Cerebral ammonia uptake and accumulation during prolonged exercise in humans
1 Department of Human Physiology, Institute of Exercise and Sport Sciences,2 Departments of Anaesthesia
3 Infectious Diseases, The Copenhagen Muscle Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Abstract
We evaluated whether peripheral ammonia production during prolonged exercise enhances the uptake and subsequent accumulation of ammonia within the brain. Two studies determined the cerebral uptake of ammonia (arterial and jugular venous blood sampling combined with Kety–Schmidt-determined cerebral blood flow; n = 5) and the ammonia concentration in the cerebrospinal fluid (CSF; n = 8) at rest and immediately following prolonged exercise either with or without glucose supplementation. There was a net balance of ammonia across the brain at rest and at 30 min of exercise, whereas 3 h of exercise elicited an uptake of 3.7 ± 1.3 μmol min–1 (mean ± S.E.M.) in the placebo trial and 2.5 ± 1.0 μmol min–1 in the glucose trial (P < 0.05 compared to rest, not different across trials). At rest, CSF ammonia was below the detection limit of 2 μM in all subjects, but it increased to 5.3 ± 1.1 μM following exercise with glucose, and further to 16.1 ± 3.3 μM after the placebo trial (P < 0.05). Correlations were established between both the cerebral uptake (r2 = 0.87; P < 0.05) and the CSF concentration (r2 = 0.72; P < 0.05) and the arterial ammonia level and, in addition, a weaker correlation (r2 = 0.37; P < 0.05) was established between perceived exertion and CSF ammonia at the end of exercise. The results let us suggest that during prolonged exercise the cerebral uptake and accumulation of ammonia may provoke fatigue, e.g. by affecting neurotransmitter metabolism.
, http://www.100md.com
Introduction
During prolonged exercise the circulating levels of ammonia may become markedly elevated (Eriksson et al. 1985; Snow et al. 2000). The enhanced ammonia production seems to be a combined effect of purine nucleotide deamination and amino acid catabolism within the active myofibrils (Katz et al. 1986; Hellsten et al. 1999) and, although some ammonia remains in the skeletal muscles, the majority is released to the circulation. Since ammonia penetrates the blood–brain barrier readily (Bachmann, 2002), high levels of systemic ammonia may eventually cause accumulation in both intra- and extracellular spaces of the brain with potential detrimental effects on neurotransmission, cerebral metabolism and circulation (Banister & Cameron, 1990).
, 百拇医药
The central nervous system (CNS) has no effective urea cycle, and the brain depends on the synthesis of glutamine from glutamate and ammonia for removal of excess ammonia (Suarez et al. 2002). However, elimination of ammonia may not only reduce the level of the excitatory neurotransmitter glutamate, but also that of -aminobutyric acid (GABA) for which glutamate is a precursor. Such disturbance of the cerebral neurotransmitter homeostasis combined with ammonia-induced perturbations of intracellular metabolism seem to be main factors underlying cerebral dysfunction during hyperammonaemia in acute liver failure (Butterworth et al. 1988; Ott & Larsen, 2004). In agreement, during prolonged exercise, increased levels of circulating ammonia may provoke cerebral perturbation that could influence central fatigue (Banister & Cameron, 1990; Davis & Bailey, 1997).
, http://www.100md.com
In support of this notion, cerebral accumulation of ammonia and exercise-induced changes in the levels of glutamate, glutamine and GABA are observed in rats (Guezennec et al. 1998). Conversely, in humans a cerebral ammonia uptake is reported during short-lasting maximal exercise without accumulation in the cerebrospinal fluid (CSF; Dalsgaard et al. 2004b), suggesting that the capacity of the brain for elimination of ammonia is sufficient during brief exercise. However, during prolonged exercise, a continuous uptake by the brain may eventually exhaust the cerebral capacity for ammonia removal. Therefore, two studies were conducted to quantify the cerebral uptake of ammonia during prolonged exercise, and to determine whether such uptake is associated with accumulation in the CSF. Glucose supplementation attenuates perceived exertion and the systemic ammonia response to prolonged exercise (Snow et al. 2000; Nybo, 2003), and trials were conducted both with and without glucose ingestion to evaluate whether there is an association between the perception of fatigue and the cerebral accumulation of ammonia.
