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Validation of the Use of Urinary Phenylacetyl-Glutamine to Assess the Labeling Pattern of Liver Citric Acid Cycle Intermediates in Monkeys
Copyright e IFAC Mode.lin& md Coolrd in Biomedical Systems. Galvestoo. Texas. USA. 1994
Validation of the use of urinary phenylacetylglutamine to assess the labeling pattern of liver citric acid cycle intermediates in monkeys D. Yano· , S.F. Previs·, C.A. Fernandez b , M.V. Soloviev·, F. David·, S. Duoelay· and H. Brunenoraber". Departments of Nutrition· and Biomedical Engineering b , Case Western Reserve University, Cleveland, Ohio 44106.
liver, kidney and muscle were quick-frozen for determining 13C-labeling patterns of metabolites. The amount of l3C on each carbon of urinary PAGN was determined by NMR. GC-MS of PAGN and of its deamidated product phenylacetylglutamate (PAG) yielded isotopomer distributions of 4 fragments. Their isotopomer distributions were modeled as the sum of 32 possible carbon positional isotopomers. The mass isotopomer distribution (MID) of a compound does not uniquely identify its positional isotopomer distribution (PlO). Thus, it is necessary to measure the MID of fragments of the compound and from this to reconstruct the PlO of the unfragmented molecule. This was accomplished using a model that expressed the mass isotopomer abundance of a fragment as a isotopomers of the sum of positional unfragmented molecule. Since PAGN contains 5 potentially labeled mass isotopomers (M + 0 to M + 6), there are 32 possible positional isotopomers.
Phenylacetylglutamine (PAGN) is formed in the liver of humans and primates from the condensation of glutamine with phenylacetylCoAt The latter derives from phenylacetate, a secondary metabolite of phenylalanine. PAGN's basal plasma concentration is about 1 pM; it is actively excreted in urine (1 mmol/24hr in adult humans). PAGN has been recently used (1,2) to investigate the labeling pattern of liver citric acid cycle (CAC) metabolites in humans infused with phenylacetate and l4C-substrates (the ·noninvasive chemical biopsy of the human liver·). This technique assumes that the labeling pattern of PAGN reflects that of liver a-ketoglutarate (aKG), a CAC intermediate. Therefore the labeling pattern of PAGN yields important information on the regulation of the CAC and of gluconeogenesis: ratio of activities pyruvate dehydrogenase/pyruvate carboxylase, extent of randomization of label via the reversal of CAC reactions (from oxaloacetate to fumarate), rate of cycling between pyruvate, oxaloacetate and PEP.
To calculate the abundance of these 32 positional isotopomers, we assumed that labeled aKG is made only from the condensation of a molecule of acetyl-CoA and one of OAA. The infused 13C-substrates labeled either OAA or acetyl-CoA or both before aKG formation. Since OAA contains 4 potentially labeled carbons, there are 16 possible positional isotopomers. In addition, since C-1 of OAA is lost at isocitrate dehydrogenase, OAA effectively contributes only three carbons to aKG. There are 2 3 = 8 possible positional isotopomers for this three-carbon moiety. The two carbons of AcCoA label C-4 and C-5 of aKG. There are 22 = 4 possible positional isotopomers for the AcCoA moiety of aKG. Thus, from the isotopomer profiles of OAA and AcCoA, one can calculate the abundances of the 32 positional isotopomers of aKG as products of the abundances of the appropriate isotopomers of OAA and AcCoA. Therefore, the relative abundances of the isotopomers are dependent on one another because of the constraints imposed by the metabolic pathway.
