Molecular Genetics and Metabolism 81 (2004) S79 S85
`
`www.elsevier.com/locate/ymgme
`
`Effect of alternative pathway therapy on branched chain amino
`acid metabolism in urea cycle disorder patients
`
`Fernando Scaglia,a Susan Carter,a,b William E. OÕBrien,a and Brendan Leea,b,*
`
`a Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Rm 635E, Houston, TX 77030, USA
`b Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Rm 635E, Houston, TX 77030, USA
`
`Received 3 October 2003; received in revised form 23 October 2003; accepted 4 November 2003
`
`Abstract
`
`Urea cycle disorders (UCDs) are a group of inborn errors of hepatic metabolism caused by the loss of enzymatic activities that
`mediate the transfer of nitrogen from ammonia to urea. These disorders often result in life threatening hyperammonemia and
`hyperglutaminemia. A combination of sodium phenylbutyrate and sodium phenylacetate/benzoate is used in the clinical manage
`ment of children with urea cycle defects as a glutamine trap, diverting nitrogen from urea synthesis to alternatives routes of ex
`cretion. We have observed that patients treated with these compounds have selective branched chain amino acid (BCAA) deficiency
`despite adequate dietary protein intake. However, the direct effect of alternative therapy on the steady state levels of plasma
`branched chain amino acids has not been well characterized. We have measured steady state plasma branched chain and other
`essential non branched chain amino acids in control subjects, untreated ornithine transcarbamylase deficiency females and treated
`null activity urea cycle disorder patients in the fed steady state during the course of stable isotope studies. Steady state leucine levels
`were noted to be significantly lower in treated urea cycle disorder patients when compared to either untreated ornithine tran
`scarbamylase deficiency females or control subjects (P < 0:0001). This effect was reproduced in control subjects who had depressed
`leucine levels when treated with sodium phenylacetate/benzoate (P < 0:0001). Our studies suggest that this therapeutic modality has
`a substantial impact on the metabolism of branched chain amino acids in urea cycle disorder patients. These findings suggest that
`better titration of protein restriction could be achieved with branched chain amino acid supplementation in patients with UCDs who
`are on alternative route therapy.
`Ó 2004 Elsevier Inc. All rights reserved.
`
`Keywords: Branched chain amino acids; Sodium phenylacetate; Sodium phenylbutyrate; Urea cycle disorders
`
`Introduction
`
`Urea cycle disorders (UCDs) are a group of inborn
`errors of hepatic metabolism that result in often life-
`threatening hyperammonemia and hyperglutaminemia
`[1]. The management of hyperammonemic episodes in
`these disorders is achieved by dietary protein restriction,
`supportive management of catabolic stress, and the use
`of compounds that remove nitrogen by alternative
`pathways. Alternative pathway therapy includes the use
`of sodium phenylacetate/benzoate (Ucephan) or sodium
`phenylbutyrate (Buphenyl) to stimulate the excretion of
`nitrogen as phenylacetylglutamine and hippuric acid
`
`* Corresponding author. Fax: +713-798-7773.
`E-mail address: blee@bcm.tmc.edu (B. Lee).
`
`1096-7192/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
`doi:10.1016/j.ymgme.2003.11.017
`
`(in the case of Ucephan) [2,3]. Phenylbutyrate does not
`accumulate in plasma, and within minutes, it is first
`activated to its CoA ester, and then converted to
`phenylacetylCoA via b-oxidation [2]. This ultimately is
`conjugated with glutamine in the liver and kidney to
`yield phenylacetylglutamine, which is excreted in the
`urine. Hence, it replaces urea as a means of eliminating
`excess nitrogen compounds. We have previously shown
`that
`the treatment with Ucephan in both control
`and UCD subjects will
`increase glutamine flux and
`decrease total body urea flux directly proportional to
`the molar conversion of phenylacetate to phenylace-
`tylglutamine [4].
`We have observed in our UCD patient population
`that this therapy leads to a marked fall in serum bran-
`ched chain amino acids (BCAA) concentrations in spite
`of apparently adequate levels of total protein intake.
