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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2013 Dec 18;34(3):460–466. doi: 10.1038/jcbfm.2013.218

Effect of glutamine synthetase inhibition on brain and interorgan ammonia metabolism in bile duct ligated rats

Andreas W Fries 1, Sherry Dadsetan 2, Susanne Keiding 1,3, Lasse K Bak 2, Arne Schousboe 2, Helle S Waagepetersen 2, Mette Simonsen 1, Peter Ott 3, Hendrik Vilstrup 3, Michael Sørensen 1,3,*
PMCID: PMC3948122  PMID: 24346692

Abstract

Ammonia has a key role in the development of hepatic encephalopathy (HE). In the brain, glutamine synthetase (GS) rapidly converts blood-borne ammonia into glutamine which in high concentrations may cause mitochondrial dysfunction and osmolytic brain edema. In astrocyte-neuron cocultures and brains of healthy rats, inhibition of GS by methionine sulfoximine (MSO) reduced glutamine synthesis and increased alanine synthesis. Here, we investigate effects of MSO on brain and interorgan ammonia metabolism in sham and bile duct ligated (BDL) rats. Concentrations of glutamine, glutamate, alanine, and aspartate and incorporation of 15NH4+ into these amino acids in brain, liver, muscle, kidney, and plasma were similar in sham and BDL rats treated with saline. Methionine sulfoximine reduced glutamine concentrations in liver, kidney, and plasma but not in brain and muscle; MSO reduced incorporation of 15NH4+ into glutamine in all tissues. It did not affect alanine concentrations in any of the tissues but plasma alanine concentration increased; incorporation of 15NH4+ into alanine was increased in brain in sham and BDL rats and in kidney in sham rats. It inhibited GS in all tissues examined but only in brain was an increased incorporation of 15N-ammonia into alanine observed. Liver and kidney were important for metabolizing blood-borne ammonia.

Keywords: alanine, amino acids, hepatic encephalopathy, hyperammonemia, methionine sulfoximine (MSO)

Introduction

Hepatic encephalopathy (HE) is a neuropsychiatric complication of liver failure with symptoms ranging from subtle changes in conscious level to somnolence, coma, and death. Liver failure with decreased production of urea and/or portosystemic shunting leads to hyperammonemia, which in turn leads to increased brain uptake of ammonia from blood.1, 2 It is widely accepted that hyperammonemia has a central role in the development of HE3 although the precise mechanisms remain unsolved. When blood-borne ammonia enters the brain, it is rapidly incorporated into glutamine by glutamine synthetase (GS) which is located in astrocytes.4, 5, 6 Glutamine concentrations were increased in brains of animals with experimental liver failure7, 8, 9 and in patients with acute liver failure studied with cerebral microdialysis.10 Glutamine acts directly as an osmolyte causing brain edema11 and studies suggest that high intracellular concentrations of glutamine may also compromise mitochondrial function12 and lead to oxidative stress.13 It is thus likely that the detrimental effects of ammonia on brain function in liver failure are primarily attributable to excess cerebral production of glutamine.

Methionine sulfoximine (MSO) is a well-known inhibitor of GS and caused a significant reduction in incorporation of 13N-ammonia into brain glutamine in healthy rats.6 Interestingly, studies in astrocyte-neuron cocultures revealed increased alanine synthesis from [U-13C]glucose when exposed to ammonia14 and the synthesis was further increased when MSO was added.15 A recent study showed increased fixation of 15NH4+ in alanine in brain of healthy rats and astrocyte-neuron cocultures during MSO inhibition of GS and in the cocultures the fixation in alanine was found to be dose dependent concerning the ammonia concentration.16These studies point to a potential role of alanine synthesis in brain as an alternative ammonia-scavenging pathway, especially during inhibition of GS with MSO. The median lethal dose (LD50) of acutely administrated ammonia was 50% higher in mice pretreated with MSO than in mice treated only with ammonia17 and MSO reduced brain edema in hyperammonemic rats.18, 19, 20, 21 It is thus likely that MSO is able to protect the brain against ammonia toxicity by inhibiting excess production of glutamine and increasing ammonia fixation in alanine. In support of this, increased blood ammonia and cerebral glutamine concentration, but not increased cerebral alanine concentration, correlated with the intracranial pressure and risk of incarceration in patients with fulminant hepatic failure.10 In the brain, GS is predominantly, if not exclusively located to astrocytes5 but otherwise found ubiquitously in extracerebral tissues. However, except for one study showing reduced GS activity in liver tissue of healthy rats,22 there are no studies showing how MSO may affect interorgan ammonia metabolism.

