SPECIAL COMMUNICATION
L-2-[13C]oxothiazolidine-4-carboxylic
acid: a probe for precursor mobilization for glutathione synthesis
Naomi K.
Fukagawa1,2,
Eswin
Hercules2, and
Alfred M.
Ajami3
1 University of
Vermont/Fletcher Allen Health Care General Clinical Research Center
and Department of Medicine, Burlington, Vermont 05405-0068;
2 Rockefeller University, New York, New York
10021; and 3 MassTrace Inc., Woburn, Massachusetts
01801
 |
ABSTRACT |
L-2-Oxothiazolidine-4-carboxylic acid (OTZ),
a 5-oxoproline analog, is metabolized by 5-oxoprolinase and converted
to cysteine, the rate-limiting amino acid for GSH synthesis, with the
release of CO2.
[13C]OTZ (1.5 mg/kg)
was used in 12 healthy men and women (ages 23-73 yr) to indirectly
assess precursor mobilization for GSH synthesis when stores were
reduced by 2 g acetaminophen. Expired breath samples were analyzed for
13CO2,
and results were analyzed using noncompartmental and two-compartment open minimal models. Results show an increase in
13C excretion (higher OTZ
hydrolysis) when GSH stores were reduced and 5-oxoprolinase substrate
utilization patterns, consequently, were altered
(P < 0.01). A metabolic rate index
(MRI) of the OTZ probe was found to be significantly higher after
reduction of GSH content by acetaminophen
(P < 0.05). The difference in
adaptive capacity (difference between control and postacetaminophen
metabolic rate indexes) was two times as large in the young than the
old subjects (P < 0.01). These data
support the use of
[13C]OTZ as a probe to
identify individuals who may be at risk for low GSH stores or who have
an impaired capacity to synthesize GSH.
cysteine prodrug; breath test; 5-oxoprolinase
 |
INTRODUCTION |
L-2-OXOTHIAZOLIDINE-4-CARBOXYLIC ACID
(OTZ) is a prodrug of cysteine, the amino acid believed to be rate
limiting for GSH synthesis (15, 16). OTZ is a 5-oxoproline analog and
therefore a substrate for the ubiquitous intracellular enzyme called
5-oxoprolinase (15, 16, 32). 5-Oxoprolinase catalyzes the reaction
converting 5-oxoproline, also known as pyroglutamate, to glutamate,
which is required for the first step for GSH synthesis (Fig.
1). 5-Oxoprolinase links the synthesis pathway and the
catabolism pathway of GSH in the
-glutamyl cycle (15, 16). OTZ, when
acted upon by 5-oxoprolinase, is converted to L-cysteine
with the release of CO2 (Fig.
2). A number of studies have demonstrated that OTZ
administration can stimulate hepatic GSH formation both in vitro and in
vivo (2, 6, 26, 31). OTZ has also been used to increase intracellular GSH stores in normal and human immunodeficiency virus-infected human
volunteers (8, 19) and, more recently, to sensitize tumor cells to
melphalan, a chemotherapeutic agent (3).

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Fig. 1.
GSH is synthesized in two steps from glutamate,
cysteine, and glycine by 2 enzymes:
1) -glutamylcysteine synthetase
and 2) GSH synthetase. GSH controls
its production by negative feedback on reaction
1 ( ). If -glutamylcysteine is not converted to
GSH, it follows an alternate pathway to produce cysteine and
5-oxoproline, which is converted to glutamate via 5-oxoprolinase
(reaction 3).
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Fig. 2.
Top: normal activity of 5-oxoprolinase
(5-OPase). Bottom: metabolism of
L-2-[13C]oxothiazolidine-4-carboxylic
acid ([13C]OTZ), a
5-oxoproline analog and hence a substrate for the ubiquitous
intracellular enzyme called 5-oxoprolinase. OTZ, when acted upon by
5-oxoprolinase, is converted to L-cysteine with the release
of CO2. * 13C label
in molecule; HS, hydrogen and sulfur component of
L-cysteine.
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GSH, a ubiquitous cellular constituent, accounts for 90% of nonprotein
thiols in mammalian tissues and plays an important role in the
detoxification of endogenous and exogenous compounds [e.g., free
radicals, peroxides, chemotherapeutic agents such as cisplatin or
doxorubicin, or drugs such as acetaminophen (APAP); see Ref. 15].