`
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`This observed fall has often preceded a metabolic de-
`compensation. One predicted effect of these compounds
`might be the dysregulation of global protein synthesis.
`Phenylbutyrate-induced glutamine depletion in healthy
`subjects has been shown to exert a profound effect on
`leucine metabolism including the lowering of plasma
`leucine concentrations and increasing leucine oxidation
`[5]. In addition, several conditions leading to hyperam-
`monemia, including liver cirrhosis and idiopathic portal
`hypertension, a condition characterized by extensive
`portal-systemic shunting, are invariably associated with
`a decline in the plasma levels of BCAA [6,7]. This sug-
`gests that hyperammonemia and intracellular glutamate
`depletion may contribute to BCAA deficiency through
`the stimulation of BCAA transamination. The present
`study was designed to achieve a deeper understanding of
`the effect of alternative pathway therapy on branched
`chain amino acid metabolism in fed UCD patients,
`asymptomatic untreated ornithine transcarbamylase
`(OTC)-deficient females, and normal controls.
`
`Materials and methods
`
`The study received prior approval from the Institu-
`tional Review Boards for Human Subjects of Baylor
`College of Medicine. The studies were carried out while
`the patients were admitted for nitrogen flux studies to
`assess in vivo urea cycle activity [4]. The use of [18O]urea
`and [15N]glutamine in these stable isotope studies should
`not have affected the levels of plasma amino acids based
`on the low total dose infused [4].
`
`Five null UCD patients (three patients with OTCD
`and two patients with carbamoylphosphate deficiency
`type I) on sodium phenylbutyrate treatment and two
`patients with partial OTCD on sodium benzoate were
`studied retrospectively and their leucine and phenylala-
`nine levels were followed over a period of three months.
`
`Clinical protocol
`
`The clinical protocol was approved by the Baylor
`College of Medicine Human Subjects
`Institutional
`Review Board. The subjects were admitted into the
`Texas ChildrenÕs Hospital General Clinical Research
`Center and were started on the study protocol after
`physical examination and informed consent. Each
`subject was started on the assigned level of protein
`intake and medication (if indicated) for a two-day pe-
`riod of stabilization. Protein intake was monitored by
`weighing portions before and after each meal. On the
`third day of the study, after an overnight fast and after
`a preinfusion blood sampling for the determination of
`baseline plasma amino acids, the subjects consumed the
`first of four twice-hourly meals that each supplied 1
`12 of
`their prior daily protein intake. Blood for plasma
`amino acids and ammonia was obtained at 0 h prein-
`fusion, and then at 4, 6, and 7.5 h during the infusion
`of stable isotopes. The levels of branched chain and
`non-branched chain essential amino acids in the dif-
`ferent groups were evaluated in fed individuals at
`steady state on the third day of the study after a two-
`day period of stabilization.
`
`Study subjects
`
`Statistical analysis
`
`Eleven healthy adult control subjects (six males, ages
`19 39 years, and five females, ages 25 42 years) were
`enrolled. Five subjects with disorders affecting the urea
`cycle were also enrolled. This group was comprised of
`two male patients with severe, neonatal-onset ornithine
`transcarbamylase deficiency (OTCD) and three patients
`with argininosuccinate synthetase deficiency (ASSD).
`We
`also investigated six OTC-deficient
`females
`(asymptomatic and untreated). All adult controls were
`initially studied on a low protein intake [0.4 g/(kg day)].
`Five of these subjects were randomly assigned to a
`group for a second study when in addition to the low
`protein diet, they received Ucephan treatment [250 mg/
`(kg day)]. Symptomatic patients with UCD continued
`on their respective medical regimens in all studies. The
`untreated asymptomatic OTCD females were studied on
`the low protein diet [0.4 g/(kg day)]. Two of the symp-
`tomatic, treated OTCD females were studied first on low
`protein intakes and then on low protein intake with
`Ucephan [250 mg/(kg day)].