The present study was designed to study interorgan ammonia metabolism in sham-operated and bile duct ligated (BDL) rats during inhibition of GS with MSO; the BDL rat is an accepted experimental model of chronic liver disease with hyperammonemia.23 Concentrations of the four amino acids glutamine, glutamate, alanine, and aspartate and incorporation of 15NH4+ into these amino acids were measured in brain, liver, muscle, kidney tissues, and plasma after an intravenous infusion of 15N-ammonia administered 3 hours after treatment with MSO or saline (vehicle). The percent 15N-labeling of metabolites provides information on the immediate metabolism (e.g., incorporation of blood-borne 15N-ammonia in glutamine) whereas the concentration measurements depend on several parameters such as substrate import/export and substrate degradation. We hypothesized that MSO would inhibit GS in all tissues leading to decreased synthesis and 15N-labeling of glutamine and increased synthesis and 15N-labeling of alanine in brain and alanine and/or aspartate in other tissues via the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT), respectively. We did not expect to find any differences between BDL rats and healthy rats.

Materials and methods

Animals and Chemicals

Female Wistar rats (body weight 235 to 288 g) were obtained from Møllegaard Breeding Centre, University of Aarhus (Denmark). Methionine sulfoximine was purchased from Sigma Chemical Co. (St Louis, MO, USA) and 15NH4Cl from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). All other chemicals used were of the purest grade available from regular commercial sources.

The protocol was approved by the Danish Animal Research Committee and conducted in accordance with Danish law on animal experiments. The rats had free access to water and standard rodent chow and were housed with two animals per cage at 22±2°C, 55±10% relative humidity in the animal facilities at Aarhus University. The animals were subjected to either sham or BDL operation. The surgical procedures have been previously described in detail.24

Methionine Sulfoximine Experiments

Six weeks after surgery, the rats were randomized to intraperitoneal injection of either MSO (150 mg/kg dissolved in 1 mL saline) or 1 mL isotone saline (vehicle). Just before administration of MSO or vehicle, a sample of eye capillary blood was collected for measurement of baseline blood concentration of ammonia.25 Three hours after injection of MSO or vehicle, the rats were subjected to inhalation anesthesia (Isoflurane) and a catheter was placed in a tail vein through which an intravenous infusion of 15N-ammonia (2 mmol/kg in 1.2 mL saline) was given over the time course of 15 minutes. Thirty seconds before the end of the infusion, a blood sample was collected by cardiac puncture and centrifuged (13,000 g, 10 minutes) and plasma was collected. Immediately after the cardiac puncture, the rats were decapitated and samples of brain cortex, liver, kidneys, and skeletal muscle (femoral muscle) were collected. The tissue samples were frozen in liquid nitrogen, the brain tissue sample within 40 to 50 seconds and the other tissue samples within 1 to 3 minutes after the blood sampling. The frozen tissue samples were extracted in ice-cold 70% v/v ethanol, homogenized, and centrifuged (20,000 g, 20 minutes). The supernatants were separated from the pellets and both were stored at −80°C until further analysis.

