Michael Sørensen

Brain metabolism of 13N‐ammonia during acute hepatic encephalopathy in cirrhosis measured by positron emission tomography

Animal studies and results from 13N‐ammonia positron emission tomography (PET) in patients with cirrhosis and minimal hepatic encephalopathy suggest that a disturbed brain ammonia metabolism plays a pivotal role in the pathogenesis of hepatic encephalopathy (HE). We studied brain ammonia kinetics in 8 patients with cirrhosis with an acute episode of clinically overt HE (I‐IV), 7 patients with cirrhosis without HE, and 5 healthy subjects, using contemporary dynamic 13N‐ammonia PET. Time courses were obtained of 13N‐concentrations in cerebral cortex, basal ganglia, and cerebellum (PET‐scans) as well as arterial 13N‐ammonia, 13N‐urea, and 13N‐glutamine concentrations (blood samples) after 13N‐ammonia injection. Regional 13N‐ammonia kinetics was calculated by non‐linear fitting of a physiological model of brain ammonia metabolism to the data. Mean permeability–surface area product of 13N‐ammonia transfer across blood–brain barrier in cortex, PSBBB, was 0.21 mL blood/min/mL tissue in patients with HE, 0.31 in patients without HE, and 0.34 in healthy controls; similar differences were seen in basal ganglia and cerebellum. Metabolic trapping of blood 13N‐ammonia in the brain showed neither regional, nor patient group differences. Mean net metabolic flux of ammonia from blood into intracellular glutamine in the cortex was 13.4 μmol/min/L tissue in patients with cirrhosis with HE, 7.4 in patients without HE, and 2.6 in healthy controls, significantly correlated to blood ammonia. In conclusion, increased cerebral trapping of ammonia in patients with cirrhosis with acute HE was primarily attributable to increased blood ammonia and to a minor extent to changed ammonia kinetics in the brain. (HEPATOLOGY 2005;43:42–50.)

Low Cerebral Oxygen Consumption and Blood Flow in Patients With Cirrhosis and an Acute Episode of Hepatic Encephalopathy

BACKGROUND & AIMS It is unclear whether patients with hepatic encephalopathy (HE) have disturbed brain oxygen metabolism and blood flow. METHODS We measured cerebral oxygen metabolism rate (CMRO(2)) by using (15)O-oxygen positron emission tomography (PET); and cerebral blood flow (CBF) by using (15)O-water PET in 6 patients with liver cirrhosis and an acute episode of overt HE, 6 cirrhotic patients without HE, and 7 healthy subjects. RESULTS Neither whole-brain CMRO(2) nor CBF differed significantly between cirrhotic patients without HE and healthy subjects, but were both significantly reduced in cirrhotic patients with HE (P < .01). CMRO(2) was 0.96 +/- 0.07 mumol oxygen/mL brain tissue/min (mean +/- SEM) in cirrhotic patients with HE, 1.34 +/- 0.08 in cirrhotic patients without HE, and 1.35 +/- 0.05 in healthy subjects; and CBF was 0.29 +/- 0.01 mL blood/mL brain tissue/min in patients with HE, 0.47 +/- 0.02 in patients without HE, and 0.49 +/- 0.03 in healthy subjects. CMRO(2) and CBF were correlated, and both variables correlated negatively with arterial ammonia concentration. Analysis of regional values, using individual magnetic resonance co-registrations, showed that the reductions in CMRO(2) and CBF in patients with HE were essentially generalized throughout the brain. CONCLUSIONS The observations imply that reduced cerebral oxygen consumption and blood flow in cirrhotic patients with an acute episode of overt HE are associated with HE and not cirrhosis as such, and that the primary event in the pathogenesis of HE could be inhibition of cerebral energy metabolism by increased blood ammonia.

Hepatic encephalopathy is associated with decreased cerebral oxygen metabolism and blood flow, not increased ammonia uptake

Studies have shown decreased cerebral oxygen metabolism (CMRO2) and blood flow (CBF) in patients with cirrhosis with hepatic encephalopathy (HE). It remains unclear, however, whether these disturbances are associated with HE or with cirrhosis itself and how they may relate to arterial blood ammonia concentration and cerebral metabolic rate of blood ammonia (CMRA). We addressed these questions in a paired study design by investigating patients with cirrhosis during and after recovery from an acute episode of HE type C. CMRO2, CBF, and CMRA were measured by dynamic positron emission tomography (PET)/computed tomography (CT). Ten patients with cirrhosis were studied during an acute episode of HE; nine were reexamined after recovery. Nine patients with cirrhosis with no history of HE served as controls. Mean CMRO2 increased from 0.73 μmol oxygen/mL brain tissue/min during HE to 0.91 μmol oxygen/mL brain tissue/min after recovery (paired t test; P < 0.05). Mean CBF increased from 0.28 mL blood/mL brain tissue/min during HE to 0.38 mL blood/mL brain tissue/min after recovery (P < 0.05). After recovery from HE, CMRO2 and CBF were not significantly different from values in the control patients. Arterial blood ammonia concentration decreased 20% after recovery (P < 0.05) and CMRA was unchanged (P > 0.30); both values were higher than in the control patients (both P < 0.05). Conclusion: The low values of CMRO2 and CBF observed during HE increased after recovery from HE and were thus associated with HE rather than the liver disease as such. The changes in CMRO2 and CBF could not be linked to blood ammonia concentration or CMRA. (HEPATOLOGY 2013)