, 百拇医药
Methods
Subjects
Study A: five endurance-trained male subjects had a mean age of 26 ± 2 years (±S.E.M.), weight of 74 ± 3 kg, height of 1.82 ± 0.03 m, and a maximal oxygen uptake of 4.9 ± 0.2 l min–1. Study B: 24 male subjects were randomly divided into three groups (rest and exercise with (CHO trial) or without glucose supplementation (placebo trial)). These subjects had an age of 24 ± 0.4 years, weight of 77 ± 2 kg, height of 185 ± 0.01 m and of 4.2 ± 0.1 l min–1.
, http://www.100md.com
Informed written consent was obtained from the subjects, and both studies were carried out in accordance with the Declaration of Helsinki as approved by the Ethical Committee of Copenhagen and Frederiksberg (KF 01-135/00).
In both studies, the subject arrived at the laboratory in the morning after an overnight (12 h) fast, and after abstaining from caffeine-containing items for 24 h. While the subjects were in a supine position, catheters were inserted in an antecubital vein, in the right internal jugular vein, and in the radial artery of the nondominant arm. Study A involved 2 days of experiments for each subject, encompassing resting measurements of cerebral blood flow (CBF), and paired arterial and jugular venous blood samples for determination of ammonia, glucose, haemoglobin and blood gases 0.5 h after catheterisation. The subject then exercised for 3 h on a cycle ergometer (Monark 829E, Sweden) at a power output (210 ± 13 W) corresponding to 60% of , either with (CHO trial) or without (placebo trial) glucose supplementation. The two trials were performed in a randomised order, and separated by 5–7 days. During the CHO trial, the arterial blood glucose level was maintained by ingestion of 250 ml of a 6% maltodextrin solution every 15th minute, while exercise with ingestion of a similar volume of a noncaloric artificially sweetened placebo resulted in progressive hypoglycaemia. Blood samples were obtained after 30 and 180 min of exercise in both trials, and CBF was measured after 180 min of exercise. Furthermore, the subject expressed his rating of perceived exertion (RPE) on the Borg scale (Borg, 1975) every 30th minute during exercise.
, 百拇医药
In study B, CSF and arterial blood samples were obtained in eight subjects at rest, and in two groups (CHO and placebo trial) of eight subjects following 2 h of exercise at 188 ± 6 W, corresponding to 60% of .
Cerebral plasma flow and blood analysis
The CBF was measured with the Kety–Schmidt technique in the desaturation mode using 133Xe as the tracer (Kety & Schmidt, 1948; Madsen et al. 1993). 133Xe dissolved in 0.9% saline was infused for 30 min via the catheter placed in the antecubital vein at 0.5 mCi min–1 at rest, and 1.5–2.5 mCi min–1 during exercise (depending on the size of the subject and his ventilation). Arterial blood samples were obtained from the radial artery, and cerebral venous blood was sampled from the internal jugular vein. To empty catheter dead space, 3 ml blood were drawn simultaneously from the catheters immediately before 1 ml of blood was drawn into syringes of known weights at exact times (t) –2, –1, 0, 0.5, 1, 2, 3, 4, 6, 8 and 10 min (t = 0 denoting the stop of infusion). After blood sampling, the syringes were re-weighed to obtain the weight of the blood sample. Subsequently, the syringes were sealed in gas-tight cylinders to avoid the escape of 133Xe, and counted for 10 min on a scintillation counter (Cobra II, Packard Instruments, Meriden, CT, USA) for determination of decay- and background-corrected 133Xe activity (counts per minute per gram) in individual samples. The CBF (millilitres per minute per gram of cerebral tissue) was calculated according to the Kety–Schmidt equation (Kety & Schmidt, 1948) with brain–blood partition coefficients for 133Xe calculated on the basis of the individual haemoglobin concentration (Hedt-Rasmussen et al. 1966). Global CBF was estimated assuming an average brain mass of 1400 g (Miller & Corsellis, 1977; Dekaban, 1978), and cerebral plasma flow was calculated on basis of the global CBF and the corresponding haematocrit. The cerebral uptake of ammonia was calculated as cerebral plasma flow multiplied by the arterial to jugular venous difference. Blood samples for determination of ammonia were drawn into glass tubes containing EDTA. These samples were immediately spun at 2200 g for 5 min at 4°C, and plasma was subsequently stored at –80°C. Plasma and CSF ammonia were analysed using spectrophotometrical determinations. Plasma glutamine and glutamate were determined by reversed-phase high performance liquid chromatography, while CSF glutamine was determined according to the methods described by Lund (1985). Arterial and jugular venous values of haemoglobin, glucose and blood gas variables were determined on an ABL 700 apparatus (Radiometer, Copenhagen, Denmark) with an accuracy of 0.1 mM, and the haematocrit and the arterial and jugular venous oxygen content were calculated on the basis of the haemoglobin concentration, the oxygen saturation and the partial pressure of oxygen (Siggaard-Andersen et al. 1988).