GSH deficiency produced in animals leads to death within a few days
(14, 15), and GSH depletion has recently been implicated in
hyperglycemia-induced embryopathy (27). GSH also serves as the
principal antioxidant for mitochondria, and low GSH levels are
associated with increased oxidative damage to mitochondria (14). Low
serum GSH levels are also reported in aging (10, 21) and are implicated
in the free radical theory of aging.
Hereditary defects have been described in four of the six enzymes in
the
-glutamyl cycle (22). A deficiency of GSH synthetase, leading to
a marked reduction in cellular levels of GSH, results in increased
formation of
-glutamylcysteine, which is efficiently converted to
5-oxoproline when not utilized for GSH synthesis or for
transpeptidation reactions. Because 5-oxoprolinase is widely distributed in human tissues, indirect measurement of its activity using a
5-[13C]oxoproline
analog such as OTZ
([13C]OTZ) and
quantitating 13C accumulation in
expired CO2 is expected to be an
index of activity within the GSH synthesis cycle. Because
5-oxoprolinase has a low Michaelis constant (8 ± 2 µM; see Ref.
17), its activity may be viewed as an indicator of the flux through the
GSH cycle (4). Under conditions of adequate GSH, precursors from the
transpeptidase reaction provide sufficient natural substrate
(5-oxoproline) for 5-oxoprolinase, resulting in a lower rate of
[13C]OTZ conversion
and lower cumulative appearance of
13CO2
in breath. When GSH demand is high (or GSH stores low), flow is shunted
toward the synthesis of
-glutamylcysteine (15), leaving the
5-oxoprolinase pathway open for OTZ metabolism (31) and resulting in
the appearance of higher amounts of
13CO2.
This paradigm is the basis for the use of
[13C]OTZ as a probe
for precursor mobilization for GSH synthesis. In this study, we used
[13C]OTZ to indirectly
assess precursor (cysteine) mobilization for GSH synthesis when GSH
stores are reduced by an acute ingestion of APAP.
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MATERIALS AND METHODS |
Materials.
[13C]OTZ (patent
pending) was obtained from MassTrace (Woburn, MA) and was administered
under investigator-sponsored Investigational New Drug (IND) application
no. 46,237. [13C]bicarbonate was
also obtained from MassTrace. Ammonium
7-fluoro-benzo-2-oxa-1,3-diazole-4-sulforate (SBD-F) was obtained from
Wako (Kyoto, Japan). GSH (reduced form) was purchased from Sigma
Chemical and was administered under investigator-sponsored IND
application no. 42,899. APAP (McNeil Laboratories, Fort Washington, PA)
is an effective analgesic and antipyretic agent available over the
counter and widely used by all age groups. Toxic does of APAP can cause
hepatocellular necrosis (20), and the mechanism for hepatic injury is
believed to be mediated by a reactive metabolite of APAP,
N-acetyl-p-benzoquinoneimine,
which depletes hepatocellular GSH (28).
Subjects. Twelve nonsmoking, nonobese
men (n = 8) and women
(n = 4), age range 23-73 yr, in
general good health volunteered to participate in these studies. They
were recruited from the community and selected on the basis of a
medical history, thorough physical examination, and normal blood and
urine tests. None was on medications known to alter GSH status. None of
the postmenopausal women were on hormone replacement therapy. The
premenopausal woman was studied in the follicular phase of her
menstrual cycle to minimize possible variation in metabolic rate and
CO2 production between studies.
Study design. Each volunteer was
studied on two separate occasions. The volunteers were admitted to the
General Clinical Research Center 2 days before the study and were
provided with a weight-maintaining diet providing 1 g
protein · kg
1 · day
1
and 45-50% of energy from carbohydrates. On the morning of the study after a 10- to 12-h overnight fast, an intravenous line was
placed for blood sampling. Baseline blood and expired breath samples
were collected and handled as previously described (7). In one study,
the volunteers received
[13C]OTZ (1.5 mg/kg or
0.101 mmol/kg) orally, and expired breath and blood samples were
obtained at timed intervals for 5 h after ingestion. In the second
study, volunteers received 2 g APAP 60 min before the
[13C]OTZ dose.
