Identification of phenylbutyrylglutamine, a new metabolite of phenylbutyrate metabolism in humans

Article abstract of DOI:10.1002/jms.316

Phenylbutyrate is used in humans for treating inborn errors of ureagenesis, certain forms of cancer, cystic fibrosis and thalassemia. The known metabolism of phenylbutyrate leads to phenylacetylglutamine, which is excreted in urine. We have identified phenylbutyrylglutamine as a new metabolite of phenylbutyrate in human plasma and urine. We describe the synthesis of phenylbutyrylglutamine and its assay by gas chromatography/mass spectrometry as a tert-butyldimethylsilyl or methyl derivative, using standards of [²H₅]phenylbutyrylglutamine and phenylpropionylglutamine. After administration of phenylbutyrate to normal humans, the cumulative urinary excretion of phenylacetate, phenylbutyrate, phenylacetylglutamine and phenylbutyrylglutamine amounts to about half of the dose of phenylbutyrate. Thus, additional metabolites of phenylbutyrate are yet to be identified. Copyright

Full text of DOI:10.1002/jms.316

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2002; 37: 581–590
Published online 7 May 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.316
Identification of phenylbutyrylglutamine, a new
metabolite of phenylbutyrate metabolism in humans
Blandine Comte,1 Takhar Kasumov,1 Bradley A. Pierce,1 Michelle A. Puchowicz,1
Mary-Ellen Scott,1 Williams Dahms,2 Douglas Kerr,2 Itzhak Nissim3 and
Henri Brunengraber1∗
1
Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106, USA
Department of Pediatrics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA
Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
2
3
Received 26 December 2001; Accepted 1 March 2002
Phenylbutyrate is used in humans for treating inborn errors of ureagenesis, certain forms of cancer, cystic
fibrosis and thalassemia. The known metabolism of phenylbutyrate leads to phenylacetylglutamine,
which is excreted in urine. We have identified phenylbutyrylglutamine as a new metabolite of
phenylbutyrate in human plasma and urine. We describe the synthesis of phenylbutyrylglutamine
and its assay by gas chromatography/mass spectrometry as a tert-butyldimethylsilyl or methyl derivative,
using standards of [2H5]phenylbutyrylglutamine and phenylpropionylglutamine. After administration of
phenylbutyrate to normal humans, the cumulative urinary excretion of phenylacetate, phenylbutyrate,
phenylacetylglutamine and phenylbutyrylglutamine amounts to about half of the dose of phenylbutyrate.
Thus, additional metabolites of phenylbutyrate are yet to be identified. Copyright  2002 John Wiley &
Sons, Ltd.
KEYWORDS: phenylacetate; phenylbutyrate; phenylacetylglutamine; phenylbutyrylglutamine; liver conjugation
which could either undergo ˇ-oxidation to PA-CoA (the
precursor of PAGN), or be conjugated with glutamine to
form a new compound, phenybutyrylglutamine (PBGN).
The latter would be excreted in urine with PAGN.
We synthesized unlabeled and 2H-labeled PBGN, and
present sensitive methods for its determination in biological
fluids by GC/MS. Further, we assayed PB and its metabolites
in plasma and urine from seven humans after an oral bolus
of Na-PB. We demonstrated the formation and urinary
excretion of PBGN.