The sensitivity of GC-MS techniques allows measuring the concentration and total l3C-labeling of physiological concentrations of PAGN in plasma. The technique of ·chemical biopsy· could therefore be used in humans infused with l3C-substrates, without having to infuse phenylacetate. To validate the PAGN chemical biopsy, one needs to compare directly the labeling pattern of plasma and urine PAGN with those of liver CAC intermediates and related compounds (citrate, aKG, glutamate, glutamine and PEP). We infused anesthetized rhesus monkeys with various tracers: [2- 13 C]acetate, [1 ,2- 13 C2]acetate, l3 13 [3- C]lactate, [U- C3]1actate, [U- 13C3]glycerol, [3- l3C]KIC or NaH 13C0 3. Blood and urine were collected every 30 min. After 5 hr, samples of
The GC-MS fragments whose abundances were used in the computations were (i) ions 265 and
417
13C on each of the 5 carbons of the glutamine moiety of PAGN. These relative distributions of label derived from GC-MS and NMR data were used to calculate the parameters of Magnusson et ai's model (1).
291 of PAGN as a trimethylsilyl (TMS) derivative, and (ii) ions 234 and 394 of PAG-TMS. For PAGN, ion 265 contains C-1 +2 and ion 291 contains C-2+3+4+5. For PAG, ion 234 contains C-2 + 3 and ion 394 contains C-1 + 2 + 3 + 4 + 5. Since none of these fragments was formed via breakage between C-4 and C-5, we were unable to distinguish between the 2 possible single labeling on C-4 + 5 (ie the acetylCoA moiety) of PAGN. Therefore isotopomers with 13C on either C-4 or C-5 were grouped together, thus decreasing the total number of . distinguishable positional isotopomers from 32 to 24. Nine parameters are needed to calculate the PlO of aKG: (i) seven for the three-carbon moiety of OAA yielding C-1 + 2 + 3 of aKG (the sum of these is constrained to be between zero and 1), (ii) one for the sum of the two M + 1 positional isotopomer abundances of the AcCoA moiety, and (iii) one for the M + 2 positional isotopomer abundance. The sum of parameters under (ii) and (iii) was constrained to fall between 0 and 1. The unlabeled positional isotopomer abundance for the AcCoA moiety was obtained as 1 minus the sum of the M + 1 and M + 2 isotopomers. Thus, the abundance of a given positional isotopomer for carbons 1 through 5 is the product of the abundances of the corresponding positional isotopomer for C-1 + 2 + 3 and C-4 + 5.
Reaction
Rates (GC-MS)
Rates (NMR)
V1(PDH)
0.202
0.229
V2(FA -> AcCoA)
0.798
0.771
V3(CS)
1.000
1.000
V4(FUM - >OAA)
8.53
9.52
V5(OM -- > FUM)
7.53
8.52
V6(PEPCK)
3.10
2.64
V7(PK)
0.905
1.47
V8(LACT -> PYR)
2.40
1.40
V9(PEP -> GLUC)
2.20
1.17
The relative rates calculated from the GC-MS and NMR data were well correlated by linear regression (slope = 1.13, r2 = 0.97) Our data show that the isotopomer distribution of PAGN and some of its fragments, measured by GC-MS, allows to calculate the distribution of 13C on each of the carbons of the glutamine moiety of PAGN. The data are similar to those obtained by NMR, but require much smaller amounts of substrate for the analysis.
The system is non-linear since the output is calculated via products of parameters. The algorithm used to obtain the best least squares fit solution was implemented in Microsoft Fortran 5.1 for DOS and utilized a reduced gradient method for estimation of linear and non-linear systems. The data from 13 experiments with monkeys infused with various 13C-substrates was used as the input to the parameter estimation algorithm.
(Supported by grants from the NIH (OK4553) and the Cleveland Mt. Sinai Medical Center) References 1. Magnusson, I, Schumann, WC, Bartsh, GE Chandramouli, V, Kumaran, K, Wahren, . J, Landau, B.(1991) J. Bioi. Chem. 266, 6975
As an example, the distribution of isotopomers in PAGN isolated from the urine of a monkey infused with [3- 13CJlactate was used to calculate the relative distribution of molecules containing
2.Esenmo, E, Chandramouli, V, Schumann WC, Kumaran, K., Wahren, J . and Landau, B.R. (1992) Am. J. Physiol. 263, E36-E41.