Baseline blood samples in study 2 were
obtained immediately before and 60 min after the APAP dose. Indirect
calorimetry was obtained four times within the 5-h study to determine
rates of CO2 production
(Deltatrac; Datex, Helsinki, Finland).
Five of the 12 volunteers underwent a third study during which time
they received a 5-h intravenous infusion of GSH (reduced form; Sigma
Chemical, St. Louis, MO) at 10 mg/min (~32 ± 2 µmol · kg
1 · h
1)
immediately after drinking the
[13C]OTZ. Three of the
12 volunteers were also studied with
[13C]bicarbonate
(0.101 mmol/kg) instead of OTZ with and without APAP.
Analytic methods. Expired breath
samples were analyzed using isotope ratio-mass spectrometry for
13C enrichment of expired
CO2 [atoms percent excess
(APE)]. Plasma amino acid concentrations were measured after
precolumn derivatization with phenylisothiocyanate and separation by
reversed-phase column HPLC with detection at 254 nm (Pico-Tag
Analysis System; Waters, Milford, MA). Plasma GSH was measured
after reduction and decoupling from plasma proteins with
tri-n-butylphosphine, derivatization with SBD-F, and separation by reversed-phase HPLC with fluorometric detection (Waters Chromatography), as previously described (1).
Calculations. The appearance of
13CO2
in breath after an oral bolus (Fig. 3) is
characterized by a rapid, almost linear rise after a short lag time to
accommodate variable gastric emptying, followed by an exponential
decay, which when plotted logarithmically appears to be at least
biphasic. The goal of this study was to describe this mass flow of
tracer from [13C]OTZ
to CO2 enrichment vs. time in a
polyexponential functional form. This would enable us to
1) determine the overall area under the curve (AUC) and net recovery of tracer as a percentage of dose,
2) describe the postabsorptive
enrichment changes independent of the lag and absorption phases, and
3) derive an index of OTZ conversion
outside of the central (plasma) compartment as the correlate for the
effect of the APAP "insult."

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Fig. 3.
Example of the change in 13C
[atom percent excess (APE) × 103] in expired
CO2 over the course of the study
in one individual.
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Pharmacokinetic analysis by nonlinear, weighted least squares (25) of
multiple exponential models showed that the metabolic process of OTZ
hydrolysis in vivo is best described by the expression for zero-order
absorption and biexponential decay (9)
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(1)
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during
tracer absorption when time (t, min)
is less than or equal to the zero-order absorption time
(T); and
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(2)
|
during tracer disappearance. Ca
and Cd are
13C enrichments in breath
expressed as APE times 1,000, lag is the time before the start of
absorption, and
C1,
C2,
k1,
k2,
T, and lag are the unknown parameters
to be solved.
This numeric model (29) was chosen over all others, including those
with first-order absorption of tracer, because it afforded the lowest
root mean squared scaled by degrees of freedom for each model (SE),
lowest Akaike Information Criterion (AIC) for adequacy of the
exponential term selection, highest
F-test value for goodness of fit, and
normally distributed residuals, especially during the absorption phase
(30). The weighting scheme selected was based on the squared inverse of
the dependent variable. Representative fitting values on a cohort of
six young male subjects, including the data depicted in Fig. 3
[average fitted parameter, average coefficient of variation (CV)
of individual weighted fits, and CV of average fits,
respectively] were 1) for the
basal or pre-APAP: C1, 66.5 (12 and
35%); k1, 0.0088 (12 and 18%);
C2, 57.3 (15 and 16%); k2, 0.0466 (13 and 47%); T, 43.2 (5 and 36%);
lag, 11.6 (3 and 100%); and 2) for
the post-APAP period:
C1, 59.2 (11 and 30%); k1, 0.0080 (15 and 20%);
C2, 31.2 (14 and
54%); k2, 0.0222 (10 and 43%); T, 48.2 (7 and 30%);
lag, 9.2 (5 and 52%). For this numeric model, the
weighted SE, AIC, and F-test (data
points, parameters) were as follows: pre-APAP, 0.06, 0.87, and 43,000; post-APAP, 0.13, 0.95, and 39,000, respectively. For the corresponding monoexponential, zero-order absorption model, the commensurate goodness-of-fit criteria were as follows: pre-APAP, 1.3, 2.6, and 1,600 and post-APAP, 2.1, 2.9, and 1,200, respectively. Similar mono-, bi-,
and triexponential models with first-order absorption on average showed
unacceptable fitting characteristics as follows: pre-APAP, >4, >3,
and <800; post-APAP, >3, >3, and <1,000.