, http://www.100md.com
Cerebrospinal fluid
CSF was obtained following local anaesthesia of the skin with 2% lidocaine (lignocaine) through a 25 gauge pencil-point cannula (Braun, Melsungen, Germany) advanced between the third and fourth lumbar vertebrae until penetration of dura. In order to avoid the subjects developing a spinal headache, subjects rested for 1 h after the puncture while isotonic saline (1 l) was administered intravenously. The procedure was well tolerated and, except for one subject, who transiently developed headache 2 days after the experiment, there were no complications.
, http://www.100md.com
Statistical analysis
Two-way (time-by-trial) repeated measures analysis of variance (ANOVA) was performed to evaluate differences between and within trials. Following a significant F-test, pair-wise differences were identified using Tukey's significance (HSD) post hoc procedure. Furthermore, simple linear regression was used to test the strength of the association between variables. Data are presented as means ± S.E.M. unless otherwise indicated.
, 百拇医药
Results
In study A, the arterial ammonia concentration increased from 22 ± 2 μM at rest to 42 ± 13 μM by the end of the CHO trial, and further to 56 ± 13 μM in the placebo trial (P < 0.05), whereas in study B including less-trained subjects, the arterial ammonia level was 116 ± 18 μM by the end of the CHO trial and 190 ± 44 μM following the placebo trial (P < 0.05 between trials and P < 0.01 compared to study A and rest). At rest and after 30 min exercise, there was a net balance of ammonia across the brain, whereas 3 h of exercise elicited a cerebral ammonia uptake of 3.7 ± 1.3 μmol min–1 in the placebo trial and 2.5 ± 1.0 μmol min–1 in the glucose trial (P < 0.05 compared to rest; not different across trials). The cerebral plasma flow was similar at rest compared to both exercise conditions (416 ± 17 ml min–1 in the placebo trial and 410 ± 18 ml min–1 during the CHO trial).
, http://www.100md.com
In all subjects, CSF ammonia was below the detection limit of 2 μM at rest, but increased (P < 0.05) to 5.3 ± 1.1 μM following exercise with glucose, and to 16.1 ± 3.3 μM after the placebo trial. Moderate to high correlation coefficients were observed between the arterial ammonia level and both the cerebral uptake (Fig. 1) and the CSF level (Fig. 2), and a weaker correlation (r2 = 0.37; P < 0.05) was observed between CSF ammonia and perceived exertion during the final stage of the exercise trials (Fig. 3). Following exercise, CSF glutamine was not different between treatments (539 ± 16 μM in placebo and 529 ± 12 μM in the glucose trial), but there was a weak correlation (r2 = 0.30; P < 0.05) between CSF glutamine and CSF ammonia (Fig. 4), and CSF glutamine tended (P = 0.08) to be higher following exercise as compared to rest (495 ± 24 μM). In contrast, there was no correlation between the cerebral ammonia uptake and the cerebral arterial to jugular venous (a–v) differences of either glutamine or glutamate (Table 1).