Gas Chromatography Coupled to Mass Spectrometry and High Performance Liquid Chromatography Analyses

Plasma samples and tissue extracts used for determination of percent 15N-labeling of amino acids were derivatized with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide in the presence of 1% tert-butyldimethylchlorosilane26 followed by GC–MS (gas chromatography coupled to mass spectrometry). Labeling measurements were corrected for natural isotopic abundance by subtracting the mass distribution of a standard and isotopic enrichment was calculated according to Biemann27 and reported as percent labeling. Concentrations of glutamine, glutamate, alanine, and aspartate in plasma and tissue extracts were determined using reversed-phase high performance liquid chromatography. The amino acids were separated on an Agilent Eclipse AAA column (4.6 mm × 150 mm, particle size 5 μm) after precolumn o-phthaldialdehyde derivatization using citrate-phosphate/acetonitrile mobile phases and fluorescence detection (excitation 350 nm; detection 450 nm) modified after Geddes and Wood.28 Protein content was determined in the dissolved tissue pellets (1 mol/L KOH at 20°C for 24 hours) according to Lowry et al29 using bovine serum albumin as standard. Amino-acid concentrations are reported pr. mg protein.

Figure 1 shows the main metabolic pathways leading to incorporation of intravenously infused 15NH4+ (15N-ammonia) into glutamate, glutamine, alanine, and aspartate.

Figure 1.

Figure 1

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Schematic cartoon of possible pathways for 15N-labeling of glutamate, glutamine, alanine, and aspartate in brain from blood-borne 15NH4+. Under normal conditions, the majority of 15N-ammonia taken up from blood reacts rapidly with glutamate, forming mono-labeled [5-15N]glutamine in a reaction catalyzed by glutamine synthetase (GS), which in brain is found only in astrocytes.5 15N-glutamate can also react with α-ketoglutarate forming 15N-glutamate in a reaction catalyzed by glutamate dehydrogenase (GDH). If 15N-glutamate reacts with non-labeled ammonia, then mono-labeled [2-15N]glutamine is formed and if 15N-glutamate reacts with15N-ammonia, then double-labeled [2,5-15N]glutamine is formed. Alternatively, 15N-glutamate can be transaminated to α-ketoglutarate forming 15N-alanine and 15N-aspartate catalyzed by alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT), respectively.

Statistical Analysis

Differences between groups were analyzed statistically using one-way ANOVA followed by Tukey post hoc test. Data are given as median (range). P<0.05 was considered to indicate a statistically significant difference.

Results

Before MSO treatment, mean ammonia concentration in eye capillary plasma was 50±7 μmol/L in sham rats and 123±28 μmol/L in BDL rats (P<0.05). For technical reasons, plasma ammonia was not measured before 15N-ammonia infusion. However, at the end of the 15N-ammonia infusion the plasma concentration of ammonia had increased 20-fold (to range, 1,600 to 2,000 μmol/L) in all four groups of animals with no significant differences between the groups (P>0.05).

In all tissues and plasma, none of the concentrations or the extent of 15N-labeling of the analyzed amino acids were significantly different between sham+vehicle and BDL+vehicle (Table 1). This means that any differences in concentrations and/or extent of labeling when comparing sham+MSO vs. sham+vehicle and BDL+MSO vs. BDL+vehicle can be ascribed to effects of MSO and not BDL.

Table 1. Concentrations and percent 15N-enrichment of amino acids in rats.

Amino acid Sham+vehicle Sham+MSO BDL+vehicle BDL+MSO
Brain
 Glutamine
  Conc. (nmol/mg prot.) 29.7 (16.1–105.3) 31.5 (24.0–39.2) 31.5 (24.2–250.0) 50.2 (16.1–105.3)
  Mono-label (%) 28.3 (6.6–30.9) 13.7 (6.9–16.2)* 26.5 (17.3–30.1) 14.0 (5.5–17.6)***
  Double-label (%) 0.2 (0.0–2.4) 1.7 (0.4–3.6) 1.0 (0.0–3.0) 1.6 (0.0–4.0)
         
 Glutamate
  Conc. (nmol/mg prot.) 48.1 (44.2–180.1) 42.2 (34.9–47.2) 45.4 (3.6–184.1) 55.2 (30.4–147.9)
  Label (%) 0.1 (0.0–0.2) 0.8 (0.0–1.7)* 0.0 (0.0–0.4) 1.0 (0.4–1.8)***
         