`
`Three different groups (healthy control subjects, null
`patients, and asymptomatic OTCD females) were ini-
`tially compared with a one-way analysis of variance
`(ANOVA). A P (two-tailed) of less than 0.05 was taken
`as statistically significant. Within a group, treatment
`effects (use of phenylbutyrate) were assessed by paired
`t tests.
`
`Results
`
`Steady state branched chain amino acid levels in UCD
`subjects in comparison with normal controls and asymp-
`tomatic OTCD females
`
`Plasma amino acids were determined in fed control,
`asymptomatic OTCD, and treated UCD patients after
`stabilization on the standard low protein diet. Inter-
`estingly, steady state serum BCAA (leucine, valine,
`and isoleucine) levels were found to be significantly
`
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`F. Scaglia et al. / Molecular Genetics and Metabolism 81 (2004) S79 S85
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`S81
`
`lower (P < 0:0001) in null UCD subjects that were
`treated with sodium phenylbutyrate when compared to
`normal controls or untreated asymptomatic OTCD
`females that participated in nitrogen flux studies (Ta-
`ble 1). Other essential non-BCAA (lysine and phen-
`ylalanine) were evaluated and no significant differences
`were found among the three groups while on the same
`diet (Table 2).
`
`Steady state BCAA levels in control subjects and asymp-
`tomatic OTCD females treated and not treated with
`Ucephan (sodium benzoate/sodium phenylacetate)
`
`Plasma amino acids were determined in a control
`group after stabilization on a low protein diet and
`
`then again after stabilization and treatment with so-
`dium phenylacetate/benzoate. Steady state leucine,
`valine, and isoleucine levels were significantly dimin-
`ished in the treatment period as compared to the pre-
`treatment period (P < 0:0001) (Table 3). As expected,
`steady state glutamine levels were also lower while on
`(P < 0:005). Other essential non-BCAAs
`treatment
`were compared and no statistically significant differ-
`ences were found between the pre-treatment and
`treatment periods (Table 4). Similarly, branched chain
`amino acids were evaluated in two OTCD females
`before and after treatment with phenylacetate/benzo-
`ate (Table 5). The three BCAAs were decreased, while
`no differences were observed among other essential
`amino acids (Table 6).
`
`Table 1
`Steady state serum branched chain amino acids in fed control, asymptomatic OTCD females, and treated UCD patients
`
`Subjects
`
`Plasma concentrations (lmol/L)
`
`Control (N
`11)
`Asymptomatic OTCD females (N
`Treated UCD (N
`5)
`Significance
`
`6)
`
`Leucine
`109 11
`110 12
`35 6
`P < 0:0001
`
`Valine
`173 34
`180 52
`87 42
`P < 0:0001
`
`Isoleucine
`51 13
`55 14
`24 14
`P < 0:0001
`
`Table 2
`Steady state serum non branched chain amino acids in fed controls, asymptomatic OTCD females, and UCD patients
`
`Subjects
`
`Plasma concentrations (lmol/L)
`
`Control (N
`11)
`Asymptomatic OTCD females (N
`Treated UCD (N
`5)
`Significance
`
`6)
`
`Threonine
`119 31
`126 53
`140 77
`NS
`
`Lysine
`186 30
`162 29
`163 9
`NS
`
`Glutamine
`518 71
`510 55
`579 100
`P < 0:01
`
`Methionine
`21 5
`24 6
`22 6
`NS
`
`Phenylalanine
`45 7
`42 3
`38 6
`NS
`
`Table 3
`Steady state serum branched chain amino acids and glutamine in control subjects treated with phenylacetate/benzoate
`
`Subjects (N
`
`5)
`
`Plasma concentrations (lmol/L)
`
`Before treatment
`After treatment
`Treatment effect
`
`Leucine
`106 6
`40 5
`P < 0:0001
`
`Valine
`171 33
`126 33
`P < 0:001
`
`Isoleucine
`51 12
