lactate and/ or pyruvate for tracing hepatic metabolism in vivo.

The rate of gluconeogenesis (GNG)’ measured in vivo using

* This work was supported by Medical Research Council Grants MA-9575 and MT-10816 (to C. D. R.) and National Institutes of Health Grant DK35543 (to H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed Research Center, Notre-Dame Hospital, 1560 Sherbrooke St. East, Montreal PQ H2L 4M1, Canada.

The abbreviations used are: GNG, gluconeogenesis; CAC, citric acid cycle; GC-MS, gas chromatography-mass spectrometry; IE, isotopic enrichment; OAA, oxaloacetate; PEP, phosphoenolpyruvate; TBDMS, t-butyldimethylsilyl; TMS, trimethylsilyl.

14 C- or 13C-labeled three-carbon precursors is underestimated because of isotopic exchanges in the citric acid cycle (CAC). Estimations of the dilution factor between the tracer and phosphoenolpyruvate (PEP) (1-9) require (i) a labeled tracer, such as acetate, glucose, lactate, or pyruvate, and (ii) the determination of the label distribution in glucose or a CAC intermediate. Among these approaches, the use of lactate and/ or pyruvate labeled on carbon 3 and determination of the labeling pattern of a CAC intermediate provides more accu- rate estimates of the rate of GNG (10).

Recently, non-invasive sampling of liver CAC intermediates in humans by “chemical biopsy” was developed by Magnusson et al. (11). In their study, [3-14C]lactate and gram amounts of phenylacetate were administered. Hepatic conjugation of phenylacetate to glutamine forms phenylacetylglutamine which is excreted in urine. Isolation of phenylacetylglutamine followed by acid hydrolysis yields glutamate whose labeling pattern is determined by sequential degradation to five 14C02. The labeling of the glutamine moiety of phenylacetylglutamine presumably reflects that of liver a-ketoglutarate, an intermediate of the CAC. Carbons 4 + 5 of a-ketoglutarate derive from acetyl-coA, whereas carbons 1 + 2 + 3 derive from oxaloacetate (OAA) and are precursors of PEP. Using a CAC model (Fig. 1) initially proposed by Katz (6), Magnusson et al. (11) developed equations to calculate, from the labeling pattern of glutamate, fluxes of GNG and of the CAC as well as the dilution factor between pyruvate and PEP.

For future investigations of the CAC metabolism in humans, it would, however, be preferable to avoid using 14C

tracers and gram amounts of phenylacetate. In this respect, the potential of stable isotopes and isotopomer analysis using either gas chromatography-mass spectrometry (GC-MS) or nuclear magnetic resonance (NMR) has recently been dem- onstrated by several investigators (5, 7-9, 12-20). However, none of these studies report the complete determination of the labeling of each carbon of glutamate which is necessary to use the model described by Magnusson et al. (11).

In the present paper, we show how the 13C-labeling pattern of glutamate from isolated livers perfused with [3-13C]lactate and [3-13C]pyruvate can be assessed by GC-MS fragmentation patterns coupled with isotopomer analysis. Since rats do not synthesize phenylacetylglutamine, we based our study on the labeling of glutamate isolated from liver extracts. The IE of each carbon of glutamate is then used to calculate the rate of GNG and CAC along with several parameters of the CAC. Part of this work has been presented in abstract form (21, 22).

EXPERIMENTAL PROCEDURES

Materials

rich, Boehringer Mannheim (Dorval, Quebec), Fisher Scientific Enzymes, coenzymes, and biochemicals were purchased from Ald-

4170

Gluconeogenesis and Citric Acid Cycle in Liver 4171

FIG. 1. Model of gluconeogenesis and of the citric acid cycle metabolism. This model was reproduced with minor modifications and with the per- mission of Magnusson et al. (11) where V indicates rates and intermediates are designated by L1-L3 for lactate and PI- P3 for pyruvate, etc.

p o 2 f Acety l -coA

Glucose A

Pi COOH Lq COOH

P2 CO L, HCOH I ' 8 I

I I

A K G COOH Citrate T I I

y Aci

C'3

dS

(Montreal, Quebec), and Sigma. ~-[l- '~C]Glutamate (99%), L-[~-'~C] glutamate (99%), ~- [U-~~C~]glu tamate (95.7%), ~-[2,3,3,4,4-~H~]glutamate (99%), sodium [2-I3C]acetate (99.4%), sodium [1,2-l3Cz]acetate (99%), [y-amin~-l-'~CJbutyrate (99%), [y-amin0-1,4-'~C~]butyrate (99%), sodium [3-I3C]pyruvate (99%), sodium [3-13C]lactate (99%), and NaB'H, (98%) were obtainedfrom Merck Sharp & Dohme Isotopes (Pointe Claire, Quebec). ~-[U-"C]glutamate was purchased from ICN Radiochemicals (Imine, CA). N,O-bis-(trimethylsily1)-acetamide, bis-(trimethylsily1)-trifluoroacetamide (with 1% trimethylch- lorosilane), t-butyldimethylsilyl trifluoroacetamide, and pyridine (si- lylation grade; dried over Na2S0,) were obtained from Regis Chemical Company (Morton Grove, IL). NaBH4 (or NaB2H4) was dissolved in 0.1 N NaOH to a final concentration of 1 M. All solutions were made with water purified by a "Milli-Q" system (Millipore).

Liver Perfusions Livers isolated from 24-h starved Sprague-Dawley rats were per-

fused (23) at 37 "C for 35 min with non-recirculating bicarbonate buffer, pH 7.4, equilibrated with 95% 0, + 5% COz containing various substrates and I3C-labeled tracers, either [2-13C]acetate, [1,2-I3C2] acetate or [3-I3C]lactate and [3-13C]pyruvate. The tracers were chosen to maximize labeling of either C-4, C-4 + C-5, or C-2 + C-3 of glutamate: respectively. At 35 min, livers were freeze-clamped.

f3ClAcetate-Livers (200-225-g rats) were perfused at 30 ml/min in the presence of 10 mM glucose, 10 mM lactate, 1 mM pyruvate, 10

The abbreviations C-1, C-2, (2-3, C-4, C-5 refer to the carbons of glutamate and C1+ C5, C2 + C5, C2 + C4, C1+ C3, C4 + C5 and C2 refer to the various fragments containing C1 + C2 + C3 + C4 + C5, C2 + C3 + C4 + C5, C2 + C3 + C4, C1+ C2 + C3, C4 + C5, and C2, respectively.

mM OAA, 5 mM asparagine, and 0.1 mM 3-mercaptopicolinate, an inhibitor of PEP carboxykinase (24-27). At 20 min, 1 mM of unlabeled sodium acetate was infused. At 25 min, unlabeled acetate was replaced with [2-I3C]acetate or [1,2-13C2]acetate.

[3-I3C]Lactate and [3-13C]Pyruuate (28)"Livers (100-120-g rats) were perfused at 15 ml/min in the presence of 4 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 0.2 mM acetate, and where indicated 0.2 mM octanoate. At 15 min, unlabeled lactate and pyruvate were replaced with [3-13C]lactate and [3-I3C]pyruvate.

Gas Chromatography-Mass Spectrometry

All samples were analyzed on an Hewlett Packard 5988A GC-MS equipped with an HP-5 fused silica capillary column (25 m X 0.2-mm inner diameter, 0.33-pm film thickness). Split injection ratio was approximately 10. Carrier gas used was helium (0.6-0.8 ml/min). Injection port was at 270 '. The transfer line to the mass spectrometer was at 280 *. The MS operates in the positive ion electron impact mode (70 eV). Appropriate ion sets were monitored with a dwell time of 120 ms/ion. Ions monitored were limited to a maximum of five per ion set.

Analytical Procedures

Frozen livers were extracted with 6% perchloric acid (3.5 v/w). The neutralized extracts (25 ml), spiked with 5 X lo4 disintegrations/ minute [U-14C]glutamate, were diluted 4-fold and loaded onto 30 X 1.5-cm Bio-Rad AG-I-XB-Cl- column, which were developed with 100 ml of H20,50 ml of 15 mM HCl, and 110 ml of 200 mM HC1.