INTRODUCTION
Phenylbutyrate (PB) has been used as a pro-drug of
phenylacetate (PA) in the treatment of hyperammonemia
related to inborn errors of urea synthesis1 and as a drug
in the treatment of a number of malignancies,2–4 cystic
fibrosis5 and sickle cell anemia.6 The main fate of PA in
primates and humans is its conjugation as phenylacetyl-CoA
with glutamine in the liver to form phenylacetylglutamine
(PAGN), which is excreted in urine.7–9 The labeling pattern
of PAGN has been used for the non-invasive probing of the
labeling pattern of citric acid cycle intermediates in human
and primate liver.10–14 Compared with PA, PB has a more
acceptable taste and smell and is less toxic.2 However, after
administration of PB, the combined urinary excretion of PB,
PA and PAGN is less than half of the ingested amount
of PB.15 We hypothesized that by analogy with PA, PB
is activated in the liver to phenylbutyryl-CoA (PB-CoA),
EXPERIMENTAL
Materials
Chemicals and solvents were obtained from Sigma-
Aldrich Chemicals (Milwaukee, WI, USA). [2H7]Phenylacetic
acid (99%) and [2H6]benzene were purchased from Isotec
(Miamisburg, OH, USA). The derivatization agents N-
methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MtB-
STFA) and Methyl-8 (dimethylformamide dimethyl acetal)
were supplied by Regis Chemical (Morton Grove, IL, USA)
and Pierce (Rockford, IL, USA), respectively. All aqueous
solutions were made with water purified with a Milli-Q
system (Millipore).
ŁCorrespondence to: Henri Brunengraber, Department of
Nutrition, Case Western Reserve University, Cleveland, Ohio
44106-7139, USA. E-mail: hxb8@po.cwru.edu
Contract/grant sponsor: NIH.
Contract/grant sponsor: Cleveland Mt Sinai Health Care
Foundation.
Abbreviations: EI, electron ionization; GC/MS, gas
chromatography/mass spectrometry; PA, phenylacetate; PAGN,
phenylacetylglutamine; PAG, phenylacetylglutamate; PB,
4-phenylbutyrate; PBGN, 4-phenylbutyrylglutamine; PBG,
4-phenylbutyrylglutamate; PCI, positive chemical ionization; PP,
3-phenylpropionate; PPG, 3-phenylpropionylglutamate; PPGN,
3-phenylpropionylglutamine; TBDMS, tert-butyldimethylsilyl.
Preparation of unlabeled and deuterated standards
(Table 1)
PAGN and [2H7]PAGN were synthesized16 by reacting
unlabeled or [2H7]phenylacetyl chloride with glutamine, as
Copyright  2002 John Wiley & Sons, Ltd.

582
B. Comte et al.
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

Identification of phenylbutyrylglutamine
583
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

584
B. Comte et al.
described previously. Other compounds, listed in Table 1,
were prepared as follows. [2H5]PB was prepared by alu-
minum chloride-catalyzed condensation of (ꢀ-butyrolactone
with a twofold excess of [2H6]benzene.17 The yield was
94% based on 0.125 mol of (ꢀ-butyrolactone. The mass iso-
topomer distribution of [2H5]PB was 67% M5, 27% M4, and
5.4% M3 (the mass isotopomer distribution of [2H6]benzene
used for the synthesis was 88%M6, 11% M5, and 0.8%
M4). PBGN, [2H5]PBGN and 3-phenylpropionylglutamine
(PPGN) were synthesized by conjugation of glutamine with
4-phenylbutyryl chloride, 4-[2H5]phenylbutyryl chloride and
3-phenylpropionyl chloride, respectively. Unlabeled stan-
dards of glutamate conjugates (formed during the analytical
processing of the glutamine conjugates), i.e. phenylacetyl-
glutamate (PAG), 3-phenylpropionylglutamate (PPG) and
4-phenylbutyrylglutamate (PBG), were prepared by reacting
the corresponding acid chlorides with glutamate. Note that
other procedures have been described for the syntheses of
PBGN, PBG, and PPG.18–20 Except for commercial pheny-
lacetyl chloride, the unlabeled and labeled acid chlorides
used in the above syntheses were prepared by reacting the
acids with freshly distilled dichloromethyl methyl ether and
distilled under vacuum. The yields of [2H5]phenylbutyryl
chloride and [2H5]phenylbutyrylglutamine synthesis were
98% and 45%, respectively.