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and alpha-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the alpha-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the alpha-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-13C]glucose (2.5 mM) and isoleucine (1 mM) or [U-13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-13C]glucose. Labeling in glutamate and aspartate from [U-13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-13C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.

Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons

It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH(4)Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.

New findings on cerebral ammonia uptake in HE using functional (13)N-ammonia PET.

PET is a functional imaging technique suitable for studies of brain ammonia metabolism. Dynamic 13N-ammonia PET yields time-courses of radioactivity concentrations in brain (PET camera) and blood (samples). Ahl et al. (Hepatology 40:73–79, 2004) and Keiding et al. (Hepatology 43:42–50, 2006) analysed such data in patients with HE by a kinetic model accounting for transfer of 13N-ammonia across the blood–brain barrier (BBB) and intracellular formation of 13N-glutamine. Initial unidirectional 13N-ammonia transfer across BBB was characterized by the permeability-surface area product PSBBB (ml blood min−1 ml−1 tissue). There was a tendency to lower PSBBB values in patients with cirrhosis and HE than in patients with cirrhosis without HE and healthy controls but the differences were not statistically significant. Keiding et al. (Hepatology 43:42–50, 2006) also calculated PSmet (ml blood min−1 ml−1 tissue) as a measure of the combined transfer of 13N-ammonia across BBB and subsequent intracellular metabolism of 13N-ammonia; neither did this PS-value show significant difference between the groups of subjects. Net flux of ammonia from blood into intracellular metabolites was linearly correlated to arterial ammonia. In conclusion, basic brain ammonia kinetics was not changed significantly in patients with cirrhosis +/- HE compared to healthy controls. Blood ammonia seems to be the more important factor for increased brain ammonia uptake in HE.

Hepatic uptake and metabolism of galactose can be quantified in vivo by 2-[18F]fluoro-2-deoxygalactose positron emission tomography

Metabolism of galactose is a specialized liver function. The purpose of this PET study was to use the galactose analog 2-[(18)F]fluoro-2-deoxygalactose (FDGal) to investigate hepatic uptake and metabolism of galactose in vivo. FDGal kinetics was studied in 10 anesthetized pigs at blood concentrations of nonradioactive galactose yielding approximately first-order kinetics (tracer only; n = 4), intermediate kinetics (0.5-0.6 mmol galactose/l blood; n = 2), and near-saturation kinetics (>3 mmol galactose/l blood; n = 4). All animals underwent liver C15O PET (blood volume) and FDGal PET (galactose kinetics) with arterial and portal venous blood sampling. Flow rates in the hepatic artery and the portal vein were measured by ultrasound transit-time flowmeters. The hepatic uptake and net metabolic clearance of FDGal were quantified by nonlinear and linear regression analyses. The initial extraction fraction of FDGal from blood-to-hepatocyte was unity in all pigs. Hepatic net metabolic clearance of FDGal, K(FDGal), was 332-481 ml blood.min(-1).l(-1) tissue in experiments with approximately first-order kinetics and 15.2-21.8 ml blood.min(-1).l(-1) tissue in experiments with near-saturation kinetics. Maximal hepatic removal rates of galactose were on average 600 micromol.min(-1).l(-1) tissue (range 412-702), which was in agreement with other studies. There was no significant difference between K(FDGal) calculated with use of the dual tracer input (Kdual(FDGal)) or the single arterial input (Karterial(FDGal)). In conclusion, hepatic galactose kinetics can be quantified with the galactose analog FDGal. At near-saturated kinetics, the maximal hepatic removal rate of galactose can be calculated from the net metabolic clearance of FDGal and the blood concentration of galactose.

Synthesis of neurotransmitter GABA via the neuronal tricarboxylic acid cycle is elevated in rats with liver cirrhosis consistent with a high GABAergic tone in chronic hepatic encephalopathy.