 Alanine
  Conc. (nmol/mg prot.) 3.6 (0.5–9–6) 3.0 (1.3–4.4) 2.1 (0.2–10.5) 4.9 (1.1–7.2)
  Label (%) 0.0 (0.0–0.2) 4.4 (2.3–8.0)** 0.0 (0.0–1.1) 6.4 (0.0–9.1)***
         
 Aspartate
  Conc. (nmol/mg prot.) 10.6 (7.4–25–9) 8.1 (6.8–16.9) 9.6 (5.7–31.6) 9.8 (4.8–30.0)
  Label (%) 0.0 (0.0–1.7) 1.3 (0.5–2.2) 1.1 (0.0–1.9) 1.1 (0.2–2.2)
         
Liver
 Glutamine
  Conc. (nmol/mg prot.) 16.7 (6.3–28.6) 2.8 (1.5–4.0)*** 14.3 (1.1–32.3) 2.3 (1.2–5.6)***
  Mono-label (%) 24.1 (8.0–32.5) 3.3 (0.0–10.9)*** 22.5 (8.0–34.4) 1.5 (0.0–12.9)***
  Double-label (%) 1.8 (0.0–4.0) 0.2 (0.0–2.5) 1.4 (0.0–4.0) 0.2 (0.0–2.3)
         
 Glutamate
  Conc. (nmol/mg prot.) 3.6 (1.9–5.2) 3.5 (2.1–6.1) 5.2 (0.6–16.9) 5.9 (0.9–20.8)
  Label (%) 11.3 (2.1–21.3) 15.7 (0.0–26–2) 11.2 (0.0–18.5) 9.8 (0.0–25.0)
         
 Alanine
  Conc. (nmol/mg prot.) 7.4 (5.9–13.7) 10.8 (3.8–18.0) 6.7 (0.6–12.1) 8.9 (1.0–15.3)
  Label (%) 19.5 (4.3–31.2) 28.7 (3.5–37.2) 17.9 (0.0–30.4) 17.4 (2.8–33.4)
         
 Aspartate
  Conc. (nmol/mg prot.) 1.4 (0.8–2.5) 1.9 (0.7–3.2) 1.6 (0.5–3.9) 3.0 (0.9–4.7)
  Label (%) 10.1 (0.0–18.1) 10.3 (1.2–21.0) 8.7 (3.2–16.6) 7.8 (0.0–20.9)
         
Muscle
 Glutamine
  Conc. (nmol/mg prot.) 5.9 (1.9–16.1) 4.6 (2.1–10.8) 6.0 (2.1–14.4) 5.5 (0.7–12.9)
  Mono-label (%) 8.7 (2.0–14.8) 0.0 (0.0–2.0)*** 8.0 (2.6–12.1) 0.3 (0.0–1.7)***
  Double-label (%) 0.5 (0.0–1.8) 0.1 (0.0–0.7) 0.0 (0.0–0.7) 0.5 (0.0–0.8)
         
 Glutamate
  Conc. (nmol/mg prot.) 1.9 (0.2–3.3) 2.2 (0.3–8.0) 1.1 (0.5–3.1) 1.9 (0.4–5.9)
  Label (%) 0.0 (0.0–0.0) 0.0 (0.0–1.5) 0.0 (0.0–0.7) 0.0 (0.0–2.1)
         
 Alanine
  Conc. (nmol/mg prot.) 4.8 (2.2–11.1) 5.0 (1.5–21.5) 2.3 (1.5–4.7) 3.3 (2.0–8.7)
  Label (%) 0.0 (0.0–0.0) 0.0 (0.0–0.9) 0.0 (0.0–1.0) 0.0 (0.0–0.4)
         
 Aspartate
  Conc. (nmol/mg prot.) 0.3 (0.0–1.0) 1.1 (0.2–2.5)* 0.2 (0.0–0.6) 0.8 (0.2–2.4)*
  Label (%) 0.0 (0.0–8.8) 0.0 (0.0–7.4) 0.6 (0.0–3.7) 0.0 (0.0–2.3)
         