For the synthesis of N-(4-phenylbutyryl)pyroglutamic
acid and its methyl ester, procedures described for the
phenylacetyl analogs were adapted.21 Pyroglutamic acid
was converted to its ditrimethylsilyl derivative, which
was reacted with 4-phenylbutyryl chloride. Removal
of the carboxyl trimethylsilyl group yielded N-(4-
phenylbutyryl)pyroglutamic acid. For the synthesis of
the methyl ester of N-(4-phenylbutyryl)pyroglutamic acid,
pyroglutamic acid was converted to its methyl ester, which
was activated by NaH before reaction with 4-phenylbutyryl
chloride.22 Dimethyl N-(4-phenylbutyryl)glutamate was
prepared by reacting 4-phenylbutyryl chloride with
commercial L-glutamate dimethyl ester hydrochloride.
sulfosalicylic acid and 50 µl of 6 M HCl, the slurries were
saturated with NaCl and extracted three times with 5 ml of
ethyl acetate. The extracts were pooled, dried with Na2SO4,
evaporated and the residues were reacted with 70 µl of
°
Methyl-8 and incubated at 60 C for 20 min.
For the assay of PAGN and PBGN in urine, we tested two
different methods. In the first assay, samples (0.2 ml) were
adjusted at pH 12 with 1 M NaOH, spiked with 0.5 µmol
of [2H7]PAGN and 0.25 µmol of [2H5]PBGN and incubated
°
at 75 C for 4 h to convert PAGN and PBGN to their respective
glutamate conjugates (PAG and PBG).12 Then, the samples
were acidified with 50 µl of 6 M HCl, saturated with NaCl
and extracted three times with 5 ml of ethyl acetate. As
previously described,23 the extracts were dried with Na2SO4
°
and reacted with 70 µl of MtBSTFA at 60 C for 20 min and
analyzed as their TBDMS derivatives. In the second assay,
samples (0.2 ml) were spiked with 1 µmol of PPGN and
treated as for the first assay, except that PAG and PBG were
derivatized with Methyl-8.
GC/MS methods
All the metabolites were analyzed as their TBDMS or methyl
derivatives on a Hewlett-Packard Model 5890 gas chro-
matograph equipped with an HP-5 capillary column (30 mð
0.2 mm i.d., 0.5 mm film thickness; Hewlett-Packard) and
coupled to a Model 5989A mass-selective detector. Sam-
ples (0.2–1 µl) were injected with a splitting ratio of 20–50 : 1.
The carrier gas was helium (1 ml minꢀ1) and the column
head pressure was 32 kPa. The injector port temperature
°
°
was at 250 C, transfer line at 305 C, source temperature
°
°
at 230 C and quadrupole at 150 C. For the analysis of
the TBDMS derivatives, the column temperature program
ꢀ1
°
°
was initial temperature 150 C, increased at 10 C min to
240 C, held for 1 min at 240 C, increased at 30 C min to
ꢀ1
°
°
°
°
°
305 C and held for 18 min at 305 C. After automatic cali-
bration, the mass spectrometer was operated in the electron
ionization (EI) mode (70 eV). Appropriate ion sets were
monitored with a dwell time of 25–45 ms per ion, at
m/z 193/200 (PA/[2H7]PA), 221/226 (PB/[2H5]PB), 304/311
(PAG/[2H7]PAG cyclic form), 436/443 (PAG/[2H7]PAG lin-
ear form), 332/337 (PBG/[2H5]PBG cyclic form) and 464/469
(PBG/[2H5]PBG linear form).