`34 14
`P < 0:001
`
`Glutamine
`495 33
`438 34
`P < 0:005
`
`Table 4
`Steady state serum non branched chain essential amino acids in control subjects treated with phenylacetate
`
`Subjects (N
`
`5)
`
`Plasma concentrations (lmol/L)
`
`Before treatment
`After treatment
`Treatment effect
`
`Methionine
`22 3
`22 4
`NS
`
`Lysine
`177 36
`173 33
`NS
`
`Threonine
`128 28
`134 24
`NS
`
`Phenylalanine
`46 7
`48 4
`NS
`
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`F. Scaglia et al. / Molecular Genetics and Metabolism 81 (2004) S79 S85
`
`Table 5
`Effects of sodium phenylacetate on BCAA and glutamine in two asymptomatic OTCD female
`
`Subjects (N
`
`2)
`
`Plasma concentrations (lmol/L)
`
`Before treatment
`After treatment
`
`Leucine
`104 9
`58 13
`
`Isoleucine
`52 5
`30 10
`
`Valine
`199 12
`129 9
`
`Glutamine
`544 40
`505 63
`
`Table 6
`Effects of sodium phenylacetate on non branched chain essential amino acids on two asymptomatic OTCD female
`
`Subjects (N
`
`2)
`
`Plasma concentrations (lmol/L)
`
`Before treatment
`After treatment
`
`Phenylalanine
`40 2
`40 6
`
`Threonine
`114 6
`131 3
`
`Methionine
`25 1
`21 1
`
`Lysine
`218 17
`192 6
`
`Retrospective study of serum leucine and phenylalanine
`levels in UCD subjects treated with sodium phenylbuty-
`rate and sodium benzoate
`
`A retrospective study was conducted on five null
`UCD patients (three OTCD males, two male patients
`with carbamoylphosphate synthetase deficiency) over a
`period of
`three months after initiation of sodium
`phenylbutyrate therapy. Serum leucine levels declined
`over a period of three months despite increasing protein
`supplementation. The patientÕs protein sufficiency was
`appropriate as measured by growth and biochemical
`markers. For example, the lysine and phenylalanine
`levels remained stable (Figs. 1A and B). These patients
`also exhibited normal albumin and prealbumin levels
`(data not
`shown). Moreover, when leucine levels
`were evaluated over the same period of time in partial
`UCD patients who were treated with only sodium
`benzoate, a similar effect on BCAAs was not observed
`(Figs. 2A and B).
`
`Discussion
`
`We report that UCD patients treated with either so-
`dium phenylacetate/benzoate or with sodium phen-
`ylbutyrate exhibit a selective depression of steady state
`serum BCAA levels in the face of adequate protein in-
`take as measured by steady state levels of non-branched
`chain essential amino acids and albumin. In UCD pa-
`tients, phenylbutyrate is used as a ‘‘glutamine trap’’
`causing a decrease in plasma glutamine. This compound
`decreased plasma glutamine levels even in infants in
`whom plasma glutamine levels were within normal
`limits [2]. This effect is achieved via molar conversion of
`glutamine to phenylacetylglutamine. Glutamine is the
`most abundant amino acid in the body and comprises
`two-thirds of the intracellular amino acid pool
`[8].
`Glutamine can be synthesized de novo from glutamate
`
`and ammonia in a wide variety of tissues containing
`glutamine synthetase, and hence,
`it is considered a
`non-essential amino acid. However evidence has accu-
`mulated that glutamine might also play a role in the
`regulation of protein homeostasis.
`In hypercatabolic dogs adapted to a normocaloric,
`low protein diet, enteral glutamine supplementation
`
`A
`
`B
`
`Fig. 1. Retrospective study of serum leucine and phenylalanine levels in
`five UCD patients treated with sodium phenylbutyrate at one, two,
`and three months after initiation of treatment. (A) Serum leucine
`levels. (B) Serum phenylalanine levels.
