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Vol. 28, Issue 7, 857-864, July 2000
Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences (S.K., H.W., S.A.Y., K.W.R., F.S.A.) and B.C. Research Institute of Children's and Women's Health, Department of Obstetrics and Gynecology, Faculty of Medicine (D.W.R.), The University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Metabolism and renal excretion of valproic acid (VPA) were
examined in maternal, fetal, and newborn sheep to identify the underlying reasons for the previously observed reduced VPA clearance in
newborn lambs. Plasma and urine from VPA infusion studies in maternal,
fetal, and newborn sheep were analyzed for VPA and its metabolites
[VPA-glucuronide; 
-oxidation products: (E)-2-ene, (E)-3-ene, and 3-keto VPA; hydroxylated metabolites:
3-hydroxy, 4-hydroxy, and 5-hydroxy VPA (5-OH VPA); and 4-ene VPA,
4-keto VPA, 2-propylglutaric acid, and 2-propylsuccinic acid] using
gas chromatography-mass spectrometry. All measured metabolites were detectable in maternal and fetal plasma, with 3-keto and 5-OH VPA being
at higher concentrations in the fetus. Plasma concentrations of
(E)-2-ene, (E)-3-ene, 3-keto, and 5-OH
VPA were higher in the newborn compared with the mother, whereas those
of the other metabolites were similar. A smaller percentage of the dose
was excreted as VPA-glucuronide in newborn lamb urine (28.3 ± 12.0%) compared with the mother (77.0 ± 7.8%). Similarly, a
lower fraction of the dose was excreted unchanged in newborn urine
(11.0 ± 5.8%) relative to the urine of the mother
(19.3 ± 5.8%); however, significantly larger percentages were
excreted as (E)-2-ene (0.11 ± 0.04 versus 0.02 ± 0.01%), 3-keto (11.6 ± 3.5 versus 1.6 ± 0.8%), 4-hydroxy (6.1 ± 3.2 versus 2.3 ± 1.3%), and 5-OH
VPA (2.2 ± 0.6 versus. 0.8 ± 0.6%). The major reason for
the reduced VPA elimination in newborn lambs appears to be impaired
renal excretion and glucuronidation capacity. As a result, a larger
fraction of the dose is channeled to -oxidation and hydroxylation
pathways. The -oxidation activities are high at birth; this may
explain the high plasma concentrations of (E)-2-ene and
3-keto VPA observed in newborn lambs and human newborns exposed to VPA.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
antiepileptic, valproic acid
(VPA)2, is a
simple branched short-chain fatty acid that exhibits an extremely
complex metabolic fate (Fig. 1). There
are approximately 50 known VPA metabolites, of which at least 16 are
observed consistently in humans (Baillie and Sheffels, 1995
). The major
routes of VPA metabolism can be divided into three categories:
glucuronidation, mitochondrial -oxidation, and microsomal oxidative
metabolism including desaturation and 
- and -1 oxidations (Fig.
1). Glucuronidation is the major route of VPA metabolism and results in
the formation of
1-o-acyl--D-ester-linked glucuronide (Dickinson et al., 1979). In different studies, 10 to 70%
of the dose has been recovered in urine as VPA-glucuronide in humans
(Gugler et al., 1977; Dickinson et al., 1989; Levy et al., 1990). A
significant fraction of the VPA dose is also metabolized via the
-oxidation pathway in humans. The VPA metabolites formed via the
mitochondrial -oxidation pathway include
2-n-propyl-2-pentenoic acid (2-ene VPA) (predominantly as
the E-isomer), 2-n-propyl-3-pentenoic acid (3-ene
VPA) (predominantly the E-isomer),
2-n-propyl-3-hydroxypentanoic acid (3-OH VPA), and
2-n-propyl-3-oxopentanoic acid (3-keto VPA) (Bjorge and
Baillie, 1991; Li et al., 1991). The 3-OH VPA metabolite may also be
formed via cytochrome P-450-mediated microsomal hydroxylation in
addition to the -oxidation pathway (Rettenmeier et al., 1987). The
3-keto VPA metabolite is a prominent urinary metabolic product of VPA
in humans, and may account for 10 to 60% of the total administered dose (Dickinson et al., 1989; Levy et al., 1990; Sugimoto et al., 1996). Small amounts (~1-3% of dose) of other -oxidation
metabolites, (E)-2-ene and 3-OH VPA, are also detectable in
human urine. The (E)-3-ene VPA metabolite arises from the
isomerization of (E)-2-ene VPA, and may be further
metabolized to (E,E)-2,3'-diene VPA via -oxidation (Fig. 1) (Bjorge and Baillie, 1991). The products of
microsomal - and -1 hydroxylation of VPA are
2-n-propyl-5-hydroxypentanoic acid (5-OH VPA) and
2-n-propyl-4-hydroxypentanoic acid (4-OH VPA), respectively
(Rettenmeier et al., 1987). Additional oxidation of 5-OH VPA leads to
2-propylglutaric acid (2-PGA), whereas that of 4-OH VPA results in the
formation of 2-n-propyl-4-oxopentanoic acid (4-keto VPA) and
2-propylsuccinic acid (2-PSA) (Granneman et al., 1984).