The fractions containing glutamate, eluted with 15 mM HCl, were identified by [14C]glutamate counting, pooled, and assayed for glutamate (29). Five aliquots of the pooled fractions containing about 0.05

4172 Gluconeogenesis and Citric Acid Cycle in Liver

(I), 0.25 (II), and 0.5 (111-V) pmol of glutamate were treated as follows. Glutamate-Aliquot I was dried under air at 55 'C before addition

of 20 pl of t-butyldimethylsilyl trifluoroacetamide + 60 pl of acetonitrile followed by heating for 2 h at 80 "C.

For GC-MS analysis, the column temperature initially set at 200 "C for 4 min was increased by 5 "C/min until 270 "C and kept at this temperature for 10 min. The tris-t-butyldimethylsilyl (TBDMS) derivative of glutamate (glutamate-(TBDMS)3) elutes a t 13-14 min. Ion sets monitored are m/z 432-437 and m/z 330-334.

Glutamate is then converted, in aliquots 11-V, to y-aminobutyrate (aliquot 11), a-hydroxyglutarate (aliquot 111), isocitrate (aliquot IV), and glutamine (aliquot V) as follows.

y-Aminobutyrate-Aliquot I1 was adjusted to pH 5 and evaporated to 200 pl, before addition of 0.2 unit of glutamic decarboxylase (Escherichia coli suspended in 4.2 M (NH,),SO,) in 0.5 ml of 50 mM HEPES, pH 5. After 15 min at 37 "C, the solution was neutralized with KOH and loaded onto 10 X 0.5-cm Bio-Rad AG-50-X8-H' columns, which were developed with 5 ml of H,O and 10 ml of 3 N NH,OH. The fractions containing [y-amino-"Clbutyrate were neutralized and then lyophilized. The dried residue was reacted with 50 /*1 of bis-(trimethylsily1)-trifluoroacetamide and 150 pl of pyridine.

For GC-MS analysis, the column temperature initially set a t 70 "C for 1 min was increased by 10 "C/min until 250 "C and kept at this temperature for 10 min. The tris-trimethylsilyl (TMS) derivative of y-aminobutyrate (y-aminobutyrate-(TMS)3) elutes at 13 min. Ion sets monitored are m/z 304-309 and m/z 174-175.

a-Hydronyglutarate-Aliquot I11 was evaporated to 200 pl before addition of 10 &mol of pyruvate and 30 units of glutamate-pyruvate transaminase in 0.5 ml of 2 M Tris-acetate, pH 7.4. After 30 min, the p H was raised to 9.0 and a-ketoglutarate was reduced to a-hydroxyglutarate by addition of 2-fold molar excess of NaBH4 or NaB2H4. After 45 min, the solution was acidified to pH 2.0, saturated with NaC1, and extracted three times with 5 ml of ethyl acetate. The extract was dried with anhydrous Na2S04 and evaporated to dryness under N, a t 45 "C. The dried residue was reacted with 40 pl of N,O- bis-(trimethylsily1)-acetamide.

For GC-MS analysis the column temperature initially set at 70 "C for 1 min was increased by 10 "C/min until 250 "C, and kept at this temperature for 10 min. The tris-TMS derivative of a-hydroxyglutarate (a-hydr~xyglutarate-(TMS)~) elutes a t 13-14 min. Ion sets monitored are m/z 349-354, m/z 247-251, and m/z 129-132 when NaBH4 was used and m/z 350-355, m/z 248-252, and m/z 130-133 when NaB2H4 was used. a-Ketoglutarate was reduced to a-hydroxyglutarate with NaBH, for the standard curve of C-1 and (2-5. For liver perfusions, a-ketoglutarate was reduced to cu-hydro~y-[2-~H] glutarate with NaBZH4 because of a contaminant interfering with the measurement of the ion m/z 349. Similar IEs have been obtained when monitoring ion sets at (i) m/z 129-132 or m/z 130-133 and (ii) m/z 247-251 or m/z 248-252.

Zsocitrate-The procedure of Chen and Plaut (30) was modified as follows. Aliquot IV was evaporated to 200 p1, before addition of 10 pmol of pyruvate and 30 units of glutamate-pyruvate transaminase in 0.5 ml of 2 M Tris-acetate, pH 7.4. After 30 min, 2 pmol of MnCL, 1.5 pmol of NADPH, 10 pmol of glucose 6-phosphate, 125 pmol of NaHC03, and 1 unit of isocitrate dehydrogenase was added in a total volume of 1.2 ml. After 45 min, 14 units glucose-6-phosphate dehydrogenase was added to regenerate NADPH. After 15 min, the solution was acidified to pH 2.0, saturated with NaC1, and extracted three times with 5 ml of ethyl acetate. The extract was dried with anhydrous Na,S04 and evaporated to dryness under Nz a t 45 "C. The dried residue was reacted with 10 pl of bis-(trimethylsily1)-trifluoroacetamide and 30 pl of pyridine.

For GC-MS analysis the column temperature initially set at 150 "C for 3 min was increased by 5 "C/min until 250 "C, and kept a t this temperature for 10 min. The tetrakis-TMS derivative of isocitrate (isocitrate-(TMS)4) elutes a t 13-14 min. Ion sets monitored are m/z 465-470, m/z 363-367, m/z 273-277, m/z 245-248, and m/z 204-206.

Glutamine-Aliquot V was evaporated to 200 pl, before the addition of 0.5 ml of 50 mM imidazole-HC1, pH 7.0, 50 pmol of NH,Cl, 50 pmol of MgCl,, 10 pmol of KC1,7.2 pmol of ATP, 0.35 pmol of NADH, 1.2 pmol of PEP, 20 units of pyruvate kinase, 55 units of lactate dehydrogenase, and 1 unit of glutamine synthetase (enzymes were centrifuged and resuspended in KC1 0.1 M ) in a total volume of 1.0 ml (31). After 1 h, the reaction mixture was loaded onto 10 X 0.5-cm Bio-Rad AG-50-X8-H' column, which were developed with 5 ml of H20 and 10 ml of 4 N NH,OH. The fractions containing glutamine were identified by enzymatic assay (32) and then lyophilized. The lyophilized residue was reacted with 50 pl of t-butyldimethylsilyl

trifluoroacetamide and 100 p1 of acetonitrile and heated for 2 h at 85 "C.

For GC-MS analysis the column temperature initially set at 200 "C for 4 min was increased by 5 "C/min until 270 "C, and kept a t this temperature for 10 min. Glutamine silylated with t-butyldimethylsilyl trifluoroacetamide/acetonitrile forms a tris-TBDMS derivative of glutamine (gl~tamine-(TBDMS)~), eluting a t 15-16 min, and a tetrakis-TBDMS derivative (glutamine-(TBDMS),), eluting a t 18-19 min. The glutamine-(TBDMS)s is more abundant than glutamine- (TBDMS),; formation of the latter increases with time (33). Ion sets monitored are m/z 431-436, m/z 329-333, and m/z 271-275 for gl~tamine-(TBDMS)~ and m/z 545-550 and m/z 258-261 for glutamine-(TBDMS),. As shown in Fig. 2 and spectrum G of "Appendix 11," the relative intensity of the ion m/z 545 of glutamine-(TBDMS), can be quite low when working a t low concentrations of glutamate. An alternative is to simply monitor the ion m/z 431 of glutamine- (TBDMS)3 which has a relative intensity 10-fold greater than the equivalent ion in glutamine-(TBDMS),.

The fractions eluted with 200 mM HCl, containing PEP, were pooled and adjusted to pH 8.5 with KOH. The pooled fractions were evaporated under air a t 65 "C to 3-5 ml. PEP was converted to pyruvate under the following conditions: 50 mM triethanolamine, pH 7.4, 10 mM MgSO,, 5 mM EDTA, 25 pmol of ADP, and 2 units of pyruvate kinase were added. After 30 min, pH was adjusted to 8.5 with 0.3 N NaOH, a 2-fold molar excess of hydroxylamine hydrochlo- ride was added and heated a t 60 "C for 1 h. The reaction mixture was acidified to pH 2, saturated with NaC1, and extracted three times with 5 ml of diethyl ether. The extract was dried with anhydrous Na2S04 and evaporated to dryness under N, a t 37 "C. The dried residue was reacted with 25 pl of t-butyldimethylsilyl trifluoroacetamide.