J. Neurochem. (2011) 117, 824–832.

[N-Methyl-11C]Cholylsarcosine, a Novel Bile Acid Tracer for PET/CT of Hepatic Excretory Function: Radiosynthesis and Proof-of-Concept Studies in Pigs

Excretion of conjugated bile acids into bile is an essential function of the liver, and impairment of canalicular bile acid secretion leads to cholestatic liver injury. However, hepatic excretory function cannot be quantified in vivo because of the lack of suitable methods. Cholylsarcosine is an analog of the endogenous bile acid conjugate cholylglycine and exhibits characteristics in vivo that led us to hypothesize that the 11C-labeled form, that is, [N-methyl-11C]cholylsarcosine (11C-cholylsarcosine), would be a suitable PET tracer for quantification of hepatic excretory function. Methods: A method for radiosynthesis of 11C-cholylsarcosine was developed involving 11C-methylation of glycine followed by conjugation with cholic acid. Blood-to-liver uptake and liver-to-bile excretion were investigated in vivo by dynamic 11C-cholylsarcosine PET/CT of 2 anesthetized pigs. In pig 1, a second dynamic 11C-cholylsarcosine PET/CT examination was preceded by a high dose of the endogenous bile acid conjugate cholyltaurine to investigate possible inhibition of the transhepatocellular transport of 11C-cholylsarcosine. In pig 2, a second 11C-cholylsarcosine administration was given to determine the biodistribution of the tracer by means of 5 successive whole-body PET/CT recordings. Possible formation of 11C-metabolites was investigated by analysis of blood and bile samples from a third pig. Results: The radiochemical yield was 13% ± 3% (n = 7, decay-corrected) and up to 1.1 GBq of 11C-cholylsarcosine was produced with a radiochemical purity greater than 99%. PET/CT studies showed rapid blood-to-liver uptake and liver-to-bile excretion of 11C-cholylsarcosine, with radioactivity concentrations being more than 90 times higher in the bile ducts than in liver tissue. Cholyltaurine inhibited the transhepatocellular transport of 11C-cholylsarcosine, indicating that the tracer is transported by one or more of the same hepatic transporters as cholyltaurine. 11C-cholylsarcosine underwent an enterohepatic circulation and reappeared in liver tissue and bile ducts after approximately 70 min. There were no detectable 11C-metabolites in the plasma or bile samples, indicating that the novel conjugated bile acid 11C-cholylsarcosine was not metabolized in the liver or in the intestines. The effective absorbed dose of 11C-cholylsarcosine was 4.4 μSv/MBq. Conclusion: We have synthesized a novel conjugated bile acid analog, 11C-cholylsarcosine, and PET/CT studies on anesthetized pigs showed that the hepatic handling of tracer uptake from blood and excretion into the bile was comparable to that for the endogenous bile acid cholyltaurine. This tracer may be valuable for future studies of normal and pathologic hepatic excretory functions in humans.

Metabolic fate of isoleucine in a rat model of hepatic encephalopathy and in cultured neural cells exposed to ammonia.

Hepatic encephalopathy is a severe neuropathological condition arising secondary to liver failure. The pathogenesis is not well understood; however, hyperammonemia is considered to be one causative factor. Hyperammonemia has been suggested to inhibit tricarboxylic acid (TCA) cycle activity, thus affecting energy metabolism. Furthermore, it has been suggested that catabolism of the branched-chain amino acid isoleucine may help curb the effect of hyperammonemia by by-passing the TCA cycle block as well as providing the carbon skeleton for glutamate and glutamine synthesis thus fixating ammonia. Here we present novel results describing [U-13C]isoleucine metabolism in muscle and brain analyzed by mass spectrometry in bile duct ligated rats, a model of chronic hepatic encephalopathy, and discuss them in relation to previously published results from neural cell cultures. The metabolism of [U-13C]isoleucine in muscle tissue was about five times higher than that in the brain which, in turn, was lower than in corresponding cell cultures. However, synthesis of glutamate and glutamine was supported by catabolism of isoleucine. In rat brain, differential labeling patterns in glutamate and glutamine suggest that isoleucine may primarily be metabolized in the astrocytic compartment which is in accordance with previous findings in neural cell cultures. Lastly, in rat brain the labeling patterns of glutamate, aspartate and GABA do not suggest any significant inhibition by ammonia of TCA cycle activity which corresponds well to findings in neural cell cultures. Branched-chain amino acids including isoleucine are used for treating hepatic encephalopathy and the present findings shed light on the possible mechanism involved. The low turn-over of isoleucine in rat brain suggests that this amino acid does not serve the role of providing metabolites pertinent to TCA cycle function and hence energy formation as well as the necessary carbon skeleton for subsequent ammonia fixation in hyperammonemia. The higher metabolism of isoleucine in muscle could, however, contribute to ammonia fixation and thus likely be of value in the treatment of hepatic encephalopathy.