In terms of a physiological, compartmental description, the functional
form described above is consistent with the kinetics of xenobiotic
disposal shown by the minimal two-compartment open model with central
compartment elimination, insensible losses by alternate routes of
disposal, and zero-order input, depicted in Fig.
4.

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Fig. 4.
Minimal two-compartment open model consists of a metabolic compartment
(compartment 2) and a central or
plasma compartment (compartment 1).
Lag is the time immediately before absorption and accommodates variable
gastric emptying. "Breath" and "other disposal" represent
pathways for the metabolism of the OTZ tracer. Details of the use of
the model are presented in the text.
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According to Wagner's derivation (29, 30), the
postabsorptive tracer kinetics of OTZ metabolism are readily described by stripping away the effects of lag and absorption. Thus rearranging and combining Eqs. 1 and 2 yields Eq. 3
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(3)
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in
which Cm represents the rate of
tracer disappearance from compartments
1 and 2 that would
result from an instantaneous dose input into
compartment 1.
The percentage of the OTZ dose recovered in breath was computed from
the cumulative integral of Eqs. 1 and 2 taken as a continuous function from
0 to 300 min multiplied by the CO2
production rate of each volunteer measured by indirect calorimetry.
This recovery factor (Rf) was
used to adjust the amount of the input tracer dose in subsequent
calculations for the decrementing effect of insensible losses (urinary
excretion of OTZ, biological fixation of
13CO2)
upon the mass of
13CO2
metabolite in the system. We defined the metabolic rate index (MRI,
nmol · kg
1 · min
1)
for OTZ as the dose per kilogram body weight (D) of OTZ, adjusted for
insensible losses (Rf),
multiplied by the fractional catabolic rate outside of the
central compartment (FCRmc,
min
1), a parameter equivalent to
the inverse of the mean residence time (MRTmc,
min) of tracer outside the central compartment, and determined it
by noncompartmental manipulation of Eq. 3, in accordance with Eq. 4
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(4)
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For noncompartmental analysis, the area under the curve (AUC) was
computed for the
13CO2
enrichment versus time graph by numeric integration, and the apparent
replacement rate (RR, mmol/min; or clearance equivalent of
CO2 derived from OTZ) was computed
as the tracer dose divided by AUC. The apparent metabolic distribution
pool (MD, mmol) was calculated as the tracer dose times AUMC divided by
AUC2, and system mean residence
time (MRT, min) was calculated as AUMC divided by AUC where AUMC is the
area under the moment curve of time multiplied by enrichment vs. time.
Statistics. Results are presented as
means ± SD. Comparisons between tests were made using the paired
t-test. Group differences based on age
or gender were compared using a two-sample
t-test.
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RESULTS |
The volunteers (8 men and 4 women) ranged in age from 23 to 73 yr. The
women were older (58 ± 14 vs. 36 ± 16 yr) and shorter (163 ± 3 vs. 174 ± 5 cm) than the men but of similar weight (72 ± 7 vs. 78 ± 6 kg) and body mass index (27 ± 3 vs. 26 ± 3 kg/m2). When the subjects were
grouped according to age (young 23-41 yr; old
53 yr), they
differed only in height but not weight or body mass index.
Consequently, all volunteers received approximately the same dose of
[13C]OTZ (0.75 mmol).
Indirect calorimetry results were similar in both studies, as shown in
Table 1. Expected differences in the rate
of CO2 production and energy
expenditure between young and old or men and women were found.
With regard to the noncompartmental kinetics over the entire time
course (absorption plus disposition phase), there was a significant
increase in the AUC of
13CO2
excretion and a slight decrease in the apparent RR (clearance) of
CO2 derived from the OTZ after
APAP compared with the study in which
[13C]OTZ was ingested
alone (control; Fig. 5). The percentage of the dose excreted 5 h after ingestion did not significantly differ among subjects or studies, averaging ~75%. The mass of exchangeable bicarbonate (apparent whole body distribution pool, MD) did not differ
between the two studies (1,155 ± 181 vs. 1,116 ± 199 mmol) but
was significantly lower in women compared with men (962 vs. 1,230 mmol,
respectively, P < 0.05) and old
compared with young subjects (1,004 vs. 1,230 mmol, respectively,
P < 0.01), reflecting the age- and
gender-related differences in body metabolic "space" and
bicarbonate volume of distribution. Plasma levels of APAP 4 h after
ingestion averaged 12 ± 6 µg/ml, with one individual contributing
to the variability with a level of 28 µg/ml. Time-to-peak 13CO2
excretion averaged 45 min in both the OTZ alone (45 ± 10) and OTZ
plus APAP (45 ± 14) studies, and MRT did not differ (99 ± 10 vs. 102 ± 10 min, respectively).