For GC-MS analysis the column temperature initially set at 70 "C for 1 min was increased by 10 "C/min to 250 "C, and kept at this temperature for 10 min. The bis-TBDMS derivative of oxime-pyruvate elutes a t 13-14 min. Ion sets monitored are mlz 274-277. This fragment is formed by the loss of the t-butyl group'(M-57) from the TBDMS radical.

Concentrations of acetoacetate and P-hydroxybutyrate (34), pyruvate and lactate (35), glutamate (29), and aspartate (36) were determined enzymatically in neutralized perchloric acid extracts of perfusate. Concentration of octanoate was determined by GC-MS using 2-butyl heptanoate as external standard (28).

Calculations

Peak areas, determined by computer integration, were corrected for naturally occurring isotopes at higher masses (natural abundance) and isotopic impurities in tracers occurring at lower masses. This correction, previously described in detail (37, 38), involves solving a set of n equations with n unknowns, where n is the number of ions monitored. Equations were solved using the matrix function of Lotus 123 (release 3.1). Whenever possible, experimentally determined correction factors were used in area calculations instead of theoretical values. These were determined by SIM at all ions sets of TMS or TBDMS derivatives of standard solutions of unlabeled and labeled glutamate ([5-13C], [1,5-13C2], and [U-'3Cs]glutamate) and of derived compounds. TBDMS derivatives of standard solutions of [3-I3C] lactate and [3-13C]pyruvate were also analyzed to determine the correction factors for light isotopic impurities of the tracers. All IEs are calculated using corrected areas.

Appendix I presents equations developed to calculate the IE of each carbon of glutamate. Equations developed by Magnusson et al. (11) used to calculate rates of GNG and of the CAC have been solved by computer using a Lotus 123 spreadsheet program (release 3.1). Comparisons were performed using Student's paired t test.

RESULTS

Fig. 2 shows characterist ic fragment ions of unlabeled glutamate, [1-l3CC], [5-13C], and [U-'3C6]glutamate, analyzed either as g I~ tama te - (TBDMS)~ , y -aminobu ty ra t e - (TMS)3 , a-hydro~y-[2-~H]glutarate-(TMS)~ (or a-hydroxyglutarate- (TMS),) , isocitrate-(TMSh, glutamine-(TBDMSh, or glutamine-(TBDMS)4. The proposed structure for each of these fragments is also shown. The electron impact mass spectra of unlabeled TMS or TBDMS derivatives of glutamate and derived compounds are shown under "Appendix 11." Ion clusters chosen correspond to single fragments as characterized


P

L lZC, I

432

330

304

174

350 (349)

248 (247 1

130 (129)

465

363

273

245

204

431

329

271

545

258

GLUTAMATE

: 1-'3C] - 433

330

304

174

(350) 351

248 (247 1

(129) 130

466

363

2 1 3

245

204

432

329

271

546

259

: 5- '3C]

433

331

305

174

351 (350)

249 (248 1

130 (129)

466

364

274

245

205

432

330

2 1 2

546

258

u"3c,1

437

334

308

175

355 (354)

252 (251)

133 (132)

471

367

277

248

206

436

333

275

550

261

i= CARBON NUMBERING

5 4 3 2 1

TBDMSOOC-CH,-CH,-CH -COOSF (Me) p

I

N

I

II

I

II

II

NHTBDMS

TBDMSOOC-CH,-CH,-CH

NHTBDMS

(Me),SiSOOC-CH,-CH,-CH,

N(TMS)z

CH2

N(TMS),

TMSOOC-CH,-CH,-C'H-COOSi (Me),

OTMS

TMSOOC-CH,-CH,-C~H

OTMS

CH,=CH -C2H

OTMS

TMSOOC-CH,-CH -CH -COOSi (Me) , I I

I II

I

I I

TMSOOC OTMS

TMSOOC-CH,-CH -CH

TMSOOC OTMS

TMSOOC-CH,-C =CH

TMSOOC

CH,-C =CH

TMSOOC OTMS

(TMSO) ,C=CH,

OC-CH,-CH,-CH-COOSi(Me), I I

I II

I II

II I

I

NH-TBDMS NH-TBDMS

OC-CH,-CH,-CH

NH-TBDMS NH-TBDMS

OC-CH,-CH,-CH

NH-TBDMS N - S i (Me) 2

TBDMSO-C-CH,-CH,-CH-COOSi (Me) ,

N-TBDMS NH-TBDMS

CH,=C -COOS i ( Me ) , NH-TBDMS

- %BP

- 80

100

35

100

10

61

100

9

5

100

75

19

100

40

20

33

100

FIG. 2. GC-MS analysis of unlabeled and '%-labeled gl~tarnate-(TBDMS)~, y-aminobutyrate-(TMS)a, a-hydroxy-[Z-'H] gl~tarate-(TMS)~ (or a-hydroxyglutarate-(TMS)3), i~ocitrate-(TMS)~, gl~tamine-(TBDMS)~, and glutamine-(TBDMS)r. Stand- ard solutions of unlabeled and labeled glutamate were analyzed by GC-MS in the SCAN mode as glutamate-(TBDMS)3 and after enzymatic conversion to y-aminobutyrate-(TMS)3, a-hydro~y-[2-~H]glutarate-(TMS)3 (or a-hydroxyglutarate-(TMS)3), isocitrate-(TMS),, glutamine- (TBDMS), and glutamine-(TDBMS), (see Analytical Procedures). The mass spectra of unlabeled glutamate and derived compounds revealed characteristic ions ("Appendix 11"). The identity of each ion was confirmed using [1-13C], [5-I3C], and [U-13C6]glutamate. The proposed chemical structure for each ion is shown. For ol-hydro~y-[2-~H]glutarate-(TMS)~, the values shown inparenthesis represent the characteristic ions of a-hydroxyglutarate-(TMS)3 (that is a-ketoglutarate reduced with NaBH4). Note that the numbering of each carbon atom corresponds to that of the glutamate molecule. %BP, % base peak.


by normal natural isotopic abundance and no evidence for (”1) ions and were separated from heavier clusters by at least n mass units where n equals the number of glutamate carbons in the molecule or fragment.

The spectrum of gl~tamate-(TBDMS)~ (Fig. 2 and spectrum A of Appendix 11) includes two major ions at m/z 330 and m/ z 432. The base peak at m/z 330 is formed by the loss of C-1 (“159) (33). The ion at m/z 432, formed by the loss of a t- butyl group (“57) from the molecular ion of mass 489, includes all five carbons of glutamate. This is confirmed by the shifts of the 330 and 432 ions (i) to m/z 334 and 437 in the spectrum of [U-’3Cs]glutamate, (ii) to m/z 330 and 433 in the spectrum of [1-13C] glutamate, and (iii) to m/z 331 and 433 in the spectrum of [5-13C]glutamate. Similar shifts in mass were followed to identify characteristic ions pro- duced by the fragmentation of y-amin~butyrate-(TMS)~, ~~-hydroxy-[2-~H]glutarate-(TMS)3 (or a-hydroxyglutarate- (TMS)& isocitrate-(TMS)r, gl~tamine-(TBDMS)~, and glutamine-(TBDMS)4. The identity of the ion at m/z 174 of ~-aminobutyrate-(TMS)~ corresponding to C-2 of glutamate was confirmed using [1-13C]- and [1,4-’3Cz]-labeled y- aminobutyrate.

To validate the determination of the labeling pattern of glutamate, extracts of livers perfused without tracer were spiked with labeled glutamate to obtain an IE of 1-25%. The labeled standards used were [l-’3C]glutamate, [5-13C]glutamate, [U-’3C5]glutamate, and [2,3,3,4,4-2Hs]glutamate. Fig. 3 shows a standard curve for the IE of each carbon of glutamate

25 -

20 -

- x v

15 - w - 0 C i;

10 -

5 -

0 1 2 3 4

n I I I I I

0 5 10 15 20 25

Init ial IE (%)

FIG. 3. Standard curves for the percent IE of each carbon of glutamate. Neutralized perchloric acid extracts of livers containing unlabeled glutamate were spiked with 0.06-1.5 pmol of [1-’3C], [5-I3C], [U-”CC,], or [2,3,3,4,4-2Hs]glutamate. The horizontal scak shows the initial IE of glutamate in the spiked extracts measured at the ion sets of m/z 432-437. The uertical scak shows the final IE of C-1, C-2, C-3, C-4, and C-5 of glutamate measured after isolation (C- 1 and C-3 measured at m/z 432-437 and at m/z 330-334, and +, respectively) and enzymatic conversion to y-aminobutyrate (C-2 measured at m/z 174-175, V), a-hydroxyglutarate (C-1 measured at mfz 349-354 and at m/z 247-251, & C-5 measured at m/z 247-251 and at m/z 129-132, A), and isocitrate (C-1 measured at (i) m/z 465- 470 and at m/z 273-277 (0) and (ii) m/z 465-470 and at m/z 363- 367 (0); C-4 measured at m/z 204-206 (m); C-5 measured at (i) m/z 273-277 and at m/z 245-248 (V); and (ii) measured at m/z 363-367 and at m/z 245-248 (0)). See Fig. 2 and “Appendix 11” for fragment identities and electron impact mass spectra. The IE of each carbon of glutamate is calculated using the Equations detailed under “Ap- pendix I.”