Sample preparation
For the determination of the concentrations of the free
acids (PA and PB) in blood, plasma samples (0.5 ml)
were spiked with 0.17 µmol of [2H7]PA, [2H5]PB and 3-
phenylpropionate (PP) before deproteinization with 25 µl
of saturated sulfosalicylic acid. The slurries were saturated
with NaCl, acidified with one drop of 6 M HCl and extracted
three times with 5 ml of diethyl ether. For the assays in urine,
0.2 ml samples were spiked with 0.68 µmol of [2H7]PA and
0.16 µmol [2H5]PB, acidified to pH 3 with HCl, saturated with
NaCl and extracted three times with 3 ml of diethyl ether. The
combined extracts were dried with Na2SO4 and evaporated
before reacting the residues with 70 µl of MtBSTFA or
For the analysis of the methylated derivatives, the column
°
program was slightly modified to initial temperature 90 C
ꢀ1
ꢀ1
°
°
held for 1 min, increased at 15 C min to 190 C, then at
ꢀ1
°
°
°
°
5 C min to 225 C, then at 25 C min to 305 C and held
°
°
for 6 min at 305 C. The injector port was at 280 C, transfer
°
°
line at 305 C, source temperature at 200 C and quadrupole at
°
100 C. The mass spectrometer was operated in the positive
chemical ionization mode (CI, ammonia, 133 eV) with the
appropriate selected ions at m/z 168/175 (PA/[2H7]PA), 182
(PP), 196/201 (PB/[2H5]PB), 279/311 (PAG cyclic and linear
form, respectively), 293/325 (PPG cyclic and linear) and
307/339 (PBG cyclic and linear forms). All samples were
injected two or three times.
°
Methyl-8 at 60 C for 20 min. PA and PB were analyzed
as their TBDMS or methyl derivatives.
For the assay of the glutamine conjugates (PAGN
and PBGN) in plasma, samples (0.5 ml) were spiked with
0.8 µmol of PPGN, alkalinized to pH 12 and incubated at
Clinical investigation
The protocol was reviewed and approved by the Institutional
Review Board of Case Western Reserve University and Uni-
versity Hospitals of Cleveland. All subjects were free from
°
75 C for 4 h to convert the glutamine conjugates to the
glutamate derivatives. After the addition of 25 µl of saturated
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

Identification of phenylbutyrylglutamine
585
any chronic or acute illness. Women had a negative preg-
nancy test and were not breastfeeding. Seven subjects (three
men, four women; age 31.7 š 5.0 years; height 171.3 š 3.4 cm;
weight 79.5 š 5.9 kg) received detailed information on the
purpose of the investigation and signed an informed consent
form. After an overnight fast, the subjects were admitted
to the Clinical Research Center at 7.30 a.m. They remained
fasting until completion of the study. An intravenous line
derivatives, i.e. a di-TBDMS, a tri-TBDMS and a dimethyl
derivative (Table 2, row 3, lines 2 and 3). Similar cyclic and
open derivatives were obtained starting from [2H5]PBGN
and [2H5]PBG (the latter derived from mild hydrolysis of the
former; see Table 2, rows 2 and 4).
To confirm further the identity of the cyclic
derivatives of PBGN and PBG, we reacted the N-(4-
phenylbutyryl)pyroglutamic acid that we had synthesized
(Table 1, row 4) with MtBSTFA and with Methyl-8 and
obtained the same cyclic derivative (Table 2, compare row 5
with rows 1 and 3). In addition, we prepared the methyl ester
of N-(4-phenylbutyryl)pyroglutamic acid (Table 1, row 5),
which after injection without further processing into the
GC/MS system yielded the same spectrum as PBGN, PBG
and N-(4-phenylbutyryl)pyroglutamic acid derivatized with
Methyl-8 (Table 2, compare row 6 with rows 1, 3 and
5). Lastly, to confirm the identity of the linear dimethyl
derivative obtained by reacting PBG with Methyl-8, we
prepared dimethyl N-(4-phenylbutyryl)glutamate (Table 1,
row 6), which after injection without further processing into
the GC/MS system yielded the same spectrum as PBG
derivatized with Methyl-8 (Table 2, compare row 7 with
line 2 of row 3 in the methyl derivative column).
was installed in the forearm with a saline infusion (20 ml hꢀ1
)
and a short blood sampling catheter was inserted in a super-
ficial vein of the contra-lateral hand. The hand was placed
°
in a heating box at 60 C for sampling of arterialized venous
blood. At 8.00 a.m, after baseline blood and urine sam-
pling, each subject ingested 0.36 mmol kgꢀ1 (5 g per 75 kg)
of Na-PB. This dose corresponds to 15–25% of the dose com-
monly used in the treatment of patients with inborn errors
of urea synthesis (0.2–0.4 g kgꢀ1 per day). Water intake was
adjusted to induce a diuresis of at least 100 ml in 30 min.