, 百拇医药
The rather large interindividual variation in the systemic and cerebral ammonia responses to prolonged cycling may relate to differences in the subjects training. Thus, the subjects with no or only a very small increase in arterial plasma ammonia after 3 h of moderate intense cycling (<40 μmol l–1) were competitive cyclists with a maximal oxygen consumption of 70 ml min–1 kg–1 and a training volume above 20 h per week. All data points in the plot were used to derive the r2 value.
, 百拇医药
The symbols represent individual values from study B. All data points in the plot were used to derive the r2 value.
RPE values represent the individual scores during the last 15 min of the prolonged cycle trial, whereas the CSF sample was obtained immediately after the trial. All data points in the plot were used to derive the r2 value.
The symbols represent individual values from study B. All data points in the plot were used to derive the r2 value.
, 百拇医药
Discussion
The present paper demonstrates that prolonged exercise provokes a cerebral uptake of ammonia and an increased concentration in the CSF, especially when conducted without glucose supplementation. The cerebral ammonia uptake and CSF levels following exercise displayed relatively large interindividual variation, being most pronounced in subjects with high levels of circulating ammonia. These subjects also experienced the largest difficulty in completing the exercise trials, indicating that ammonia could constitute a link between muscle metabolism and central fatigue.
, 百拇医药
Considering a CSF volume of 150–300 ml (Lüders et al. 2002), the exercise-induced elevation of CSF ammonia by 5–16 μM corresponds to the accumulation of 1–5 μmol in the CSF. This value approximates the amount of ammonia taken up by the brain every minute during the later stages of the exercise trials and, even with the assumption that ammonia is distributed equally in all brain tissues, the cerebral accumulation accounts only for the ammonia taken up by the brain during a few minutes of exercise. Consequently, a major part of the ammonia taken up by the brain seems to be metabolised or removed from the CSF by other means. The CNS has no effective urea cycle, as it lacks carbamoyl-phosphate synthase I and ornithine transcarbamylase, and the predominant pathway for removal of excess ammonia is formation of glutamine on the basis of glutamate and ammonia (Felipo & Butterworth, 2002). The weak correlation between CSF ammonia and glutamine following exercise may support the idea that some ammonia is removed via glutamine synthesis. In accordance, during liver failure, both acute and chronic hyperammonaemia is associated with reductions of CNS glutamate (Tyce et al. 1981; Mans et al. 1994) and elevated glutamine levels (Vossler et al. 2002), and corresponding observations have been made following exercise in rats (Guezennec et al. 1998). On the other hand, the results should be interpreted with caution since there were no changes in the a–v differences of glutamate and glutamine, and CSF glutamine following exercise was not significantly different across trials.
, 百拇医药
A cerebral uptake of ammonia, but without changes in the CSF level of ammonia, has previously been reported during maximal exercise with a duration of 12 min (Dalsgaard et al. 2004b). In contrast, the cerebral uptake of ammonia during the prolonged exercise trials elevated CSF ammonia several fold, suggesting that it takes a certain time, and thereby ammonia load, before the capacity for ammonia removal is exhausted (Meeusen & De Meirleir, 1995; Guezennec et al. 1998).
, http://www.100md.com
Activation of the brain either by a mental challenge or when exercise requires a determined effort provokes a cerebral uptake of glucose out of proportion to that of oxygen (Nybo & Secher, 2004). During prolonged exercise part of this ‘surplus’ glucose could combine with ammonia in the process of amino acid synthesis explaining some of the ammonia uptake (Dalsgaard et al. 2004a,b), which seemingly is metabolised by the brain. During a comparable bout of prolonged exercise the cerebral ‘surplus’ uptake of glucose that is not accounted for by oxidation (or by lactate release from the brain) is calculated to 35 μmol min–1 (Nybo et al. 2002, 2003). If the ammonia uptake in that study equates to 1–5 μmol min–1 as in the present study, amino acid synthesis may explain some 3–14% of the ‘surplus’ glucose uptake. These calculations confirm that amino acid synthesis explains only a fraction of the ‘surplus’ carbohydrate uptake during brain activation as demonstrated with brief maximal exercise in man (Dalsgaard et al. 2004a,b). The other way around, elevated ammonia levels in the brain may also influence the cerebral oxygen to carbohydrate uptake ratio as ammonium stimulates glycolysis in glia cells (Tsacopoulos et al. 1997).