(slope = 0.99 -+ 0.01; P = 0.99). This method was then used for metabolic investigations in

livers perfused with various tracer/substrate combinations to favor labeling of either C-4 + 5 of glutamate ([2-13C]acetate or [1,2-l3C2]acetate + aspartate + mercaptopicolinate) or C-2 + 3 of glutamate ([3-13C]lactate and [3-13C]pyruvate f octanoate).

Table I shows the IE of the various fragments obtained in the analysis of glutamate and derived compounds isolated from livers perfused with [2-13C]acetate or [1,2-13C2]acetate. The coefficient of variation of replicate injections and replicate analyses is 0.04 and 2.4%, respectively. Note the agreement of redundant analyses of IE of carbon groups, C1- C5, C2 + C5, and C2 + C4 of glutamate. From the IE of each fragment in Table I, one calculates using the equations under “Appendix I” the IE of each carbon of glutamate. For glutamate labeled from [2-13C]acetate3: C-1 = not detected; C-2 = 1.2 f 0.2%; C-3 = 1.4 k 0.1%; C-4 = 17.6 f 0.1%; C-5 = not detected. For glutamate labeled from [1,2-’3C2]acetate3: C-1 =

f 0.3%; C-5 = 12.6 f 0.3%. These labeling patterns were expected since livers were perfused with aspartate and mercaptopicolinate to favor labeling of C-4 + 5 of glutamate from [13C]acetate. At each turn of the CAC, the IE of OAA is greatly diluted by unlabeled OAA derived from aspartate. This is confirmed by the low IE of C-1 to C-3 of glutamate.

The complete determination of the labeling pattern of glutamate requires 1.8 or 0.75 pmol of substrate, depending on whether one uses redundant determinations (Table I) or single determinations of each carbon’s IE. In the latter case, one needs to analyze only two compounds, y-aminobutyrate and either isocitrate or glutamine. Depending on the carbon of interest, one needs only 0.05-0.5 pmol of glutamate. Thus, the method is much more sensitive than chemical degradation and somewhat more sensitive than NMR. If one limits oneself to the analysis of two compounds, analysis of glutamine should be favored over isocitrate when (i) one expects an IE less than 5% for the fragment C4 --$ C5, and (ii) when the 13C tracer is such that one expects only an M + 1 isotopomer of this fragment. Under those conditions, we noted greater var- iability (14 uersus 2%) in the measurement of the IE of this fragment. This interference is due to the presence of another fragment, as evidenced by a high m/z ratio 205204. Analysis of isocitrate-(TMS)( derived from various ‘3C-labeled gluta- mates suggests that this interfering fragment includes C-1.

Table I1 shows the IE of each carbon of glutamate from livers perfused with [3-13C]lactate and [3-13C]pyruvate f 0.2 mM octanoate. The labeling pattern is compatible with metabolism of pyruvate through pyruvate dehydrogenase and pyruvate carboxylase since glutamate is labeled in C-4 as well as C-1 + 2 + 3 (39). Note that with these tracers, we detected only M + 1 isotopomers of glutamate. This results from the low IEs of acetyl-coA and OAA and the low probability of condensation of labeled OAA with labeled acetyl-coA molecules to form M + 2 citrate.

The total IE of pyruvate in the effluent perfusate (97.5 k 0.4%) was similar to that in the influent. In livers perfused without octanoate, we measured ketone body production (0.09 k 0.01 pmol/min x g wet weight), the total IE of effluent ketone bodies (2.8 -+ 0.4%) and of liver PEP (65 f l%), lactate and pyruvate uptake (0.71 f 0.08 pmol/min X g wet weight) and the following ratios, P-hydroxybutyratelacetoacetate (0.95 f 0.06) and lactate/pyruvate (17.2 f 1.7). In livers perfused with octanoate, ketone body production (0.83 f 0.07

IE are mean f S.E. of values calculated using various sets of isotopomers except for the IE of C-2, obtained by direct measurement, which represents mean & S.E. of three GC-MS injections.

0.4 f 0.1%; C-2 = 0.9 f 0.1%; C-3 = 1.1 f 0.2%; C-4 = 12.6


TABLE I Fragment analysis of liver glutamate and derived compounds

Two livers from overnight fasted rats were perfused with either [2-13C]acetate or [1,2-%2]acetate. Glutamate isolated from the liver was converted to y-aminobutyrate, ~u-hydroxy-[2-~H]glutarate, isocitrate, and glutamine. The indicated ion sets corresponding to a fragment of a given molecule were monitored as detailed under “Experimental Procedures.” The IE of each fragment is shown as a mean f S.E. of three GC-MS injections. On line 3 (*), the IE represents only one GC-MS injection.

IE (96) . ~. Fragments Molecules analyzed monitored [2-13C]acetate [1,2-13C2]acetate

Ion sets

(n = 1) (n = 1) ~

C5-C4-C3-C2-C1 Glutamate ~Hydroxy-[2-*H]glutarate Isocitrate Glutamine Glutamine

c4-c3-c2

Glutamate a-Hydro~y-[2-~H]glutarate Isocitrate Isocitrate y-Aminobutyrate Glutamine Glutamine

oi-Hydroxy-[2-2H]glutarate Isocitrate

C3-C2-C1 Glutamine

c5 -c4 Isocitrate

c2 v-Aminobutvrate

mlz 432-437 350-355 465-470 431-436 545-550

330-334 248-252 363-367 273-277 304-308 329-333 271-275

130-132 245-248

258-261

204-206

174-175

20.5 f 0.2 19.9 f 0.5 20.7’ 20.1 f 0.2 20.2 f 0.1

Mean 20.3 f 0.6

20.6 f 0.2 19.8 f 0.5 20.7 f 0.2 20.6 f 0.2 21.2 f 0.1 20.0 f 0.1 20.0 f 0.3

Mean 20.4 f 0.7

20.7 f 0.2 20.6 f 0.2

Mean 20.7 f 0.2

2.4 f 0.1

17.3 f 0.2

1.2 f 0.2

15.3 f 0.1 15.0 f 0.3 15.7* 15.0 f 0.1 14.6 f 0.1

Mean 15.1 f 0.4

14.6 f 0.2 13.9 & 0.4 14.9 f 0.2 14.9 f 0.1 15.5 f 0.1 14.1 f 0.1 15.0 f 0.1

Mean 14.7 f 0.5

15.9 f 0.1 15.7 f 0.1

Mean 15.8 & 0.1

1.8 f 0.04

11.8 f 0.3

0.9 f 0.1

TABLE I1 Percent IE of glutamate isolated from livers perfused with [3-’3CI

lactate and [3-’3C]pyruuate f 0.2 mM octanoate Livers from 24-h starved rats were perfused with [3-I3C]lactate and

[3-’3C]pyruvate, f 0.2 mM octanoate. The IE of each carbon of glutamate was calculated using the equations detailed under “Appen- dix I.” Data are presented as mean f S.E. of the indicated number of perfusions (n) except for the IE of C-2 (without octanoate), which was available for one liver perfusion, and thus represents mean f S.E. of three GC-MS injections.