Kidney
 Glutamine
  Conc. (nmol/mg prot.) 2.5 (1.2–4.3) 0.6 (0.4–1.0)*** 3.3 (1.5–4.4) 0.6 (0.4–1.2)***
  Mono-label (%) 17.6 (13.1–36.7) 5.3 (0.0–8.9)*** 30.2 (13.3–36.6) 3.0 (0.0–12.8)***
  Double-label (%) 0.7 (0.0–9.4) 0.1 (0.0–6.7) 3.5 (1.5–6.4) 0.0 (0.0–6.4)
         
 Glutamate
  Conc. (nmol/mg prot.) 22.8 (22.3–23.3) 19.9 (14.3–28.5) 22.0 (18.4–37.5) 19.9 (10.3–22.1)
  Label (%) 11.7 (3.0–17.8) 18.8 (14.1–23.5)** 15.0 (7.7–21.0) 15.5 (5.7–21.7)
         
 Alanine
  Conc. (nmol/mg prot.) 5.2 (3.8–6.0) 4.8 (4.4–5.6) 4.1 (3.6–4.9) 4.8 (2.9–5.5)
  Label (%) 3.6 (0.7–9.6) 8.2 (5.4–12.7)* 3.7 (0.5–10.9) 5.7 (1.2–12.4)
         
 Aspartate
  Conc. (nmol/mg prot.) 4.4 (3.7–6.1) 5.6 (4.6–7.4) 3.9 (2.5–7.4) 4.7 (2.8–6.4)
  Label (%) 9.5 (1.1–13.6) 16.6 (12.1–23.6)** 9.5 (4.7–18.2) 12.4 (6.1–20.8)
         
Plasma
 Glutamine
  Conc. (μmol/L) 743 (533–963) 171 (110–221)*** 815 (522–886) 202 (106–299)***
  Mono-label (%) 21.1 (14.2–33.8) 4.3 (2.1–15.9)*** 20.2 (12.9–26.8) 9.4 (2.0–27.8)***
  Double-label (%) 1.7 (0.0–2.7) 0.7 (0.0–8.2) 1.2 (0.0–2.3) 0.3 (0.0–4.1)
         
 Glutamate
  Conc. (μmol/L) 52.4 (26.6–61.2) 80.8 (53.2–93.0) 56.4 (33.8–79.6) 80.1 (64.6–249)
  Label (%) 2.1 (0.2–5.1) 4.4 (2.3–12.8) 2.2 (0.0–14.4) 3.1 (0.0–8.7)
         
 Alanine
  Conc. (μmol/L) 530 (438–589) 779 (501–994)* 640 (505–874) 851 (585–1,275)**
  Label (%) 1.5 (0.0–4.1) 1.0 (0.0–6.2) 0.9 (0.0–2.4) 3.3 (0.0–7.0)
         
 Aspartate
  Conc. (μmol/L) ND ND ND ND
  Label (%) ND ND ND ND
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BDL, bile duct ligated; MSO, methionine sulfoximine; ND, not detectable.

Rats were treated with vehicle or MSO 3 hours before 15N-ammonia was administered. Number of rats in each group is 6 to 10. Values are given as median (range).

*P<0.05, **P<0.01, and ***P<0.001 indicate statistically significant difference from respective vehicle group.

No statistical significant differences were found in concentrations or percent 15N-labeling of amino acids between sham+vehicle and BDL+vehicle or between sham+MSO and BDL+MSO in any tissue except for kidneys.

All concentration and labeling measurements are presented in Table 1.