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In addition to 2-ene and 3-ene VPA, another monounsaturated metabolite
of VPA is 2-n-propyl-4-pentenoic acid (4-ene VPA). In
contrast to 2-ene and 3-ene VPA, 4-ene VPA is formed via a distinct
microsomal cytochrome P-450-mediated desaturation reaction (Rettie et
al., 1987). The study of 4-ene VPA formation and its subsequent fate
has received considerable attention because of its possible involvement
in VPA-induced idiosyncratic hepatotoxicity. The 4-ene VPA metabolite
can be subsequently metabolized via mitochondrial -oxidation to form
the diunsaturated metabolite (E)-2,4,-diene VPA (Kassahun
and Baillie, 1993). One hypothesis of VPA-induced hepatotoxicity is
that subsequent oxidative metabolism of 4-ene VPA, possibly via
(E)-2,4-diene VPA formation, leads to the generation of
chemically reactive and potentially toxic intermediates that are
capable of reacting with and depleting mitochondrial glutathione stores
(Kassahun and Abbott, 1993; Kassahun and Baillie, 1993; Tang and
Abbott, 1996). This may eventually result in mitochondrial alterations
and inhibition of -oxidation, two characteristic features of
VPA-induced hepatotoxicity (Kesterson et al., 1984).
In our companion article on VPA pharmacokinetics in pregnant sheep and
newborn lambs (Kumar et al., 2000), striking differences in the
pharmacokinetics of VPA in newborn lambs as compared with maternal
sheep have been outlined. These include a reduced clearance, a longer
elimination half-life, and a pronounced Michaelis-Menten type nonlinear
pharmacokinetic profile. Interestingly, these differences also appear
to exist in human newborns exposed to VPA either via direct neonatal
administration for control of seizures or via in utero placental
transfer and persistence of the drug in the neonatal circulation after
birth. Thus it was of interest to elucidate the underlying cause(s) of
these differences. This includes possible differences in the functional
capacity of various VPA metabolism pathways and the renal elimination
of VPA in the newborn lamb compared with maternal sheep. In addition,
the possible involvement of VPA metabolites in its idiosyncratic
hepatotoxicity and pharmacological activity necessitates detailed study
of fetal and newborn exposure to these compounds as compared with the
adult. In this study, we have examined comparative maternal, fetal, and
newborn metabolism and renal elimination of VPA in sheep.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The experimental details regarding the surgical procedures
performed on animals are described in the companion manuscript (Kumar
et al., 2000).
Drug Administration Protocols.
The detailed protocols for drug administration are also described in
Kumar et al. (2000). Briefly, the following experiments were conducted.
Pregnant Sheep Experiments. Two sets of experiments were carried out on five pregnant sheep in a randomized manner and with an appropriate washout period in between.
Maternal administration. A bolus loading dose of VPA (Sodium Valproate; Sigma Chemical Co., St. Louis, MO) equivalent to 20.1 mg VPA/kg maternal body weight was administered to the ewe via the maternal femoral venous catheter over 1 min; this was followed immediately by a 24-h continuous infusion of the drug at 138.3 µg/min/kg via the same route.
Fetal administration. The fetal experimental protocol was similar to that for maternal experiments described above, except that doses were administered via the fetal lateral tarsal vein and were reduced to one-fourth of the maternal doses (i.e., 5.0 mg/kg bolus and 34.6 µg/min/kg infusion rate based on maternal body weight).