418
Validation of the use of urinary phenylacetylglutamine to assess the labeling pattern of liver citric acid cycle intermediates in monkeys D. Yano· , S.F. Previs·, C.A. Fernandez b , M.V. Soloviev·, F. David·, S. Duoelay· and H. Brunenoraber". Departments of Nutrition· and Biomedical Engineering b , Case Western Reserve University, Cleveland, Ohio 44106.
liver, kidney and muscle were quick-frozen for determining 13C-labeling patterns of metabolites. The amount of l3C on each carbon of urinary PAGN was determined by NMR. GC-MS of PAGN and of its deamidated product phenylacetylglutamate (PAG) yielded isotopomer distributions of 4 fragments. Their isotopomer distributions were modeled as the sum of 32 possible carbon positional isotopomers. The mass isotopomer distribution (MID) of a compound does not uniquely identify its positional isotopomer distribution (PlO). Thus, it is necessary to measure the MID of fragments of the compound and from this to reconstruct the PlO of the unfragmented molecule. This was accomplished using a model that expressed the mass isotopomer abundance of a fragment as a isotopomers of the sum of positional unfragmented molecule. Since PAGN contains 5 potentially labeled mass isotopomers (M + 0 to M + 6), there are 32 possible positional isotopomers.
Phenylacetylglutamine (PAGN) is formed in the liver of humans and primates from the condensation of glutamine with phenylacetylCoAt The latter derives from phenylacetate, a secondary metabolite of phenylalanine. PAGN's basal plasma concentration is about 1 pM; it is actively excreted in urine (1 mmol/24hr in adult humans). PAGN has been recently used (1,2) to investigate the labeling pattern of liver citric acid cycle (CAC) metabolites in humans infused with phenylacetate and l4C-substrates (the ·noninvasive chemical biopsy of the human liver·). This technique assumes that the labeling pattern of PAGN reflects that of liver a-ketoglutarate (aKG), a CAC intermediate. Therefore the labeling pattern of PAGN yields important information on the regulation of the CAC and of gluconeogenesis: ratio of activities pyruvate dehydrogenase/pyruvate carboxylase, extent of randomization of label via the reversal of CAC reactions (from oxaloacetate to fumarate), rate of cycling between pyruvate, oxaloacetate and PEP.
To calculate the abundance of these 32 positional isotopomers, we assumed that labeled aKG is made only from the condensation of a molecule of acetyl-CoA and one of OAA. The infused 13C-substrates labeled either OAA or acetyl-CoA or both before aKG formation. Since OAA contains 4 potentially labeled carbons, there are 16 possible positional isotopomers. In addition, since C-1 of OAA is lost at isocitrate dehydrogenase, OAA effectively contributes only three carbons to aKG. There are 2 3 = 8 possible positional isotopomers for this three-carbon moiety. The two carbons of AcCoA label C-4 and C-5 of aKG. There are 22 = 4 possible positional isotopomers for the AcCoA moiety of aKG. Thus, from the isotopomer profiles of OAA and AcCoA, one can calculate the abundances of the 32 positional isotopomers of aKG as products of the abundances of the appropriate isotopomers of OAA and AcCoA. Therefore, the relative abundances of the isotopomers are dependent on one another because of the constraints imposed by the metabolic pathway.