Glutamate [3-’’C]lactate and [3-’3C]pyruvate

carbons Without octanoate With octanoate

1 2.7 f 0.4 1.3 f 0.3

2 20.4 f 0.5 19.1 f 1.8

3 11.4 f 1.2 6.7 f 1.7

4 7.8 f 0.9 5.1 t 0.7

5 0.3 f 0.1 Not detected

( n = 6 ) (n = 4)

pmol/min X g wet weight), lactate and pyruvate uptake (0.85 f 0.10 pmol/min X g wet weight), and lactate/pymvate ratio (77.2 +- 12.9) increased, the total IE of effluent ketone bodies (0.3 & 0.2%) and of liver P E P (53 k 3%) decreased, whereas the P-hydroxybutyrate/acetoacetate ratio (1.05 & 0.02) did not change. Eighty-three percent of influent octanoate was taken up at a rate of 0.41 & 0.02 pmol/min X g wet weight. However, octanoate did not affect the rate of transfer of label from [3-’3C]pyruvate to ketone bodies (5.01 & 0.45 nmol/min X g wet weight), calculated from the production and the total IE of effluent ketone bodies, which was equivalent to 1/200 of the measured lactate and pyruvate uptake.

In agreement with Haussinger’s data (40), our perfused livers released glutamate, presumably from endogenous proteolysis, at a rate of 0.049 & 0.002 and 0.062 & 0.004 @mol/ min X g wet weight, in the absence or presence of octanoate, respectively. Aspartate was not detected, by enzymatic assay, in the effluent.

DISCUSSION

In this paper, the 13C-labeling pattern of glutamate was determined by GC-MS combined with isotopomer analysis. This requires taking into account both mass and positional isotopomers of glutamate, where the sum of all mass isotopomers is equal to that of all positional isotopomers. We have identified various glutamate carbon fragments by the GC-MS analysis of TMS or TBDMS derivatives of glutamate and derived compounds. Equating each mass isotopomer of the various glutamate carbon fragments to a set of positional isotopomers yields 24 equations and allows solving for all glutamate isotopomers. Although there are 32 possible positional glutamate isotopomers, a maximum of 24 can theoretically be formed from any given 13C-labeled tracer. Our GC- MS procedure for determining the labeling pattern of glutamate is simpler and less time consuming than the chemical degradation method (11,39) and allows the parallel processing of many samples. Multiple injections of the derivatized sample increase the precision of the measurements.

The I3C-labeling pattern of glutamate isolated from livers perfused with [3-I3C]lactate and [3-I3C]pyruvate & octanoate (Table 11) was introduced into the equations developed by Magnusson et al. (Fig. 1, see equations in Ref. 11) based on a CAC model proposed by Katz (6). The model assumes that glutamate and a-ketoglutarate have the same labeling pattern. All fluxes, calculated from ratios of labeling of individual


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% IE C-5 of gutamate

FIG. 4. Rates of gluconeogenesis and of the citric acid cycle as a function of the IE of C-5 of glutamate. Absolute rates were calculated from the I3C-labeling pattern of glutamate obtained from livers perfused with [3-I3C]lactate and [3-13C]pyruvate without (panel A ) or with octanoate (panel B ) using the equations developed by Magnusson et al. (11) and the uptake of lactate and pyruvate ( V8). The choice of the value of IE for C-5 of glutamate is discussed in the text. The IE of individual carbons were proportionately adjusted to keep total IE constant. Flux rates are designated by as follows: pyruvate dehydrogenase (0); conversion of fatty acids to acetyl-coA (V); CAC (+); conversion of fumarate to OAA (V); conversion of OAA to fumarate (0); pyruvate carboxylase and PEP carboxykinase (0); pyruvate kinase (A); conversion of lactate to pyruvate (A); and GNG (0). The S.E. of the mean of calculated flux rates from all liver perfusions were below 10% for pyruvate dehydrogenase, pyruvate carboxylase, PEP carboxykinase, conversion of lactate to pyruvate and GNG, and between 20 and 36% for the remaining flux rates. Pyruvate kinase, pyruvate carboxylase, and PEP carboxykinase are represented by a dotted line since these rates vary when the IE of C-5 varies from 0.01 to 0.4%.

carbons, were originally expressed relative to citrate synthesis set to 10 pmol/unit of time (11). Since we measured the uptake of lactate and pyruvate ( Va), we can calculate absolute fluxes. The model includes the rate of pyruvate kinase activity ( V7) which, in the presence of [3-13C]lactate and [3-13C] pyruvate, would introduce label on C-2 of pyruvate, C-1 of acetyl-coA, and C-5 of glutamate. Note that C-2 of pyruvate, C-1 of acetyl-coA, and C-5 of glutamate could also become labeled via malic enzyme. Since the measured IE of C-5 of glutamate is at the limit of detection and imprecise (Table 11), fluxes were calculated using a range of low values of C-5 IE (0.01-0.4%, Fig. 4, A and B). Our values for IE of C-5 of glutamate are somewhat lower than those reported by Mag- nusson et al. (11) in 60-h fasted humans (C-5 ranges from 0.5 to 2.3%). Note that in uiuo, one cannot distinguish whether labeling of C-5 occurs through liver pyruvate kinase or inter- organ Cori cycling. In contrast, in perfused livers isolated from fasted rats, where the activity of malic enzyme is presumably very low, V7 is equated to pyruvate kinase flux.

In spite of the analytical uncertainties of the low level of labeling of C-5 of glutamate, we feel confident discussing most of the rates calculated from the model, since these rates vary only slightly as shown in Fig. 4, A and B. This is not the case for pyruvate kinase ( V7), pyruvate carboxylase (Ve), and PEP carboxykinase (v6) where these rates increase on average 14- fold (without octanoate) and 45-fold (with octanoate) when the IE of C-5 of glutamate varies from 0.01 to 0.4%. Magnus- son et al. (11) had pointed out that "some estimates are sensitive to small changes in percentages in the distributions." For further comparison of fluxes between experiments without and with octanoate, only IEs of C-5 of glutamate reported in Table I1 are considered. Since IE of C-5 of glutamate was undetectable in the presence of octanoate, we assume the rates are at the left end of Fig. 4B.

The flux ratio pyruvate carboxylase ( V6)/pyruvate dehydrogenase (VI) is 13 and 34 in the absence and presence of 0.2 mM octanoate, respectively. Flux through pyruvate dehydrogenase is decreased %fold by octanoate, probably because of accumulation of acetyl-coA (41-44) rather than an increase in the mitochondrial (NADH/NAD+) ratio, since the effluent

@-hydroxybutyrate/acetoacetate ratio is not significantly affected by 0.2 mM octanoate. The decrease in pyruvate dehydrogenase translates into a 25% increase in GNG ( Vs). Our data agree with those of several authors (42, 45-53) who showed that GNG from lactate or pyruvate is stimulated by fatty acids. This stimulation results probably from the acti- vation of pyruvate carboxylase by increased mitochondrial acetyl-coA derived from &oxidation (42, 50, 53-56). The small stimulation of GNG by octanoate is consistent with the slight, but not significant, increase in the uptake of lactate and pyruvate.

Note that in this model, as pointed out by Katz (6) and by Magnusson et al. ( l l ) , VI is the rate of conversion of pyruvate to acetyl-coA that condenses with OAA to form citrate and is oxidized in the CAC. The model does not include equations for the conversion of pyruvate to acetyl-coA that ends up in fatty acids, ketone bodies, and free acetate. However, under our conditions, the impact of these processes on the balance of the [3-13C]pyruvate label is probably very small. First, fatty acid synthesis is negligible in livers from 24-h starved rats, perfused with 4 mM glucose (23) in the absence of albumin (57). In addition, the rate of transfer of label from [3-13C] pyruvate to ketone bodies is no more than 1/200 of the measured lactate and pyruvate uptake, 1/10 of the calculated flux of pyruvate + acetyl-coA + citrate ( Vl), and 1/200 of the calculated fluxes through pyruvate carboxylase (Va) and P E P carboxykinase (v6). Lastly, although we have not measured the rate of acetate production and its IE in the effluent p e r f ~ s a t e , ~ we feel that the transfer of label from [3-13C] pyruvate to free acetate is negligible. This is because free acetate derives from hydrolysis of extramitochondrial acetyl- CoA (59, 60), whose production from [3-13C]pyruvate should be minimal under our non-lipogenic conditions.