Brain

Methionine sulfoximine treatment did not affect the concentrations of glutamine, glutamate, alanine, or aspartate in the brains of sham or BDL rats when compared with their respective control groups, i.e., rats treated with vehicle (Table 1). Methionine sulfoximine reduced mono-labeling of glutamine in both sham and BDL rats to approximately half the value in respective vehicle groups (Table 1; Figure 2); double-labeling was low and unaffected by MSO (Table 1). A significant inhibition of brain GS by MSO was accordingly observed. The discrepancy between a significant inhibition of incorporation of blood-borne ammonia in glutamine and no effect on the glutamine concentration indicates that the glutamine pool in the brain has a slow turnover and that the brain is able to maintain a constant glutamine concentration even when glutamine synthesis from blood-borne ammonia is inhibited. Methionine sulfoximine increased 15N-labeling of glutamate and alanine in both sham and BDL rats (Table 1; Figure 3) and had no effect on the labeling of aspartate (Table 1). These observations indicate that during MSO inhibition of GS in the brain, the concerted action of GDH and ALAT becomes important for ammonia fixation in alanine whereas ASAT has no role as a potential ammonia fixation in aspartate.

Figure 2.

Figure 2

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Effect of methionine sulfoximine (MSO) on mono-labeling of glutamine from 15N-ammonia in (A) brain tissue, (B) liver tissue, (C) muscle tissue, and (D) kidneys. Median values are indicated by horizontal lines. Rats were treated with vehicle (saline) or MSO 3 hours before administration of 15N-ammonia. NS, no significant difference (P>0.05). BDL, bile duct ligated.

Figure 3.

Figure 3

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Effect of methionine sulfoximine (MSO) on labeling of alanine from 15N-ammonia in (A) brain tissue, (B) liver tissue, (C) muscle tissue, and (D) kidneys. Median values are indicated by horizontal lines. Rats were treated with vehicle (saline) or MSO 3 hours before administration of 15N-ammonia. NS, no significant difference (P>0.05). BDL, bile duct ligated.

Liver

In the liver, MSO treatment reduced the concentration of glutamine in both sham and BDL rats when compared with vehicle (Table 1). Methionine sulfoximine reduced mono-labeling of glutamine in both sham and BDL rats (Table 1; Figure 2); double-labeling was low and unaffected by MSO (Table 1). The observation that both concentration and mono-labeling of glutamine in liver tissue decreased significantly after MSO treatment indicates that the glutamine pool in liver tissue has a fast turnover and is closely related to metabolism of blood-borne ammonia. Methionine sulfoximine did not affect the concentrations or the extent of 15N-labeling of glutamate, alanine, or aspartate (Table 1; Figure 3).

Muscle

Methionine sulfoximine treatment did not affect the concentration of glutamine in muscle tissue in neither sham nor BDL rats when compared with vehicle (Table 1). It reduced mono-labeling of glutamine (Table 1; Figure 2); double-labeling was low and unaffected by MSO (Table 1). As observed for the brain, this indicates that the glutamine pool in muscle tissue is not dependent on blood ammonia. The concentrations and extent of labeling of glutamate and alanine were unaffected by MSO in both sham and BDL rats (Table 1; Figure 3). The concentration of aspartate was increased and the labeling unaffected in both sham and BDL rats (Table 1). Although the concentrations and labeling of alanine and aspartate were low, the data indicate that the ammonia pathway through muscle ASAT is more important than through ALAT when the ammonia concentration is high due to inhibited GS.

Kidney

Methionine sulfoximine treatment reduced the concentration of glutamine in kidney tissue in both sham and BDL rats (Table 1; Figure 2). It reduced mono-labeling of glutamine in both groups of animals (Table 1; Figure 2); double-labeling was low and unaffected by MSO (Table 1). As for the liver, this indicates a fast turnover of the glutamine pool in kidney tissue which is closely related to metabolism of blood-borne ammonia. Methionine sulfoximine did not affect the concentrations of glutamate, alanine, and aspartate in neither sham nor BDL rats (Table 1). Its treatment increased 15N-labeling of glutamate, alanine, and aspartate in the sham rats but not in the BDL rats (Table 1; Figure 3) indicating an increased fixation of blood-borne ammonia in both alanine and aspartate via the concerted actions of GDH and ALAT and GDH and ASAT, respectively, in healthy but not in BDL rats.