Newborn Lamb Experiments. Newborn lamb experiments were begun the day after birth. Drug administration involved a 10-mg/kg bolus administered over 1 min via the lateral tarsal vein, followed immediately by a continuous 6-h infusion at 138.3 µg/min/kg via the same route.
In all pregnant sheep experiments, serial blood samples were collected from the fetal (2 ml) and maternal (3.0 ml) femoral arterial catheters at 5 min, and 0.5, 1, 3, 6, 9, 12, 20, 22, and 24 h during the infusion, and at 0.5, 1, 3, 6, 9, 12, 24, 36, 48, 60, and 72 h postinfusion. All fetal blood removed for sampling during the experiment was replaced, at intervals, by an equal volume of blood obtained from the mother before the start of the experiment or from another ewe (after the first day). Samples of maternal and fetal urine, amniotic fluid, and fetal tracheal fluid were also collected at predetermined intervals. Cumulative maternal urine was collected via a Foley bladder catheter inserted via the urethra of the ewe on the morning of the experiment, and attached to a sterile polyvinyl bag. Fetal urine flow rate was estimated using a computer-controlled roller pump assembly developed in our laboratory. Fetal bladder catheter was allowed to drain by gravity into a sterile reservoir (10-ml syringe barrel) to which a disposable DTX transducer was connected. When the pressure in the reservoir increased above a preset level (usually 3 mm Hg) as a result of urine accumulation, the computer activated a roller pump (DIAS, Ex154; DIAS Inc., Kalamazoo, MI), which pumped a calibrated volume of urine from the reservoir back to the amniotic cavity (via the amniotic catheter). The cumulative volume pumped per min, which equals fetal urine production rate per min, was stored on diskette. During the experimental period and at specified sampling time intervals, fetal urine samples (~5 ml) were collected by attaching a sterile sample collection syringe to the amniotic catheter via a 3-way stop-cock. During the newborn lamb experiments, serial femoral arterial blood samples (2 ml) were collected at 5 min, and 0.5, 1, 2, 3, 4, 5, and 6 h during the infusion, and at 0.5, 1, 2, 4, 6, 18, 30, 42, 54, 66, 78, and 90 h postinfusion. Cumulative urine samples were also collected for 96 h. All maternal, fetal, and newborn blood samples were placed into heparinized Vacutainer tubes (Becton-Dickinson, Rutherford, NJ) and centrifuged at 2000g for 10 min. The plasma supernatant was removed and placed into clean borosilicate test tubes with polytetrafluoroethylene-lined caps. Samples were stored frozen at
20°C until the time of analysis.
Determination of VPA Plasma Protein Binding.
The unbound plasma concentrations of VPA were measured ex vivo in all
fetal, maternal, and newborn plasma samples by an ultrafiltration procedure at 1000g for 30 min using Centrifree
micropartition devices (Amicon, Inc., Danver, MA) (see Kumar et al.,
2000).
Drug and Metabolite Analysis.
Concentrations of VPA and its metabolites were measured using a
previously developed sensitive and specific gas chromatography-mass spectrometry assay method (Yu et al., 1995). Earlier validation studies
have demonstrated that the variability and bias of this assay for all
compounds does not exceed 15% (Yu et al., 1995). The concentrations of
VPA-glucuronide in maternal, fetal, and newborn urine were measured
using a base hydrolysis procedure as follows. The samples were adjusted
to pH 12.5, incubated at 60°C for 1 h, and the total VPA
(unconjugated + conjugated) was quantified by the above gas
chromatography-mass spectrometry analysis method. The difference of
total and unconjugated (unhydrolyzed) VPA concentrations gave the
concentration of VPA-glucuronide. This procedure was preferred over
hydrolysis with -glucuronidase because VPA-glucuronide has been
shown to rearrange to at least six -glucuronidase-resistant
structural isomers via migration of the acyl moiety away from the C-1
position and subsequent ring opening, mutarotation, and lactone
formation (Dickinson et al., 1984). These rearrangements are
pH-, temperature-, and storage time-dependent (Dickinson et al., 1989).
The hydrolysis with alkali, however, is capable of measuring total
VPA-glucuronide in spite of these possible rearrangements (Dickinson et
al., 1989).