`
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`
`S83
`
`improved nitrogen balance in conditions of protein
`wasting [12,13]. It has been shown that a short course
`treatment with large doses of phenylbutyrate in healthy
`adults with an intact urea synthetic pathway, could be
`used to create a model of glutamine depletion [5]. The
`observed decline in plasma glutamine was associated
`with decreased estimates of whole body protein
`synthesis.
`In this same study, the effect of phenylbutyrate on
`leucine kinetics was analyzed. Compared with the con-
`trol study day, plasma leucine concentrations were lower
`on the phenylbutyrate treatment day. Leucine oxidation
`increased in a statistically significant way on the phen-
`ylbutyrate day, and non-oxidative leucine disposal de-
`creased. Plasma leucine concentration was at near
`steady state during each of the isotope infusions. The
`possibility of any factors affecting the recovery of la-
`beled CO2 that would affect the measured rates of leu-
`cine oxidation was ruled out in this study. Of note is that
`the observed rise in leucine oxidation occurred at a time
`when plasma leucine concentration was lowered. Be-
`cause neither bicarbonate retention nor the ratio of
`plasma [2H3]ketoisocaproic (KIC) to [2H3]leucine en-
`richment was affected by phenylbutyrate, it was thought
`that the change in measured rates of leucine oxidation
`must have resulted from an actual change rather than
`from a change in the leucine-to-KIC isotopic ratio.
`A plausible hypothesis that might explain the cause of
`BCAA depletion by ammonia scavengers could be based
`on the excessive formation of phenylacetylglutamine
`and the subsequent reduction of the intracellular gluta-
`mate pool. This pool might be partly restored by in-
`tensified BCAA transamination of a-ketoglutarate. To
`support this hypothesis, a decrease in BCAA levels has
`been observed in cases of idiopathic portal hypertension,
`a condition characterized by extensive portal-systemic
`shunting, and by hyperammonemia and hypergluta-
`minemia [6,7]. Further support comes from the fact that
`glutamate production from a-ketoglutarate utilizes
`BCAA-derived amino groups [14,15] and that hyper-
`ammonemia increases the activity of muscle BCAA
`aminotransferase [16]. It has been shown that the ad-
`ministration of BCAA to rats with hepatic failure has
`raised the amount of glutamate in brain tissue [17].
`Since exogenous hyperammonemia is known to de-
`crease the plasma levels of BCAA, an animal study with
`ammonium-infused rats was conducted to investigate
`whether changes in intracellular amino acid concentra-
`tions of muscle are associated with that effect [18]. The
`intracellular amino acid concentrations assessed in this
`particular study supported the concept under discussion
`since increased values of glutamine and decreased values
`of glutamate and alanine were seen with the deficiency
`of plasma BCAA. The fall in the alanine level could be
`explained by a decreased availability of glutamate for
`transamination with pyruvate [19]. The fall in muscle
`
`Fig. 2. Retrospective study of serum leucine and phenylalanine levels in
`two UCD patients treated only with sodium benzoate at one, two, and
`three months after initiation of treatment. (A) Serum leucine levels. (B)
`Serum phenylalanine levels.
`
`decreased leucine oxidation. It did so by improving net
`leucine balance, and thus preserving body protein [9].
`This was associated with an approximately 26% reduc-
`tion in leucine oxidation (P < 0:05) with no change in
`protein release from protein breakdown.
`In vitro studies in differentiated Caco-2 enterocyte-
`like cells demonstrated that inhibition of glutamine
`synthesis slowed down cell protein synthesis, and a
`supply of glutamine under these conditions acutely re-
`stored protein synthesis in a dose-dependent fashion.
`This suggests that the maintenance of intracellular glu-
`tamine plays a significant physiological role in the con-
`trol of protein synthesis in a cell line of human origin
`that exhibits an enterocytic differentiation in vitro [10].