Plasma

Methionine sulfoximine treatment reduced plasma concentration of glutamine in both sham and BDL rats (Table 1) and reduced mono-labeling of glutamine in both sham and BDL rats; double-labeling was low and unaffected by MSO (Table 1). Plasma concentration of alanine was increased by MSO and the concentration of glutamate was unaffected in both sham and BDL rats (Table 1). The extent of labeling of alanine and glutamate was unaffected by MSO in both groups of animals (Table 1). Aspartate was not detectable in plasma.

Discussion

The high mono-labeling of glutamine in brain of ∼25% and low double-labeling after the 15-minute 15N-ammonia infusion show that blood-borne ammonia is readily incorporated in brain glutamine by GS in both sham and BDL rats not treated with MSO. This finding is in accordance with previous findings using the tracer 13N-ammonia and shows, together with the similar concentration of glutamine and glutamate and the relatively low incorporation of 15N-ammonia into glutamate, that in the brain, GS is more important in metabolizing blood-borne ammonia than is GDH.6 Interestingly, incorporation of 15N-ammonia into glutamate was increased 7- to 10-fold by MSO, which implies increased flux of 15N-ammonia via GDH when GS is inhibited. This is probably a direct result of inhibition of GS in astrocytes that allows blood-borne ammonia to diffuse from the astrocytes into neurons and thus become available for neuronal GDH also.6

Methionine sulfoximine treatment caused a dramatic increase in percent 15N-labeling of alanine in brain which must be synthesized de novo via the concerted action by GDH and ALAT (transreamination) since alanine is not transported from blood to brain in rats in measurable amounts.30 This supports that brain alanine synthesis may be an alternative ammonia-scavenging pathway in BDL rats, confirming the hypothesis based on previous findings in astrocyte-neuron cocultures and healthy rats, especially during inhibition of GS with MSO.14, 15, 16 We did not, however, observe an increase in alanine concentration in brain tissue after MSO treatment which is in contrast to what was expected from the study in astrocyte-neuron cocultures.15 The most plausible explanation for this apparent discrepancy is efflux of alanine from brain to the blood stream in vivo,31 which is an interesting observation because it suggests that during MSO inhibition of GS, the brain is able to fix ammonia entering from the blood in alanine which then leaves the brain. The lack of any effect of MSO on the 15N-labeling of aspartate in brain tissue indicates that ASAT does not represent an alternative ammonia-scavenging pathway in brain during inhibited GS.

Methionine sulfoximine inhibited GS significantly in all four tissues examined as illustrated by the significant decrease in percent 15N-labeling of glutamine but a significant decrease in the concentration of glutamine was only observed in liver and kidney. This suggests that the glutamine pools in these tissues have high turnovers and are closely related to metabolism of blood-borne (exogenous) ammonia. In muscle, the concentration of glutamine was not affected by MSO but the extent of labeling from 15N-ammonia decreased from almost 8% to nearly 0% showing a practically complete inhibition of glutamine production from blood-borne ammonia. The dramatic decrease in both concentration and extent of labeling of glutamine in plasma indicates that plasma glutamine primarily derives from liver and kidneys. This underlines the role of these two organs in removing both endogenous and blood-borne ammonia.