Heparinized blood (10 ml) and urine samples were collected
at 30 min intervals for the first 3 h and then every hour
until 8 h. Plasma and urine samples were quickly frozen and
°
stored at ꢀ80 C until analysis.
The combination of information obtained from the
derivatives listed in Table 2, rows 1–11, confirms the identity
of the ions used to assay the concentration of PBGN in
biological fluids.
Table 2 (rows 10 and 11) also presents the derivatives
used to assay the concentration of phenylacetylglutamine
using [2H7]PAGN as internal standard. Mild hydrolysis of
the analyte and internal standard yielded the corresponding
unlabeled and deuterated PAG.
In addition to the use of deuterated internal standards,
we used unlabeled PPGN to compute the concentrations of
PAGN and PBGN. Mild hydrolysis of PPGN yielded PPG
which, after reaction with MtBSTFA and with Methyl-8,
yielded the derivatives listed in Table 2, row 13. Figure 1(A)
shows the ion chromatogram of a mixture of cyclical
methylated derivatives of PAG, PPG and PBG resulting
from the hydrolysis of the corresponding standards of
glutamine conjugates before derivatization with Methyl-8.
Figure 1(B) and (C) show similar ion chromatograms of
identical derivatives formed by treatment of the urine from a
subject who had ingested phenylbutyrate. The urine sample
was spiked with a standard of phenylpropionylglutamine.
The calibration graphs were linear from 10 to 700 nmol for
both derivatives (r2 D 0.99).
RESULTS
Synthesis and assay of PBGN
To test our hypothesis that PBGN is formed in human
subjects who have ingested PB, we synthesized unlabeled
and [2H5]PBGN (see above). PBGN is a white, crystalline
solid, with a melting-point of 126–127 C (103–104 C for
PAGN). The structure of PBGN and [2H5]PBGN were
confirmed by NMR spectroscopy (1H and 13C, Table 1, rows 1
and 2).
°
°
When planning our analytical strategy, we took into
account that PBGN would probably have characteristics
similar to those of the previously known PAGN. The lat-
ter can be isolated from biological fluids either (i) as
such by ion-exchange chromatography or (ii) more con-
veniently by solvent acid extraction following mild alka-
line hydrolysis to PAG. This is why we also synthesized
PBG (Table 1, row 3). In addition, since methyl deriva-
tization of PAG yields the methyl derivative of cyclic
N-(phenylacetyl)pyroglutamic acid, and also the open-
chain dimethyl N-(phenylacetyl)glutamate, we synthesized
(Table 1, rows 4–6) N-(4-phenylbutyryl)pyroglutamic acid,
methyl N-(4-phenylbutyryl)pyroglutamate and dimethyl N-
(4-phenylbutyryl)glutamate. Lastly, we synthesized PPGN
(Table 1, row 7) to serve as an analytical standard in addition
to [2H7]PAGN and [2H5]PBGN.