Pharmacokinetic Analysis. Renal clearance values for the total and unbound VPA in the ewe and the newborn lamb were calculated by dividing the total amount of unconjugated VPA excreted in urine by the respective plasma area under the curve from time zero to infinity of the total or unbound drug. Fetal renal clearance of total and unbound drug was calculated by dividing the urinary excretion rate determined at each sampling point by the corresponding total or unbound fetal plasma drug concentration at that time. Urinary excretion rate of the drug at each sampling point was determined from the concentration of the drug in fetal urine and the average urine flow rate during the 0.5 h preceding the sampling time point. Fetal renal clearances calculated at each sampling point were averaged to obtain a mean value for this parameter.
Statistical Analysis. All data are reported as mean ± S.D. The maternal and fetal maximal plasma concentrations (Cmax) of various VPA metabolites (during both maternal and fetal infusion) were compared against each other using a paired t test. Because maternal and fetal Cmax values of various VPA metabolites are not completely independent and are influenced by each other due to placental transfer of metabolites, they were compared with newborn plasma Cmax values independently, using unpaired t tests. The significance level was P < .05 in all cases.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Plasma Concentrations of VPA Metabolites in Maternal, Fetal, and
Newborn Sheep.
A number of metabolites of VPA such as those formed via fatty acid
-oxidation [(E)-2-ene, (E)-3-ene, and 3-keto
VPA], cytochrome P450-mediated desaturation (4-ene VPA), and
hydroxylation and subsequent oxidation (3-OH, 4-OH, 5-OH, and 4-keto
VPA, 2-PSA, 2-PGA) were measured in maternal and fetal plasma during
and after VPA administration (Table 1).
Maternal and fetal plasma concentrations of 3-OH VPA and 2-PSA were
generally below the lower limit of quantitation (LOQ) of the
assay (see footnote b in Table 1 for exceptions).
Also, the 2-PGA metabolite was below the LOQ in all fetal plasma
samples. During fetal infusion experiments, measurable concentrations
of 2-PGA were detected in maternal plasma samples from only one animal.
Concentrations of all VPA metabolites, except (E)-2-ene and
3-keto VPA, appeared to be relatively stable in maternal as well as in
fetal plasma during the 20- to 24-h period of infusion and declined
relatively rapidly to below LOQ levels within 12 to 24 h
postinfusion (the figures of plasma profiles of these metabolites are
not shown due to an apparent similarity between their shapes and time
course). However, plasma concentrations of (E)-2-ene and
3-keto VPA were still increasing at the end of the infusion period and
the maximal concentrations were typically observed a few hours later
(Fig. 2). These two metabolites were detectable in maternal and fetal plasma for up to 36 to 72 h after the end of infusion (Fig. 2). Table 1 presents the average maternal and
fetal Cmax values of all metabolites during
maternal and fetal administration experiments. Maternal plasma
Cmax of VPA after maternal administration
occurred at 5 min (i.e., in the first sample after the i.v. bolus
loading dose), whereas that after fetal administration occurred within
1 to 3 h. Similarly, the fetal plasma Cmax
of VPA after fetal administration occurred at 5 min, whereas that after
maternal administration occurred within 1 to 3 h. Maternal and
fetal Cmax values of all metabolites, except (E)-2-ene and 3-keto VPA, occurred during the 20- to 24-h
period of infusion; the Cmax values of these two
metabolites occurred within 1 to 12 h after the end of VPA
infusion (Fig. 2).
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; Kassahun et al., 1990).
Excretion of VPA Metabolites in Maternal, Fetal, and Newborn Urine. Table 2 presents the percent fractions of the VPA dose excreted as unchanged VPA and various VPA metabolites in maternal urine during maternal VPA infusion. The majority of VPA metabolites detected in maternal plasma were also excreted in maternal urine in significant quantities. However, the excretion of unchanged VPA and VPA-glucuronide alone accounted for ~95% of the administered dose (Table 2). Unchanged VPA, VPA-glucuronide, and all other VPA metabolites were also detectable in fetal urine, albeit in much lower amounts compared with the mother. Cumulative collection of fetal urine was not performed, hence, the total amounts of these compounds excreted as a fraction of the administered dose could not be estimated.