, http://www.100md.com
There is substantial evidence supporting that ammonia toxicity plays a role in the encephalopathy associated with both acute and chronic liver failure, which may elevate arterial ammonia to between 200 and 500 μM and lead to altered blood–brain permeability, cerebral oedema and impaired regulation of brain metabolism and circulation (Ott & Larsen, 2004; Butterworth et al. 1987). However, global CBF was not different across the two trials and it remained unchanged compared to rest, although both the plasma level and the cerebral uptake of ammonia were increased by exercise. It should be considered, though, that while some subjects following exercise in study B had blood and CSF ammonia levels of similar magnitude to that reported during acute liver disease (Ott & Larsen, 2004), the systemic and cerebral ammonia responses were modest in study A for which CBF was determined. However, in study A, exercise without glucose supplementation reduced the cerebral metabolic rate of glucose (see Table 1), but presumably this perturbation of the cerebral metabolism relates to inadequate substrate (glucose) availability rather than to the elevated level of ammonia (Nybo et al. 2003).
, 百拇医药
Glucose ingestion lessened perceived exertion at the same time as it attenuated the exercise-induced systemic and cerebral ammonia responses. This finding taken together with the relation between CSF ammonia and perceived exertion during the final stage of exercise supports the idea that cerebral ammonia accumulation may provoke central fatigue (Banister & Cameron, 1990; Guezennec et al. 1998). However, whether exercise elevates the cerebral ammonia level to an extent that influences glutamatergic neurotransmission and other CNS functions may depend on the exercise mode and individual differences. For instance, both glucose supplementation (Snow et al. 2000) and aerobic training (Baldwin et al. 2000) attenuate ammonia production during prolonged exercise. Accordingly, the highest levels of plasma and CSF ammonia were observed in the subjects with the lowest following exercise without glucose supplementation. On the other hand, some of the endurance-trained subjects had either no or negligible cerebral ammonia uptake during prolonged exercise (especially when they were supplemented with glucose; Fig. 1); yet, their perception of effort increased during the CHO trial from ‘fairly light’ at 30 min to ‘somewhat hard’ at 180 min (2 units on the RPE scale) demonstrating that other factors are also important for the development of fatigue. Therefore, we stress that fatigue should be acknowledged as a complex phenomenon influenced by both central and peripheral factors (cf. Fitts, 1994; Davis & Bailey, 1997; Nybo & Secher, 2004), and it has been noted that ammonia may also have detrimental effects on skeletal muscle metabolism (Brouns et al. 1990). However, the present data demonstrate that prolonged exercise has profound effects on the cerebral ammonia balance, supporting the notion that accumulation of ammonia in the brain may provoke fatigue, especially when untrained subjects exercise for a prolonged period without glucose supplementation.
, 百拇医药
References
Bachmann C (2002). Mechanisms of hyperammonemia. Clin Chem Lab Med 40, 653–662.
Baldwin J, Snow R & Febbraio M (2000). Effect of training status and relative exercise intensity on physiological responses in men. Med Sci Sports Exerc 32, 1648–1654.
Banister E & Cameron B (1990). Exercise-induced hyperammonemia: peripheral and central effects. Int J Sports Med 11, 129–142.
, http://www.100md.com
Borg G (1975). Simple rating for estimation of perceived exertion. In Physical Work and Effort, ed. Borg G, pp. 39–46. Pergamon, New York.
Brouns F, Beckers E, Wagenmakers A & Saris W (1990). Ammonia accumulation during highly intensive long-lasting cycling: individual observations. Int J Sports Med 11, 78–84.