When PBGN was reacted with MtBSTFA or Methyl-
8, each reagent yielded one derivative which, by analogy
with PAGN, was tentatively identified as the TBDMS and
methyl derivative, respectively, of the cyclic compound N-
(4-phenylbutyryl)pyroglutamic acid (Table 1, row 4; Table 2,
row 1). The formation of the cyclic compound was confirmed
by reacting PBG with the same reagents, leading to the
formation of the same cyclic derivatives (Table 2, compare
row 1 with the first line of row 3). However, reaction of
PBG with MtBSTFA or Methyl-8 also yielded non-cyclic
PA and PB were assayed using deuterated analogs and
PP as standards. The derivatives are listed in Table 2, rows
14–18. The calibration graphs of PA and PB concentrations
were linear in the range 0.4–500 nmol. Assays of methylated
PA and PB in 84 samples of plasma yielded identical data
when computed using the deuterated internal standards
versus the PP standard. The correlation slopes were 1.06 and
1.05 with r2 D 0.99 and 0.97 for PB and PA, respectively.
Clinical investigation
In seven normal adults, the baseline plasma concentrations
of PA (0.09 š 0.07 (SE) mM) and PAGN (0.6 š 0.6 µM) were
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

586
B. Comte et al.
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

Identification of phenylbutyrylglutamine
587
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

588
B. Comte et al.
8
7
6
5
4
3
2
1
0
A
PAG, m/z 279
PBG, m/z 307
PPG, m/z 293
13.0
13.5
14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
8
7
6
5
4
3
2
1
0
B
C
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
13.0
13.5
14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
Time (min)
Figure 1. (A) Ion chromatogram of a mixture of phenylacetylglutamate (PAG), phenylpropionylglutamate (PPG) and
phenylbutyrylglutamate (PBG) resulting from the hydrolysis of the corresponding standards of glutamine conjugates before
derivatization with Methyl-8. (B) and (C) similar ion chromatograms of derivatives formed by treatment of the urine from a subject
who had ingested phenylbutyrate. The urine sample was spiked with a standard of phenylpropionylglutamine.
1.8
PA
PB
PAGN
PBGN
1.5
1.2
0.9
0.6
0.3
0.0
0
100
200
300
400
500
Time (min)
Figure 2. Profile over 8 h of phenylacetate (PA), phenylbutyrate (PB), phenylacetylglutamine (PAGN) and phenylbutyrylglutamine
(PBGN) concentrations in human plasma (mean š SE, n D 7) after an oral ingestion of 0.36 mmol kgꢀ1 of Na-PB.
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

Identification of phenylbutyrylglutamine
589
20
10
0
A
B
80
60
40
20
0
240
300
360
420
480
-60
0
30 60 90 120 150 180
Time (min)
Figure 3. Profile over 8 h of the urinary excretion of phenylacetate (PA) and phenylbutyrate (PB) in the same patients as in Fig. 2.
2.5
A
2
1.5
1
0.5
0
2.5
B
2
1.5
1
0.5
0
-60
0
30 60 90 120 150 180
240
300
360
420
480
Time (min)
Figure 4. Profile over 8 h of the urinary excretion of phenylacetylglutamine (PAGN) and phenylbutyrylglutamine (PBGN) in the same
patients as in Fig. 2.
similar to previously published data.23 PB and PBGN were
not detected in basal plasma. Figure 2 shows the profiles
of PA, PB, PAGN and PBGN concentrations in plasma
after an oral bolus of Na-PB. The concentrations of PB and
PBGN peaked at 50–90 min and those of PA and PAGN at
150–180 min. The ratios of the areas under the curve were
2.3 š 0.4 for PB/PA and 2.3 š 0.7 for PBGN/PAGN.
Figure 3 shows the profiles of the urinary excretion of
PB and PA. Eight hours following the ingestion of PB,
the cumulative excretions of PB (278 š 61 µmol) and PA
(79 š 12 µmol) amounted to 0.97 š 0.23 and 0.26 š 0.06% of
the dose, respectively.
By then, excretion of PBGN was terminated, whereas
excretion of PAGN was still ongoing, albeit at a low rate.
The total recovery of the ingested dose of phenylbutyrate
as identified urinary compounds (PACPBCPAGNCPBGN)
was 53.4 š 4.5% after 8 h.