The rate of the CAC (V3) decreases 2-fold in the presence of octanoate. I t is likely that part of the ATP requirement for GNG is generated from NADH derived from @oxidation of

Based on earlier experiments (58), the concentration of acetate in the effluent perfusate was at 0.03 mM. There is no reliable method for measuring the IE of acetate at low concentration.


octanoate. Thus, less ATP needs to be generated by the CAC. &Oxidation of octanoate yields mainly ketone bodies, as evidenced by the decrease in V, in the presence of octanoate. Note that V2 represents the sum of the conversion of endogenous and exogenous fatty acids, as well as of acetate, to acetyl-coA which is converted to citrate. It does not include ketogenesis from fatty acids or acetate which, in the presence of octanoate, increases &fold.

The rate of OAA to fumarate interconversion (V4 and V,) is at least six times greater than that of the CAC, as was also found by Magnusson et al. (11) in humans. A factor of a t least three was reported by Rognstad et al. (61) in rat kidney cortex using [2-'4C]pyruvate and by Katz et al. (8) in live rats infused with [U-'3C]glucose. Using NMR, Cohen (12) reported that the rate of exchange between mitochondrial OAA and fumarate is 1.3 times the net rate of the CAC in the rat liver perfused with [2-13C]pyruvate, [1,2-'3C2]ethanol, and NH4'. Note that NMR differentiates between mitochondrial and cytosolic malate dehydrogenase activities.

The flux ratio pyruvate carboxylation ( V6)/CAC ( V3) is three and six in the absence and presence of octanoate, respectively. Values of at least two were reported by Magnus- son et al. (11) in 60-h fasted subjects. Katz et al. (8) reported a ratio of one in live rats infused with [U-'3C]glucose, but half with [2-14C]acetate infusion (62), as was also reported by Consoli et al. (63) in humans. As discussed by Schumann et al. (64), labeled acetate is extensively metabolized in peripheral tissues. Labeled compounds derived from peripheral metabolism of [2-I4C]acetate are released in blood and trans- ported to the liver, where they alter the labeling profile of metabolites derived locally from [2-14C]acetate. Thus, labeling of hepatic metabolites from [2-14C]acetate in vivo does not reflect only hepatic metabolism. The agreement between our data obtained in perfused livers and those of Magnusson et al. (11) obtained in vivo suggests that labeled lactate and/or pyruvate are more suitable tracers of liver metabolism i n vivo.

The calculated rates of GNG reflect only the conversion of lactate and pyruvate to glucose. In our conditions, it is un- likely that amino acids released by proteolysis contribute significantly to GNG. Minimal contribution of amino acids t o GNG does not exclude, however, isotopic exchanges between amino acids and CAC intermediates. Such exchanges are not included in the model. The low enrichment of glutamate, compared to that of effluent pyruvate and tissue PEP, as well the release of glutamate by the perfused liver strongly suggest dilution of CAC intermediates at the level of 01-

ketoglutarate. We do not imply that there is a net flux of glutamate carbon into the CAC, but merely a partial isotopic exchange between a-ketoglutarate and glutamate. This should not affect flux rates which are derived from ratios of IEs of carbons of glutamate, especially when pyruvate carboxylase activity is higher than that of the CAC. This is the case under our conditions since the total IE of P E P is higher than that of glutamate.

The calculated flux rates might be affected by other factors which are not taken into account by the model. These factors include metabolic zonation (65), metabolic channeling between the CAC enzymes (66), or the possible existence of two nonequilibrating pools of OAA (67).

In conclusion, our procedure for measuring the 13C-labeling pattern of glutamate should permit performing chemical biopsy of liver CAC in humans without using radioactive substrates. Also, the sensitivity of the GC-MS assay should remove the need for administering large doses of phenylacetate. Since phenylacetate is physiologically present in plasma, the base-line production of phenylacetylglutamine might be sufficient to determine the labeling pattern of liver glutamate. Lastly, the close agreement between rates of various reactions

related to GNG and the CAC obtained in isolated perfused rat livers (this study) and those reported in humans i n vivo using chemical biopsy (11), support the use of labeled lactate and/or pyruvate for tracing hepatic metabolism in vivo.

Acknowledgment-We thank B. Vachon for his assistance with the figures.

APPENDIX 1

Calculation of the I E of Each Carbon of Glutamate

Theoretically, there are 32 possible glutamate isotopomers identified as Gi (Fig. AI).

The IE is expressed in percent of all labeled and unlabeled species. It is calculated using areas of GC-MS peaks corrected (37, 38) for natural abundance and isotopic impurities (CA). Thus, the IE of a given mass isotopomer (M + x) is obtained as follows

IE(M + x ) = C A W + x ) x loo (Eq. 1) n

x=o C A M + x)

where n equals the number of carbons in the molecule or fragment analyzed. The IE of each given mass isotopomer equals the IE of a given set of positional isotopomers. For a given substrate, the total IE is equal to the sum of the IE of all mass isotopomers; it is also equal to the sum of all positional isotopomers.

GC-MS analysis of glutamate and derived compounds yields the following fragments': C1 + C5, C2 + C5, C2 "-f C4, C1 + C3, C4 + C5 and C2. For each fragment, the IE of each mass isotopomer corresponds to a set of positional isotopomers. Isotopomer analysis yields 24 equations.

Analysis of fragment C1 "-f C5. MICI-C6I = Go (Eq. 2)

Gz, + Gm + G29 + G3o + G,, (Eq. 3) M + 1(CI-C51 =

M + z(cl-cS) = G17 + G I ~ + Gls + GZO

+ Gzl + Gzz + Gz3 + Gz4 + Gzs + Gz6 (Eq. 4) M + 3<Cl-C5) = G7 + Ga + G9 + Glo + GI1

+ GI, + G n + GM + GIs + G I 6 (Eq. 5)

- Gz + Ga + Gq + GS + G6 (Eq. 6) M + 4(Cl-C5l -

M + 5(Cl-CS) = GI (Eq. 7)

1 2 3 4 6 1 2 3 4 6 0-0-0-0-0 G 0 0-0-0-0-0 G l a 0 - 0 - 0 - 0 - 0 G 1 0-0-0-0-0 G17 0 - 0 - 0 - 0 - 0 G 2 0-0-0-0-0 G I , 0 - 0 - 0 - 0 - 0 G 3 0-0-0-0-0 G 1 0

0 - 0 - 0 - 0 - 0 G 4 0-0-0-0-0 G20 0 - 0 - 0 - 0 - 0 G 6 0-0-0-0-0 G27 0 - 0 - 0 - 0 - 0 G 6 0-0-0-0-0 G22 0-0-0-0-0 G 7 0-0-0-0-0 G23 0-0-0-0-0 G 8 0-0 -0 -0 -0 G24 0-0-0-0-0 G Q 0-0-0-0-0 G26 0 - 0 - 0 - 0 - 0 G1o 0-0-0-0-0 G2,3 0-0-0-0-0 G , 1 0-0-0-0-0 G,, 0-0-0-0-0 G12 0-0-0-0-0 G 2 8 0 - 0 - 0 - 0 - 0 G13 0-0-0-0-0 G ~ Q 0-0 -0 -0 -0 G14 0-0 -0 -0 -0 G30 0-0-0-0-0 G16 0-0 -0 -0 -0 G a l

FIG. AI. Listing of all 32 theoretical glutamate isotopomers,


M + 1'c4'c5' = G3 + G4 + Gs + GII + GI2 + GI, + G I 5 + GIG

+ GZO + GZ, + G 2 3 + G24 + G25 + G26 + G30 + G31 (Eq. 22)

M + 2'c4'c5' = G1 + Gz + G5

+ G6 + G7 + G g + Glo + Gls (Eq. 23)

Analysis of fragment C2.

M"" Go + G5 + G, + Ge + G g + GIs + Gls + Gzo

+ Gzl + Gzz + Gz3 + G z ~ + G P ~ + Gz9 + G30 + Gal 0%. 24)

M + = G I + Gz + G3 + G4 + Gs + GIO + GII + GI,

+ G I 3 + GI4 + G16 + GI, + GI, + G24 + G25 + Gz, (Eq. 25)

To calculate the IE of each carbon of glutamate, one must consider that the labeling on C-4 and /or C-5 of glutamate depends on the 13C tracer used. For tracers such as [U-"C] fatty acid, [2,3-13C2]pyruvate, [U-13C3]pyruvate or [U-"~C,] acetate, onlyM + 2 acetyl-coA is formed, and glutamate is labeled on both C-4 and C-5. Then, only 16 glutamate isotopomers can theoretically be formed Go, GI, Gz, Gg, GB, G7, Gg, Glo, GI31 G17, GI8, G19, G2z, G27, G28, and G29. However, for tracers such as [l-13C]acetate, [2-13C]acetate, [l-l3C1fatty acid, [l-13C]pyruvate, [2-13C]pyruvate, or [3-13C]pyruvate, only M + 1 acetyl-coA is formed, and glutamate is labeled either on

C-4 or C-5. Then, only 24 glutamate isotopomers can theoretically be formed GO, G3, G4, Gat G11, GI27 G131 Gl4, Gl57 G16, G17, G19, GZ0, Gzl, GZ2, G23, G24, G26, G26, G27, Gza, G29, G30, and

IE of Each Curbon-The IE of each carbon of glutamate is G31.

calculated using the following equations.