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Renal Clearance of VPA in Mother, Fetus, and Newborn. Table 3 presents the average renal clearance of the unbound and total VPA in maternal, fetal, and newborn sheep. Maternal renal clearances were determined during maternal drug infusion, whereas fetal renal clearances were determined during fetal drug administration. Maternal renal clearance data were available for all five animals; however, fetal renal clearance data were available for only four animals because bladder catheter was not implanted in one animal. Mean fetal renal clearance of the unbound and total VPA was significantly lower compared with the corresponding maternal value. Paired plasma and urine data were available in only three newborn lambs, hence, renal clearance could be calculated only in these three animals. Similar to the fetus, renal clearance of the unbound as well as total VPA in newborn lambs was much lower compared with the corresponding values in the mother (Table 3). Renal clearance of the unbound as well as total VPA in newborn lambs appears to be somewhat higher compared with the fetus (Table 3); however, due to a low n value for newborn lambs, a meaningful statistical comparison is difficult.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Plasma Concentrations of VPA Metabolites in Maternal, Fetal, and
Newborn Sheep.
The average maternal plasma Cmax values of a
number of VPA metabolites during maternal drug administration were
within or close to the plasma concentration range encountered in human
adult epileptics (Rettenmeier et al., 1989; Kassahun et al., 1990). These include 4-ene VPA (0.40 versus trace 0.64 µg/ml; sheep versus human), 4-keto VPA (0.24 versus trace 4.50 µg/ml),
4-OH VPA (3.91 versus trace 2.97 µg/ml), 5-OH VPA (0.88 versus trace 1.06 µg/ml), and 2-PGA (0.17 versus trace 0.22 µg/ml). Maternal plasma Cmax of
(E)-2-ene VPA (0.63 µg/ml) was near the low end of the
range encountered in human adults (0.554.66 µg/ml). Also, maternal
plasma Cmax values of the other two metabolites
formed via the -oxidation pathway, i.e., (E)-3-ene and
3-keto VPA, appear to be lower than the range in humans (0.13 versus
0.41 1.68 µg/ml for (E)-3-ene VPA; 0.48 versus
2.26 14.7 µg/ml for 3-keto VPA). Although these differences
may be related to interspecies variation in VPA metabolism, it has also
been suggested that pregnancy is associated with a reduced fatty acid
-oxidation activity during the later part of gestation (Grimbert et
al., 1993, 1995). In agreement with this, significantly higher plasma
concentrations of -oxidation VPA metabolites have been observed
during our studies in nonpregnant sheep (H. Wong, S. Kumar, K. Wayne
Riggs, and D.W. Rurak, unpublished data). Also, one report in humans
showed lower maternal serum concentrations of (E)-2-ene,
(E)-3-ene, and 3-OH VPA during the early second trimester
compared with the late first trimester of pregnancy (Omtzigt et al.,
1992). Similar maternal plasma Cmax values of the
-oxidation products during maternal and fetal VPA infusions, in
spite of ~3- to 4-fold different VPA plasma concentrations, indicate
a possible saturation of the -oxidation pathway (Table 1). However,
maternal plasma concentrations of VPA metabolites formed via the
cytochrome P-450 pathways (4-ene, 4-keto, 4-OH and 5-OH VPA, and 2-PGA)
were proportionately lower during fetal infusion experiments,
suggesting an overall linearity of these pathways. These observations
are similar to the data on human VPA metabolism (Granneman et al.,
1984).
-oxidation VPA metabolites compared with maternal serum are also
found in cord serum samples obtained at birth from epileptic mothers
(Nau et al., 1981, 1984; Kondo et al., 1987). A number of mechanisms
based on the fetus acting as a deep compartment, active transport of
metabolites across the placenta, and maternal and fetal differences in
plasma protein binding of these compounds, have been proposed to
explain the apparent fetal accumulation of these metabolites (Nau et
al., 1981, 1984). However, our combined fetal and newborn metabolite
data provide strong evidence that the apparent fetal accumulation of
these metabolites may result from increased fetal metabolism of VPA via
these pathways (see below).
The majority of VPA metabolites were also detectable in significant
concentrations in newborn lamb plasma, suggesting that these pathways
are likely functional in the late-gestational fetus as well. Perhaps
the most significant observation with respect to newborn VPA metabolism
was the detection of much higher plasma concentrations (up to 10-fold
for 3-keto VPA) of many metabolites [i.e., (E)-2-ene,
3-ene, 3-keto, 5-OH VPA, and possibly 4-OH VPA] in the newborn
compared with the fetus, and in some cases even compared with the
mother. Apart from the above discussed isolated reports on VPA
metabolite concentrations in cord serum samples, little data exist on
VPA disposition in human neonates because of its limited clinical use
in this population. However, VPA is commonly used to control seizures
in epileptic children (Nau et al., 1991; Kondo et al., 1992; Siemes et
al., 1993). The comparison of VPA metabolite serum profiles in human
children with our newborn lamb data reveals some striking similarities.