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Fig. 5.
Top: area under the curve (AUC) for
13CO2
excretion before acetaminophen (APAP; control) and after APAP (+APAP)
for 12 individuals. Bottom: reduction
in the replacement rate (RR) after APAP for 12 individuals can be seen.
* P < 0.01 vs. control.
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In the subset of five individuals who received the exogenous GSH
infusion, none of the calculated parameters differed significantly. The
AUC in the five individuals studied was 6,484 ± 664 APE · 10
3 · min,
RR was 12 ± 1 mmol/min, MRT was 104 ± 7 min, MD was
1,308 ± 168 mmol, peak time was 47 ± 14 min, and dose
recovery was 76 ± 2%. In the three volunteers who received
[13C]bicarbonate
instead of [13C]OTZ in
separate studies, there was no significant difference in the AUC of
13CO2
excretion (6,248 ± 733 vs. 6,355 ± 448 APE · 10
3 · min),
MRT (79 ± 4 vs. 76 ± 6 min), or dose recovery (64 ± 5 vs.
61 ± 3%) without or with APAP, respectively.
Plasma concentrations of glutamic acid, cysteine, glycine, and GSH
[54 ± 25, 34 ± 3, 163 ± 16, and 3.5 ± 0.9 µM,
respectively] did not vary significantly between studies or over
time in the studies with or without APAP. As previously reported,
plasma cysteine and glycine both increased during exogenous GSH
administration from baseline values of 31 ± 2 and 170 ± 36 to
41 ± 5 and 206 ± 21 µM, respectively, at 150 min after the
initiation of the GSH infusion. Plasma GSH concentrations nearly
doubled after 180 min of exogenous GSH administration.
From the modeling analysis, the results expressed in terms of nanomoles
of OTZ metabolized per kilogram body weight per minute for the basal
(control) and post-APAP (+APAP) conditions and the stimulatory
difference between +APAP and control are shown in Table
2. In all subjects, the MRI of the OTZ
probe was significantly higher (P < 0.05) after the GSH-depleting insult (+APAP) than before (control). The
young set showed a lower basal MRI and a higher level of OTZ
utilization after APAP, consistent with the view that GSH homeostasis
was altered by the aging process, possibly because of a more prolonged
exposure to oxidant stress with advancing age. The difference in
adaptive capacity, as measured by the stimulatory parameter, was two
times as large in the young than in the old subjects and was highly
significant (P < 0.01).
 |
DISCUSSION |
The amount or rate of appearance of isotopically labeled
CO2 generated from a substrate
containing the labeled carbon may be indicative of different
physiological functions related to the labeled substrate. A classic
application is the use of
[13C]aminopyrine to
distinguish among different degrees of liver disease affecting the
hepatic P-450 demethylation of
[13C]aminopyrine and
subsequent production of
13CO2
(18). Investigators have recently used labeled
-keto analogs of the
branched-chain amino acids to probe hepatic mitochondrial function by
measuring the exhalation of labeled
CO2 (11). Taken as a whole in the
context of these various breath test diagnostic paradigms, the present
data support the hypothesis that the
[13C]OTZ effectively
tracks the course of GSH depletion, even after a mild insult, and can
also be used to estimate the relative capacity of the GSH production
machinery in cohorts with different oxidant stress histories. Under
basal (normal) conditions, intracellular GSH titers are sufficiently
high to inhibit
-glutamylcysteine synthesis and GSH synthesis so
that the substrate flux of glutamyl precursors can be expected to be
shunted through the transpeptidase and cyclotransferase enzymes,
producing oxoproline and presumably saturating the 5-oxoprolinase
gateway. If a xenobiotic analog of oxoproline, namely
[13C]OTZ, is
introduced under these basal conditions, a lower proportion of it will
be hydrolyzed in the presence of a surplus of endogenous oxoproline;
therefore, the appearance of label derived from OTZ will be subject to
greater dilution and correspondingly lower enrichment. However, in
response to an acute GSH-depleting stimulus, renewed synthesis of
-glutamylcysteine and of GSH draws the glutamyl precursors away from
the substrate cycling shunt, presumably to replenish both intracellular
and extracellular GSH losses. Under these circumstances, intracellular
titers of oxoproline diminish; therefore, a greater proportion of OTZ
is hydrolyzed to
13CO2
by 5-oxoprolinase. Because the expression and concentration of
5-oxoprolinase is unaffected by GSH status, a higher rate of 13CO2
appearance and concomitant increase in the area under the breath test
curve were found.