DISCUSSION
Methodological considerations
Isotope dilution mass spectrometric assays are often made
difficult by the non-availability and/or the high cost of
internal standards labeled with stable isotopes (2H or 13C). An
alternate strategy is to use a chemical analog of the analyte,
preferably commercially available or easy to synthesize at a
low cost. We were concerned that few laboratories would
consider synthesizing [2H7]PAGN and [2H5]PBGN as part of
Figure 4 shows the profile of urinary excretion of PBGN
and PAGN. The cumulative excretions of PB metabolites over
8 h are presented in Table 3. The excretions of PBGN and
PAGN amounted to 21.5 and 32.6% of the dose, respectively.
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590

590
B. Comte et al.
Table 3. Recovery of PB and its metabolites in
communication, 1999). This is why we postulated the
formation of PBGN, an analog of PAGN. The data from
our clinical investigation clearly demonstrate that PBGN is
formed and excreted by normal humans who have ingested
PB. A substantial amount (21.5 š 2.4%) of the Na-PB load
was converted to PBGN. However, the cumulative excretions
of PA, PB, PAGN and PBGN still account for only about half
of the amount of PB ingested. Therefore, part of the ingested
PB is converted to metabolite(s) which have not yet been
identified.
human urine (n D 7) after the oral ingestion of
0.36 mmol kgꢀ1 of Na-PB
Metabolite
Amount (mmol)
Percentage
PA
PB
PAGN
PBGN
Total
0.079 š 0.012
0.278 š 0.061
9.63 š 0.74
0.26 š 0.06
0.97 š 0.23
32.6 š 1.9
21.5 š 2.4
53.4 š 4.5
6.39 š 0.50
16.33 š 1.32
Acknowledgements
This work was supported by grants from the NIH and the Cleveland
Mt Sinai Health Care Foundation.
a study of the metabolism of PB, which is why we conducted
our assays using the deuterated species and the chemical
analog PPGN as standards. Similarly, we assayed PA and
PB with [2H7]PA and [2H5]PB and also with the analog PP.
Since the two types of standardization procedures yielded
identical concentrations, investigators can select either type
of standardization of the assays.
We also showed that the analytes studied can be assayed
as either methyl or TBDMS derivatives. Although TBDMS
derivatives are more stable than methyl esters, the latter
are more sensitive. Hence a decision between derivatives
should be made depending on the expected concentrations
and instrument availability.
REFERENCES
1. Brusilow SW. Pediatr. Res. 1991; 29: 147.
2. Gore SD, Carducci MA. Expert Opin. Investig. Drugs 2000; 9: 2923.
3. Gilbert J, Baker SD, Bowling MK, Grochow L, Figg WD,
Zabelina Y, Donehower RC, Carducci MA. Clin. Cancer Res. 2001;
7: 2292–2300.
4. Bar-Ner M, Thibault A, Tsokos M, Magrath IT, Samid D. Clin.
Cancer Res. 1999; 5: 1509.
5. Rubenstein RC, Zeitlin PL. Am. J. Physiol. Cell Physiol. 2000; 278:
C259.
6. Dover GJ, Brusilow S, Charache S. Blood 1994; 84: 339.
7. Ambrose AM, Sherwin CP. J. Biol. Chem. 1933; 101: 669.
8. Moldave K, Meister A. J. Biol. Chem. 1957; 229: 463.
9. Webster LL Jr, Lucas SV, Strong JM, Mieyal JJ. J. Biol. Chem. 1976;
251: 3352.
10. Landau BR, Chandramouli V, Schumann WC, Ekberg K,
Kumaran K, Kalhan SC, Wahren J. Diabetologia1995; 38: 831–838.
11. Magnusson I, Schumann WC, Bartsch GE, Chandramouli V,
Kumaran K, Wahren J, Landau BR. J. Biol. Chem. 1991; 266:
6975–6984.