When both C-4 and C-5 of glutamate are labeled IE C-1 = GI + G5 + Gs + G g + G I 3 + GI? + G z z + G27 (Eq. 26)

IE C-2 = GI + Gz + G6 + GIO

+ GI3 + G I 7 + G 1 g + Gzs (Eq. 27)

IE C-3 = G1 + Gz + G5 + G7 + GI3 + G I ~ + Gz2 + Gzg (Eq. 28)

IE C-4 = GI + Gz + G5 + '2s + G7 + G g + Glo + GIS (Eq. 29)

IE C-5 = G1 + GZ + G5 + Gs + G7 + G g + Glo + G I ~ (Eq. 30)

When either C-4 or C-5 of glutamate is labeled IE C-1 = G3 + G4 + Gs + GI1 + GI2 + GI3

+ Gib + GI7 + GZI + G z z + G 2 3 + Gz, (Eq. 31)

IE C-2 = G3 + G4 + ( 2 1 1 + GIZ + GI3 + '214

+ G I 6 + G I 7 + G I ~ + G24 + G25 + Gzs (Eq. 32)

IE C-3 = G3 + G4 + GB + GI3 + GI4 + G I 5

+ G I 6 + GI9 + Gzo + Gzz + G z 6 + G m (Eq. 33)

IE C-4 = G3 + Gs + GI1 + G16

+ Gzo + Gz3 + Gz, + G o (Eq. 34)

IE C-5 = G4 + Glz + G14 + G I 5

+ GZI + Gz5 + Gze + G I (Eq. 35)

Example-Using data obtained from a liver perfused with [2-13C]acetate (Table I), let us show the calculation of the IE of each carbon. Since only M + 1 isotopomers are detected, we simplify the above equations by omitting M + x isotopomers, where x 2 2 (Gl to G26, leaving only GO and G27 to G31).

Analysis of fragment C1 + C5. M(cl+C5) = Go = 79.7

M + l'c1"c5' = G z 7 + GB + G 2 g + G30 + GB1 = 20.3

Analysis of fragment C2 + C5. "CZ-CS) = Go + Gz, = 79.6

M + l'c2'c5' = GZs + G2, + GW + Gsl = 20.4

Analysis of fragment C2 + C4. M(c2'c4' = Go + G27 + = 79.3

M + l'c"c'' = G28 + G2, + G,o = 20.7

Analysis of fragment C1 --* C3. = Go + G30 + GB1 = 97.6

M + l ( C 1 4 3 ) = G 27 + G28 + Gzg = 2.4

Analysis of fragment C4 + C5. M(c4-c5) = Go + G2, + G28 + G2g = 82.7

M + l'c4+c5' = G30 + G,I = 17.3

Analysis of fragment C2. M"" = Go + G 2 7 + G2g + G30 + G31 = 98.8

M + = G a = 1.2


We can now calculate the IE of each carbon of glutamate, ZE C-5-From Equation 35, IE of C-5 equals Gsl which is using Equations 31-35. calculated from two sets of isotopomers.

ZE C-I-From Equation 31, IE of C-1 equals G27 which is IE G~~ = M + 1(c2-c5) - M + ~ ( c z - c ~ ) calculated from two sets of isotopomers. IE G - M + 1(CI-C5) - M + 1 ( C 2 4 5 )

27 - = (G28 + G2g + G,o + G31) - (GB + Gm + Gao) 0%. 50)

= (G27 + G z s + G a g + Gm) - (G28 + G29 + G30) (Eq. 48) = (20.4) - (20.7) = negligible \

= (20.3) - (20.4) = negligible IE G 31 - - M(c2-c4) - MICZ"c5) IE GZ7 = M(cl-.c5) - M(C2-w

= (Go + G z 7 + Gal) - (Go + Gz7) (Eq. 51) = (Go + Gz7) - Go 0%. 49)

= (79.6) - (79.7) = negligible = (79.3) - (79.6) = negligible

ZE C-2-This is measured directly from the mass spectrum ZE C-4-From Equation 34, IE of C-4 equals GW which is of y-aminobutyrate, equals 1.2 f 0.2. calculated from two sets of isotopomers.

FIG. AII. The mass spectra of glutamate-(TBDMS)s ( A ) , y-aminobutyrate- (TMS)3 ( B ) , a-hydroxyglutarate- (TMS), (C), a-hydro~y-[2-~H-]glutarate-(TMS)s (D), isocitrate-(TMS)s ( E ) , gl~tamine-(TBDMS)~ ( F ) , and glutamine-(TBDMS)4 (G).

100 150 200 250 300 350 400 450 500

50 - / 304

0 - " ' I I

1 1 I I I I I

100 150 200 250 300 350 400

100 - 129 e /

247 /

50 - 349

0 . "' " 1 '1, " ' ' , L '. ' hI / I I I

100 150 200 250 300 350 400

100 - Z \ W 130 D

248 /

50 - Z 350 - W 0 2 100

' di ' I ' ' ' I I. I . I I . / I I L

t Q

150 200 250 300 350 400

100 150 200 250 300 350 400 450 500

100 7

374 I F 50 - 271 / /

"-

0 y .L. LA I,.. _I . L . I. L. I 1 L I I I

100 '1 50 200 250 300 350 400 450 500

l%d"-J: t ' * I _ . ' I L. ' I - I LI I

545 /

0 100 150 200 250 300 350 400 450 500 550 600

4 180 Gluconeogenesis and Citric Acid Cycle in Liver

APPENDIX 11

Mass Spectra of T M S or T B D M S Derivatives of Glutamate and of Derived Compounds

Glutamate isolated from neutralized perchloric acid extracts is enzymatically or chemically converted to y-aminobutyrate, a-hydroxyglutarate, isocitrate, and glutamine (as described under Analytical Procedures). Fig. AI1 represents the mass spectra of the TMS or TBDMS derivatives of glutamate and of derived compounds analyzed by GC-MS in the SCAN mode by electron impact.

REFERENCES 1. Strisower, E. H., Kohler, G. D., and Chaikoff, I. L. (1952) J. Biol. Chem.

2. Weinman, E. O., Strisower, E. H., and Chaikoff, I. L. (1957) Physiol. Reu. 198 , 115-126

37.252-272 3. Katz,'J., and Grunnet, N. (1979) in Techniques in Metabolic Research: Part

4. Hetenyi, G., Jr. (1982) Fed. Proc. 4 1 , 104-109 5. Chance, E. M., Seeholzer, S. H., Kobayashi, K., and Williamson, J. R.

6. Katz, J. (1985) Am. J . Physiol. 248 , R391-R399 (1983) J. Bid. Chem. 2 5 8 , 13785-13794

7. Shulman, G. I., Rossetti, L., Rothman, D. L., Blair, J. B., and Smith, D. (1987) J. Clin. Inuest. 80, 387-393

8. Katz, J., Lee, W. N. P., Wals, P. A., and Bergner, E. A. (1989) J. Biol. Chem. 264 , 12994-13004

9. Lee, W. N. P., Katz, J., Sorou, S., and Bergner, E. A. (1991) Stable Isotopes

Ltd., Andover, Hampshire in Paediatric Nutritional and Metabolic Research, pp. 179-186, Intercept

I (Kornberg, H. L., ed) pp. 1-18, Elsevier/North Holland, Amsterdam

10. Kelleher, J. K. (1986) Am. J. Physiol. 2 5 0 , E29643305 11. Magnusson, I., Schumann, W. C., Bartsch, G. E., Chandramouli, V., Ku-

maran, K., Wahren, J., and Landau, B. R. (1991) J. Biol. Chem. 2 6 6 , 6975-6984

12. Cohen, S. M. (1983) J. Biol. Chem. 258 , 14294-14308 13. Jeffrey, J. M. F., Rajagopal, A., Malloy, C. R., and Sherry, A. D. (1991)

Trends Biochem. Sci. 16 . 5-10 14. Sherry, A. D., Nunnally,~R: L., and Peshock, R. M. (1985) J. Biol. Chem.