Methionine sulfoximine did not affect the concentration of alanine in brain, liver, muscle, or kidney but plasma concentration increased by 40%. 15N-labeling of plasma alanine was low and unaffected by MSO, which indicates that the major proportion of alanine in plasma was not produced from the infused 15N-ammonia but rather from endogenously produced ammonia. This agrees with the general view of GS being the main pathway for tissues to metabolize ammonia generated from breakdown of amino acids; when blocking GS by MSO, large amounts of ammonia will accumulate and result in increased flux via other ammonia-scavenging pathways. Although the highest percentage 15N-labeling of alanine was found in the liver, in accordance with the high levels of ALAT in hepatocytes, 15N-labeling of alanine did not increase after MSO. This was also the case for muscle where 15N-labeling of alanine was close to zero and did not increase after MSO revealing a limited role of alanine production in muscle tissue during inhibition of GS. In patients with cirrhosis32, 33 and patients with fulminant hepatic failure,34 the flux of alanine measured by arterio-venous balance studies across muscle tissue did not show net uptake or release of alanine and hyperammonemia induced by an oral load of branched-chain amino acids in healthy subjects and patients with cirrhosis did not affect the flux but an increase in glutamine release was observed after 3 hours, but only in healthy subjects.32 The main conclusion of this study, which aimed at unraveling effects of branched-chain amino acids on muscle ammonia metabolism, was that muscle metabolism of blood-borne ammonia is actually limited and not stimulated by branched-chain amino acids. Interestingly, in patients with cirrhosis and transjugular intrahepatic portosystemic shunt, measurements in arterial and portal venous blood showed net release of alanine from the prehepatic splanchnic tissues33 and significant amounts of alanine and ammonia were released from the hepato-splanchnic area in patients with acute liver failure.34 These results point to the intestines as a major site for alanine production. We did not include intestinal tissue in the present study because it was not possible to homogenize this tissue satisfactorily for the analyses but based on the studies in human subjects it is likely that part, if not most of the alanine observed in plasma in the present study, originated from the hepato-splanchnic area.

The concentration and extent of 15N-labeling of aspartate was unaffected in liver tissue whereas the concentration but not the percent labeling increased in muscle tissue. This finding points to a potential role of ASAT in muscle tissue for removing intrinsic but not blood-borne ammonia when muscle GS is inhibited. Interestingly, it seems that the ability of the kidneys to increase incorporation of 15N-ammonia into alanine and aspartate after treatment with MSO was lost in BDL rats, which may be a result of chronic kidney failure. Aspartate production thus does not seem to be a significant alternative ammonia-scavenging pathway during hyperammonemia with or without MSO inhibition of GS in brain, liver, muscle, or kidney. In accordance with this, plasma concentration of aspartate remained non-detectable in animals treated with MSO.

A study found a 3-fold increase in arterial blood ammonia concentration in normal rats treated with MSO.6 In the present study, the effect of MSO on blood ammonia was blurred by the use of 15N-ammonia which requires administration of a large dose of the isotope. We chose to use the stable isotope 15N-ammonia because of high analytical accuracy and precision of the GC–MS measurement of the relative 15N-labeling of amino acids in tissue samples which cannot be obtained by administration of tracer doses of for example positron-labeled 13N-ammonia.35 The use of non-tracer doses of 15N-ammonia may also have blurred potential differences in amino-acid metabolism between sham and BDL rats.

In summary, MSO inhibited GS in brain, liver, muscle, and kidney but only in liver and kidney did it affect glutamine concentrations, which suggests that these tissues are more important in the metabolism of blood-borne ammonia, also in BDL rats. The study confirmed the hypothesis that during MSO inhibition of GS, the concerted action of GDH and ALAT provides an alternative ammonia-scavenging pathway in the brain in both healthy and BDL rats. The study furthermore suggests that alanine generated from blood-borne ammonia is able to leave the brain rather fast and that the brain was able to maintain a constant glutamine concentration in spite of glutamine synthesis from blood-borne ammonia was inhibited. These results are promising for manipulating brain metabolism during hyperammonia since the evidence of glutamine playing a significant role in the development of HE is substantial7, 8, 9, 10, 11, 12, 13 and animal studies points to a beneficial effect of inhibition GS by MSO.17, 18, 19, 20, 21 In support of this, two recent reviews considered the potential use of MSO in patients with acute liver failure and limited treatment offers.36, 37 It seems unlikely, though, that MSO will be considered as a possible treatment for hyperammonemia in patients with chronic liver disease as convulsions have been observed in animals administered MSO38, 39 but the possibility of designing analogs of MSO that inhibit GS without having the undesired side effects produced by MSO should be considered.

The authors declare no conflict of interest.

Footnotes

This study was supported in part by the Lundbeck Foundation, The Danish Council for Independent Research (Medical Sciences, 11-105338), and the A.P. Møller Foundation for the Advancement of Medical Science.

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