The sensitivity of GC-MS techniques allows measuring the concentration and total l3C-labeling of physiological concentrations of PAGN in plasma. The technique of ·chemical biopsy· could therefore be used in humans infused with l3C-substrates, without having to infuse phenylacetate. To validate the PAGN chemical biopsy, one needs to compare directly the labeling pattern of plasma and urine PAGN with those of liver CAC intermediates and related compounds (citrate, aKG, glutamate, glutamine and PEP). We infused anesthetized rhesus monkeys with various tracers: [2- 13 C]acetate, [1 ,2- 13 C2]acetate, l3 13 [3- C]lactate, [U- C3]1actate, [U- 13C3]glycerol, [3- l3C]KIC or NaH 13C0 3. Blood and urine were collected every 30 min. After 5 hr, samples of
The GC-MS fragments whose abundances were used in the computations were (i) ions 265 and
417
13C on each of the 5 carbons of the glutamine moiety of PAGN. These relative distributions of label derived from GC-MS and NMR data were used to calculate the parameters of Magnusson et ai's model (1).
291 of PAGN as a trimethylsilyl (TMS) derivative, and (ii) ions 234 and 394 of PAG-TMS. For PAGN, ion 265 contains C-1 +2 and ion 291 contains C-2+3+4+5. For PAG, ion 234 contains C-2 + 3 and ion 394 contains C-1 + 2 + 3 + 4 + 5. Since none of these fragments was formed via breakage between C-4 and C-5, we were unable to distinguish between the 2 possible single labeling on C-4 + 5 (ie the acetylCoA moiety) of PAGN. Therefore isotopomers with 13C on either C-4 or C-5 were grouped together, thus decreasing the total number of . distinguishable positional isotopomers from 32 to 24. Nine parameters are needed to calculate the PlO of aKG: (i) seven for the three-carbon moiety of OAA yielding C-1 + 2 + 3 of aKG (the sum of these is constrained to be between zero and 1), (ii) one for the sum of the two M + 1 positional isotopomer abundances of the AcCoA moiety, and (iii) one for the M + 2 positional isotopomer abundance. The sum of parameters under (ii) and (iii) was constrained to fall between 0 and 1. The unlabeled positional isotopomer abundance for the AcCoA moiety was obtained as 1 minus the sum of the M + 1 and M + 2 isotopomers. Thus, the abundance of a given positional isotopomer for carbons 1 through 5 is the product of the abundances of the corresponding positional isotopomer for C-1 + 2 + 3 and C-4 + 5.
Reaction
Rates (GC-MS)
Rates (NMR)
V1(PDH)
0.202
0.229
V2(FA -> AcCoA)
0.798
0.771
V3(CS)
1.000
1.000
V4(FUM - >OAA)
8.53
9.52
V5(OM -- > FUM)
7.53
8.52
V6(PEPCK)
3.10
2.64
V7(PK)
0.905
1.47
V8(LACT -> PYR)
2.40
1.40
V9(PEP -> GLUC)
2.20
1.17
The relative rates calculated from the GC-MS and NMR data were well correlated by linear regression (slope = 1.13, r2 = 0.97) Our data show that the isotopomer distribution of PAGN and some of its fragments, measured by GC-MS, allows to calculate the distribution of 13C on each of the carbons of the glutamine moiety of PAGN. The data are similar to those obtained by NMR, but require much smaller amounts of substrate for the analysis.
The system is non-linear since the output is calculated via products of parameters. The algorithm used to obtain the best least squares fit solution was implemented in Microsoft Fortran 5.1 for DOS and utilized a reduced gradient method for estimation of linear and non-linear systems. The data from 13 experiments with monkeys infused with various 13C-substrates was used as the input to the parameter estimation algorithm.
(Supported by grants from the NIH (OK4553) and the Cleveland Mt. Sinai Medical Center) References 1. Magnusson, I, Schumann, WC, Bartsh, GE Chandramouli, V, Kumaran, K, Wahren, . J, Landau, B.(1991) J. Bioi. Chem. 266, 6975
As an example, the distribution of isotopomers in PAGN isolated from the urine of a monkey infused with [3- 13CJlactate was used to calculate the relative distribution of molecules containing
2.Esenmo, E, Chandramouli, V, Schumann WC, Kumaran, K., Wahren, J . and Landau, B.R. (1992) Am. J. Physiol. 263, E36-E41.
418