This demonstrates for the first time the possibility of monitoring the
metabolism of an oral tracer as an index for precursor mobilization in
a ubiquitous metabolic pathway, i.e., the
-glutamyl cycle leading to
GSH synthesis. These data provide support for the efficacy of larger
doses of OTZ in stimulating cysteine production for GSH synthesis in
conditions where the body's GSH stores are low (12). This unique
tracer may act as an adjunct to monitoring GSH stores in patients at
risk for GSH depletion and perhaps in following the response to
therapeutic agents designed to replete low GSH stores (5, 8).
By extending the application of the OTZ probe to therapy management, it
should be possible, in a similar manner, to characterize individuals
both according to their basal OTZ MRI and to their production capacity
in response to a controlled insult such as that provided by APAP.
Patients with a high basal MRI or a low stimulation score might be less
likely to benefit from antioxidant and related therapies aimed at
boosting the GSH pool and might be the most susceptible to harm from
treatments such as chemotherapy, whose coincident biochemical effects
include chronic GSH depletion. Patients with higher basal MRI scores or
lower stimulation scores may sustain higher morbidity and mortality.
As shown in earlier studies, we did not find a change in plasma GSH
levels in this study. The dose of OTZ administered has been shown not
to influence free and total GSH concentrations in plasma but did
increase GSH levels in the lymphocytes (19). A number of factors makes
it unlikely that there would have been a detectable change in plasma
GSH over the course of this study, including the size of the dose, the
acute nature of the study, the health status of the volunteers, and the
well-known compartmentation of GSH in the body (23).
The only significant response induced by the intravenous infusion of
GSH was a rise in plasma cysteine levels, as previously reported (7).
We previously concluded that essentially all of the exogenous GSH was
metabolized with the release of free cysteine into plasma, accounting
for the inability to influence endogenous GSH production but reflecting
minimal changes in the hydrolysis of OTZ. Designing an experiment
whereby GSH stores are in excess and therefore a reduction in OTZ
hydrolysis might be anticipated presents a biological challenge.
However, the potential for identifying individuals requiring enhanced
synthesis of GSH and who might benefit from pharmacological efforts to
increase GSH production is exciting. Future studies are needed to
address the influence of bicarbonate kinetics on modeling of OTZ
metabolism and the use of the tracer in a variety of clinical states.
In addition, more rigorous approaches that assess the reciprocal rates
of OTZ appearance in plasma and/or urine are needed.
Separate investigations will also need to focus on the kinetics of
oxoproline itself, for which OTZ is a nonrecycling surrogate. The
approach used in this first study offers a convenient shortcut that is predicated on describing the metabolism of a xenobiotic, since OTZ has
no endogenous pool but shares a pivotal enzymatic conversion step with
the natural substrate. Hence, OTZ can be used as a surrogate of
oxoproline, and quantifying its rate of hydrolysis becomes a means to
assess the flow of precursor for GSH synthesis and potentially the
status of GSH homeostasis.
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ACKNOWLEDGEMENTS |
We thank the volunteers for participation, the General Clinical
Research Center staff for assistance, and Amy Prue for help with
preparation of the manuscript.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
AG-00599 and RR-00109.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. K. Fukagawa,
Univ. of Vermont, College of Medicine, Given Bldg. Rm. C-207,
Burlington, VT 05405-0068 (E-mail: nfukagaw{at}zoo.uvm.edu).
Received 16 February 1999; accepted in final form 7 September
1999.
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