`Similar findings in human studies were found when
`the effect of enteral glutamine on whole body protein
`metabolism in healthy subjects was studied. Doubling of
`plasma glutamine concentration obtained through an
`enteral infusion of L-glutamine in healthy subjects in the
`postabsorptive state, inhibited leucine oxidation without
`affecting leucine release from proteolysis. As a conse-
`quence, it improved net leucine balance by increasing
`non-oxidative leucine disposal (NOLD), an index of
`whole body protein synthesis [11]. Replenishment of
`muscle glutamine stores has been associated with
`
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`F. Scaglia et al. / Molecular Genetics and Metabolism 81 (2004) S79 S85
`
`BCAA levels was preceded by a reduction of plasma
`levels, implying that the organism attempts to keep the
`intracellular rather than the extracellular BCAA levels
`constant. An alternative mechanism that could explain
`the replenishment of the glutamate pool could be the
`degradation of valine and isoleucine that could supply
`the a-ketoglutarate carbon skeleton via succinate.
`Control of protein synthesis by amino acid avail-
`ability is an area that continues to be investigated. One
`particular advance is the understanding of how amino
`acids, and in this particular case leucine, serve not only
`as precursors but as signaling molecules in protein
`synthesis. Earlier studies had established an important
`role for the BCAA in regulating protein synthesis in
`skeletal muscle [20]. More recent studies have shown
`that leucine is the most potent of the BCAA in en-
`hancing mRNA translation. Oral administration of
`leucine to fasted rats invokes the same stimulation of
`protein synthesis in skeletal muscle as is observed fol-
`lowing consumption of a complete meal [21]. Further
`support for a direct effect of leucine on protein synthesis,
`is the observation in the same study that the stimulating
`effect of leucine is associated with increased activity of
`the eukaryotic initiation factor 4F [21]. These data
`suggest a stimulatory effect of leucine on protein syn-
`thesis. The available evidence suggests that
`leucine
`stimulates translation initiation by increasing the phos-
`phorylation status of the translational repressor of 4E-
`BP1 and the ribosomal protein S6 kinase (S6K1) [21,22].
`The signal transduction pathway through which leucine
`promotes hyperphosphorylation of 4E-BP1 and S6K1 is
`not completely defined, nevertheless it is clear that the
`mammalian target of rapamycin protein kinase (mTOR)
`must be active in order for leucine to be effective [23].
`Therefore one of the predicted direct outcomes of the
`effects of ammonia scavengers would be to inhibit body
`protein synthesis.
`We hypothesize that the depletion of serum BCAA
`that accompanies the treatment with sodium phenylbu-
`tyrate can be a secondary effect of glutamine depletion
`per se. The assumption that a depleted glutamate pool is
`partly restored by an intensified BCAA transamination
`cannot be completely proved by the present study and
`results, although the findings obtained could be consis-
`tent with this hypothesis. An alternative hypothesis of a
`direct effect of sodium phenylbutyrate through stimu-
`lation of BCKD cannot be completely ruled out.
`In conclusion, we suggest that BCAA supplementa-
`tion might allow for further titration of protein intake in
`the dietary management of UCD patients. Further
`studies need to be conducted in order to determine
`whether phenylbutyrate might indirectly induce trans-
`amination of BCAA to restore decreased intracellular
`glutamate pools or whether an increased rate of leucine
`oxidation could be a direct effect of sodium phenylbu-
`tyrate through stimulation of BCKD, rather than a di-
`
`rect effect of intracellular glutamate depletion. Further
`stable isotope studies are underway to clarify these
`issues.
`
`Acknowledgments
`
`The authors acknowledge the skills of the nursing
`staff of the GCRC. The authors thank Olivia Hernandez
`for administrative assistance, and the excellent nursing
`staff at the Texas ChildrenÕs Hospital General Clinical
`Research Center. This work is dedicated to the memory
`of Dr. Peter Reeds. The work was supported in part
`by the Baylor College of Medicine General Clini-
`cal Research Center
`(RR00188), Mental Retarda-
`tion and Developmental Disabilities Research Center
`(HD024064),
`the Child Health Research Center
`(HD041648), and the NIH (DK54450).
`
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