Plasma concentrations of (E)-2-ene, (E)-3-ene,
3-keto, and 5-OH VPA metabolites are generally higher in children <2
years of age compared with those >2 years. In contrast, the plasma
concentrations of 4-ene and 4-keto VPA and 2-PGA metabolites are lower
in the younger age group (Nau et al., 1991). Hepatic fatty acid
-oxidation activity is low in utero and at birth in many species
(Krahling et al., 1979; Duee et al., 1985; De Vivo et al., 1991).
However, these activities increase dramatically during the first few
hours after birth and may reach values higher than the adult within 1 day; subsequently the -oxidation activities decline steadily to
adult levels until weaning. This increased -oxidation capacity after birth may be responsible for the higher plasma concentrations of VPA
metabolites in newborn lambs and younger human children. However, as
discussed above, our sheep data and limited cord serum human VPA
metabolite concentration data provide strong evidence for significant
fetal formation of the 3-keto metabolite (Nau et al., 1981), indicating
that at least VPA -oxidation is significantly functional in utero.
Excretion of Unchanged VPA and Its Metabolites in Maternal, Fetal,
and Newborn Urine.
During maternal drug infusion, the major components of the VPA urinary
metabolite profile were unchanged VPA (~19%) and VPA-glucuronide (~77%), with all other metabolites collectively accounting for <5%. There appear to be a number of differences between the sheep and
human VPA urinary metabolite profiles (Gugler et al., 1977; Dickinson
et al., 1989; Levy et al., 1990; Levy and Shen, 1995). Firstly, renal
excretion of the unchanged drug accounts for a much larger percentage
of the dose in sheep (19%) as compared with the human (1-2%),
possibly because of a higher renal clearance of VPA in sheep (0.94 versus 0.03-0.06 ml/min/kg). Secondly, the fraction of dose excreted
as VPA-glucuronide in sheep (77%) was consistently near the high end
of the range observed in humans (10-70%). The third major difference
between maternal sheep and humans appears to be the much lower
contribution of -oxidation pathway to total VPA elimination in
sheep. In addition to VPA-glucuronide, 3-keto VPA is a major human
urinary metabolite and may account for 10 to 60% of the dose;
(E)-2-ene and 3-OH VPA may each account for an additional 1 to 3% (Dickinson et al., 1989; Levy et al., 1990; Sugimoto et al.,
1996). As discussed before, the lower contribution of -oxidation to
VPA metabolism in maternal sheep may be related to pregnancy-related
reductions in -oxidation activity. In agreement with this, we have
recovered a larger percentage (10-20%) of the VPA dose as 3-keto VPA
in nonpregnant sheep urine (H. Wong, S. Kumar, K. Wayne Riggs, and D.W.
Rurak, unpublished data). The contribution of most other measured
metabolites to total elimination of VPA appears to be close to the
range in humans.
). Similar to VPA, very small amounts of the glucuronide
conjugate have also been found in fetal lamb urine for another
carboxylic acid drug, indomethacin (Krishna et al., 1995). In contrast,
the ability to glucuronidate alcoholic/phenolic moieties appears to be
much more developed in the late gestation fetal lamb as ~63, 22, and
40% of the fetal dose of morphine, ritodrine, and labetalol,
respectively, is glucuronidated (Olsen et al., 1988; Wright et al.,
1991; Yeleswaram et al., 1993). Significant amounts of acetaminophen
and para-nitrophenol glucuronides are also formed by the
fetal lamb in utero and by the isolated perfused fetal lamb liver in
vitro, respectively (Wang et al., 1986; Ring et al., 1996). These data
may indicate differential ontogenetic development of UDP-glucuronosyl
transferases involved in the glucuronidation of acidic and
alcoholic/phenolic compounds, as has been observed in the rat and the
human fetus (Wishart, 1978; Burchell et al., 1989).