260.9272-9279 15. Tsern i K. Y . , and Jin, S. J. (1991) J. Biol. Chem. 266,2924-2929 16. Hellerstein, M. K., Wu, K., Kaempfer, S., Kletke, C., and Shackleton, C.

17. Kalhan, S. C. (1990) J. Lab. Clin. Med. 116 , 615-622 H. L. (1991) J. Clin. Inuest. 87, 1841-1852

18

19.

20.

21.

22.

23.

24.

26. 25.

28. 27.

29.

30. 31.

32.

33.

. Kalderon, B., Korman, S. H., Gutman, A., and Lapidot, A. (1986) Am. J.

, Liu, K. J. M., Henderson, T. O., Kleps, R. A,, Reyes, M. C., and Nyhus, L. M. (1990) J. Surg. Res. 49,179-185

Desage, M., Guilluy, R., Brazier, J. L., Riou, J. P., Beylot, M., Normand, S., and Vidal H. (1989) Biomed. Enuiron. Moss Spectrom. 18,1010-1015

Di Donato, L., Montgomery, J. A., Des Rosiers, C., David, F., Garneau, M., and Brunengraber, H. (1990) FASEB J. 4 ,708 (Abstr. 2560)

Di Donato, L., Des Rosiers, C., David, F., Garneau, M., and Brunengraber, H. (1992) FASEE J. 6 , 113 (Abstr. 3381)

Brunengraber, H., Boutry, M., and Lowenstein, J. M. (1973) J. Bid. Chem. 248,2656-2669

DiTullio, N. W., Berkoff, C. E., Blank, B. Kostos, V., Stack, E. J., and

Goodman, M. N. (1975) Biochem. J. 150 , 137-139 Saunders, H. L. (1974) Biochem. J. 138,'387-394

Kostos, V., DiTullio, N. W., Rush, J., Cieslinski, L., and Saunders, H. L.

Robinson, B. H., and Oei, J. (1975) FEBS Lett. 5 8 , 12-15 (1975) Arch. Biochem. Biophys. 171,459-465

Des Rosiers, C., David, F., Garneau, M., and Brunengraber, H. (1991) J. Biol. Chem. 266 , 1574-1578

Bernt, E., and Bergmeyer, H. U. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed) Vol. 4, pp. 1704-1708, Academic Press, New York

Chen, R. F., and Plaut, G. W. E. (1962) Biochemistry 2,752-756 Kingdon, H. S., Hubbard, J. S., and Stadtman, E. R. (1968) Biochemistry

Lund, E. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed)

Anderson, L. W., Zaharevitz, D. W., and Strong, J. M. (1987) Anal. Bio-

Phy~iol. 2 5 7 , E346-E353

7,2136-2142

Vol. 4, pp. 1719-1722, Academic Press, New York

chem. 163. 358-368 34. Williamson, D. H., and Mellanby, J. (1974) in Methods of Enzymatic

Analysis (Bergmeyer, H. U., ed) Vol. 3, pp. 1836-1843, Academic Press, New York

35. Gutman, I., and Wahlefeld, A. W. (1974) in Methods ofEnzymatic Analysis (Bergmeyer, H. U., ed) Vol. 4, pp. 1464-1468, Academic Press, New York

36. Bergmeyer, H. U., Bernt, E., Mollering, H., and Pfleiderer, G. (1974) in Methods of Enzymatbc Amlyscs (Bergmeyer, H. U., ed) Vol. 4, pp. 1696-

37. DesRosiers, C., Montgomery, J. A., Desrochers, S., Garneau, M., David, F., 1698, Academic Press, New York

38. Des Rosiers, C., Montgomery, J. A,, Garneau, M., David F., Mamer, 0. A., Mamer, 0. A., and Brunengraber, H. (1988) Anal. Biochem. 173,96-105

Daloze, P., Toffolo. G.. Cobelli. C.. Landau. B. R.. and Brunenmaber. H.

- . . , - - . - -

(1990) Am. J. Physiol.'258, E5191E528 ' ' - ~~~ ~

39. Koeppe, R. E., Mourkides, G. A., and Hill, R. J. (1959) J. Biol. Chem. 234 , 00-n 0000 LLIY-LLLL

40. Haussinger, D. (1983) Eur. J. Biochem. 133,269-275 41. Wieland, 0. H., Patzelt, C., and Loffler, G. (1972) Eur. J . Biochem. 2 6 ,

496-422 42. Soling, H. D., Willms, B., Friedrichs, D., and Kleineke, J. (1968) Eur. J.

43. Teufel, H., Menahan, L. A,, Shipp, J. C., Boning, S., and Wieland, 0. (1967)

44. Wieland, 0. H. (1967) Eur. J. Biochem. 2 , 182-186 45. Soling, H. D. (1965) Klin. Wochenschr. 43,914

47. Struck, E., Ashmore, J., and Wieland, 0. (1966) Adu. Enzyme Regul. 4, 46. Struck, E., Ashmore, J., and Wleland, 0. (1965) Biochem. Z. 343,107-110

"- ." Biochem. 4,364-373

Eur. J. Biochem. 2 , 182-186

219-224 48. Herrera,-G. M.. Kamm, D.. Ruderman. N.. and Cahill. G. F.. Jr. (1966) Adu.

49. Williams_on, JI R.,.Kr Enzyme Regul. 4 ,2 :

Arnd

. . !5-235 'eisberg, R. A,, and Felts, W. P. (1966) Proc. Natl.

50. Williamson, J. R., Browning, E. T., and Olson, M. S. (1968) Adu. Enzyme

51. Ross, B. D., Hems, R., Freedland, R. A., and Krebs, H. A. (1967) Biochem.

52. Teufel, H., Menahan, L. A,, Shipp, J. C., Boning, S., and Wieland, 0. (1967)

53. Williamson, J. R., Browning, E. T., and Scholz, R. (1969) J. Biol. Chem.

I . . ,

Set. U . S. A. 56,247-254

Regul. 6,67-100

J. 105,869-875

EUF. J. Blochem. 2 , 182-186

244.4607-4616 54. Krebs,'H. A., and Eggleston, L. V. (1965) Biochem. J. 9 4 , 3c-4c 55. Williamson, J. R. (1967) Adu. Enzyme Regul. 5 , 229-255 56. Ruderman, N. B., Toewes, C. J., and Shafrir, E. (1971) in Regulation of

demic Press, New York Gluconeogenesis (Soling, H. D., and Willms, B., eds) pp. 194-206, Aca-

57. Brunengraber, H., Boutry, M., and Lowenstein, J. M. (1975) Methods

58. Gavino, V. C., Somma, J., Philbert, ,L., David, F., Garneau, M., BBlair, J., Enzymol. 35,597-607

59. Prass, R. L., Isohashi, F., and Utter, M. F. (1980) J. Biol. Chem. 2 5 5 , and Brunengraber, H. (1987) J. Bbol. Chem. 262,6735-6740

60. Cr&treTL-B., Souter, M. J., and Anderson, S. E. (1989) Biochem J. 257 , 5215-5223

61. Rognstad R., and Katz J. (1972) J. Biol. Chem. 247,6047-6054 62. Katz. J. (i987) FEBS Lett. 220.257-258

b l+ti'lt4

63. Consoli, A,, Kennedy, F., Miles; J i , and Gerich, J. (1987) J . Clin. Inuest.

64. Schumann, W. C., Magnusson, I., Chandramouli, V., Kumaran, K., Wahren,

65. Haussinger, D. (1990) Bcochem. J. 267,281-290

67. Baranyai, J. M., and Blum, J. J. (1989) Biochem. J. 258,121-140 66. Srere, P. A. (1987) Annu. Reu. Biocltem. 56,89-124

80, 1303-1310

J., and Landau, B. R. (1991) J. Biol. Chem. 266,6985-6990

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