12. Yang D, Previs SF, Fernandez CA, Dugelay S, Soloviev MV,
Hazey JW, Agarwal KC, Levine WC, David F, Rinaldo P,
Beylot M, Brunengraber H. Am. J. Physiol. 1996; 270: E882–E889.
13. Cline GW, Rothman DL, Magnusson I, Katz LD, Shulman GI. J.
Clin. Invest. 1994; 94: 2369.
Physiological considerations
PA has been used for many years for the treatment of inborn
errors of the urea cycle.1,24–26 Patients treated with PA excrete
waste N as PAGN instead of urea. This conjugation occurs
only in humans and primates. The conjugation of PA with
glutamine represents by far the main fate of exogenous PA.
Indeed, in rhesus monkeys infused intravenously with PA
for 5 h, the cumulative excretion of PAGN amounted to
about 95% of the dose of PA infused.12
The formation of PAGN has been used in clinical
investigations using radioactive and stable isotopic tracers
to probe non-invasively the labeling pattern of citric acid
cycle intermediates in the livers of normal and diabetic
humans.11,13,14,27 When 14C- or 13C-labeled lactate or pyruvate
are administered, the labeling pattern of urinary PAGN is
identical to that of ˛-ketoglutarate in the liver12.
14. Jones JG, Solomon MA, Cole SM, Sherry AD, Malloy CR. Am. J.
Physiol. Endocrinol. Metab. 2001; 281: E848.
15. Piscitelli SC, Thibault A, Figg WD, Tompkins A, Headlee D,
Lieberman R, Samid D, Myers CE. J. Clin. Pharmacol. 1995; 35:
368–373.
16. Williams CM, Porter AH, Greer M, Scott KN, Sweeley CC.
Biochem. Med. 1969; 3: 164.
17. Truce WE, Olson CE. J. Am. Chem. Soc. 1952; 74: 4721.
18. Garyaev AP, Kononov OV, Dumpis MA, Piotrovskii LB. Khim.
Farm. Zh. 1990; 24: 20.
19. Gunther R, Bordusa F. Chem. Eur. J. 2000; 6: 463.
20. Goldman VS, Laney JW, Slife CW. US Patent 5, 932, 198, 1999.
21. Rigo B, Lespagnol C, Pauly M. J. Heterocycl. Chem. 1988; 25: 49.
22. Rigo B, Lespagnol C, Pauly M. J. Heterocycl. Chem. 1988; 25: 63.
23. Yang D, Beylot M, Agarwal KC, Soloviev MV, Brunengraber H.
Anal. Biochem. 1993; 212: 277.
24. Batshaw ML, Thomas GH, Brusilow SW. Pediatrics 1981; 68: 290.
25. Berry GT, Steiner RD. J. Pediatr. 2001; 138: S56.
26. Burlina AB, Ogier H, Korall H, Trefz FK. Mol. Genet. Metab. 2001;
72: 351.
27. Diraison F, Large V, Brunengraber H, Beylot M. Diabetologia
1998; 41: 212.
In recent years, the foul-smelling PA was replaced by
PB for the treatment of inborn errors of the urea cycle.25,26
It was assumed that PB undergoes one cycle of ˇ-oxidation
forming PA-CoA and PA, the former being the substrate
that actually conjugates with glutamine. In addition, PB
was used extensively as (i) a cytostatic which potentiate the
effect of cytotoxic agents such as fluorouracil on malignant
tumors, probably via inhibition of histone deacetylases,2,3
and (ii) an experimental drug for the treatment of cystic
fibrosis,5 peroxisomal biogenesis disorders28 and sickle-cell
anemia.6
Investigators at the US National Cancer Institute pointed
out to us that, following ingestion of PB, the cumulative
excretions of PB C PA C PAGN amounted to less that
half of the ingested dose of PB (L. Grochow, personal
28. McGuinness MC, Wei H, Smith KD. Expert Opin. Investig. Drugs
2000; 9: 1985.
Copyright  2002 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2002; 37: 581–590