Although the fraction of dose recovered as unchanged VPA and
VPA-glucuronide in newborn lamb urine is only ~2- to 3-fold lower compared with that of the mother, the maternal-neonatal kinetic differences in these pathways are much more pronounced (Fig. 3). The
continuous excretion of small amounts of VPA and its glucuronide in
lamb urine indicates that these pathways are likely saturated over a
prolonged period of time due to their low capacity. Also, in contrast
to the adult, metabolites formed via the -oxidation and cytochrome
P-450 pathways [(E)-2-ene, (E)-3-ene, 3-keto,
and 3-OH, 4-OH, and 5-OH VPA] appear to account for a much larger fraction of the VPA dose in newborn lambs. Interestingly, however, larger amounts of the potentially hepatotoxic cytochrome P-450 metabolite, 4-ene VPA, were not excreted in newborn lambs. Higher fatty
acid -oxidation capacity, combined with impaired VPA glucuronidation and renal excretion in the newborn lamb, is likely responsible for a
larger fraction the VPA dose being metabolized via the cytochrome P-450
and -oxidation pathways. However, these metabolic routes appear to
be kinetically less efficient, resulting in apparent nonlinear kinetics
with a significantly reduced clearance in newborn lambs (Kumar et al.,
2000). In contrast to the mother, a significant portion of the VPA dose
(~30-50%) could not be accounted for in the newborn lamb,
suggesting the possibility of additional routes of VPA metabolism
during the newborn period.
Renal Clearance of VPA in Maternal, Fetal, and Newborn Sheep.
The negligible renal clearance of VPA in the fetal lamb compared with
maternal sheep is similar to the data for a number of other acidic
compounds such as indomethacin (Krishna et al., 1995), diphenylmethoxyacetic acid (Kumar et al., 1997), and
para-aminohippurate (Elbourne et al., 1990). Limited VPA
renal excretion ability also appears to exist in the immediate newborn
period, with the average unbound and total renal clearances being
~10-fold lower compared with those of the mother (Table 3).
-oxidation VPA metabolites in newborn
lambs are much higher compared with those of the mother, possibly
because of a high -oxidation capacity at birth. Glucuronidation and
renal excretion of unchanged VPA are the major determinants of VPA
elimination in maternal sheep. Both of these routes are significantly
underdeveloped in the newborn lamb and lead to a slower VPA
elimination. This also results in a larger percentage of the dose being
metabolized via the -oxidation and cytochrome P-450 pathways in
newborn lambs. Because a number of similarities have been observed in
the pharmacokinetics and metabolism of VPA in human neonates and
newborn lambs, it is tempting to speculate that reduced VPA
glucuronidation and/or renal clearance may also underlie the slow
elimination of VPA observed in human newborns.
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Footnotes |
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Received September 2, 1999; accepted February 16, 2000.
1 A part of this work was presented at the 5th International Meeting of the International Society for the Study of Xenobiotics (1998) and is abstracted in ISSX Proc 13:74, 1998.
These studies were supported by funding from the Medical Research Council of Canada. S.K. was the recipient of a University of British Columbia Graduate Fellowship. H.W. is supported by a Pharmaceutical Manufacturers Association of Canada/Medical Research Council of Canada studentship. D.W.R. is the recipient of an Investigatorship award from the British Columbia Children's Hospital Foundation.
Send reprint requests to: Dr. Dan W. Rurak, B.C. Research Institute for Children's and Women's Health, 950 West 28th Ave., Vancouver, British Columbia, Canada V5Z 4H4. E-mail: drurak{at}cw.bc.ca
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Abbreviations |
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Abbreviations used are: VPA, valproic acid; Cmax, maximal plasma concentration; LOQ, lower limit of quantification; 3-OH VPA, 2-n-propyl-3-hydroxy pentanoic acid; 4-OH VPA, 2-n-propyl-4-hydroxy pentanoic acid; 5-OH VPA, 2-n-propyl-5-hydroxy pentanoic acid; 2-PSA, 2-propylsuccinic acid; 2-PGA, 2-propylglutaric acid; 2-ene VPA, 2-n-propyl-2-pentenoic acid; 3-ene VPA, 2-n-propyl-3-pentenoic acid; 3-keto VPA, 2-n-propyl-3-oxopentanoic acid; 4-keto VPA, 2-n-propyl-4-oxopentanoic acid; 4-ene VPA, 2-n-propyl-4-pentenoic acid.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-oxidation of valproic acid in rat liver mitochondrial preparations.
Drug Metab Dispos
19:
823-829[Abstract].-D-glucuronide in fetal lambs.
J Pharmacol Exp Ther
247:
576-584
4-VPA, a toxic metabolite of valproic acid.
Science (Wash DC)
235:
890-893This article has been cited by other articles:
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