Butterworth R, Giguere J, Michaud J, Lavoie J & Layrargues G (1987). Ammonia: key factor in the pathogenesis of hepatic encephalopathy. Neurochem Pathol 6, 1–12.
, http://www.100md.com
Butterworth R, Girard G & Giguere J (1988). Regional differences in the capacity for ammonia removal by brain following portocaval anastomosis. J Neurochem 51, 489–490.
Dalsgaard M, Ogoh S, Dawson E, Yoshiga C, Quistorff B & Secher NH (2004a). The cerebral carbohydrate cost of physical exertion in humans. Am J Physiol Regul Integr Comp Physiol 287, R534–540.
Dalsgaard M, Ott P, Dela F, Juul A, Pedersen B, Warberg J, Fahrenkrug J & Secher NH (2004b). The CSF and arterial to internal jugular venous hormonal differences during exercise in humans. Exp Physiol 89, 271–277.
, 百拇医药
Davis JM & Bailey SP (1997). Possible mechanisms of central nervous system fatigue during exercise. Med Sci Sports Exerc 29, 45–57.
Dekaban AS (1978). Changes in brain weights during the span of human life: relation of brain weights to body heights and body weights. Ann Neurol 4, 345–356.
Eriksson L, Broberg S, Bjorkman O & Wahren J (1985). Ammonia metabolism during exercise in man. Clin Physiol 5, 325–336.
, 百拇医药
Felipo V & Butterworth R (2002). Neurobiology of ammonia. Prog Neurobiol 67, 259–279.
Fitts RH (1994). Cellular mechanisms of muscle fatigue. Physiol Rev 74, 49–94.
Guezennec C, Abdelmalki A, Serrurier B, Merino D, Bigard X, Berthelot M, Pierard C & Peres M (1998). Effects of prolonged exercise on brain ammonia and amino acids. Int J Sports Med 19, 323–327.
Hellsten Y, Richter E, Kiens B & Bangsbo J (1999). AMP deamination and purine exchange in human skeletal muscle during and after intense exercise. J Physiol 520, 909–920.
, 百拇医药
Hedt-Rasmussen K, Sveinsdottir E & Lassen NA (1966). Regional cerebral blood flow in man determined by intra-arterial injection of radioactive inert gas. Circ Res 18, 237–247.
Katz A, Broberg S, Sahlin K & Wahren J (1986). Muscle ammonia and amino acid metabolism during dynamic exercise in man. Clin Physiol 6, 365–379.
Kety SS & Schmidt CF (1948). The nitrous oxide method for the quantitative determination of cerebral blood flow in man: theory, procedure and normal values. J Clin Invest 27, 476–483.
, http://www.100md.com
Lüders E, Steinmetz H & Jancke L (2002). Brain size and grey matter volume in the healthy human brain. Neuroreport 13, 2371–2374.
Lund P (1985). UV-method with glutaminase and glutamate dehydrogenase. In Methods of Enzymatic Analysis, 3rd edn, chap. 3, ed. Bergmeyer, HU, pp. 357–363. Verlag Chemie, Weinheim.
Madsen PL, Sperling BK, Warming T, Schmidt JF, Secher NH, Wildschidtz G, Holm S & Lassen NA (1993). Middle cerebral artery blood velocity and cerebral blood flow and O2 uptake during dynamic exercise. J Appl Physiol 74, 245–250.
, 百拇医药
Mans AM, DeJoseph MR & Hawkins R (1994). Metabolic abnormalities and grade of encephalopathy in acute hepatic failure. J Neurochem 63, 1829–1838.
Meeusen R & De Meirleir K (1995). Exercise and brain neurotransmission. Sports Med 20, 160–188.
Miller AK & Corsellis JA (1977). Evidence for a secular increase in human brain weight during the past century. Ann Human Biol 4, 253–257.
Nybo L (2003). CNS fatigue and prolonged exercise – effect of glucose supplementation. Med Sci Sports Exerc 35, 589–594.
, 百拇医药
Nybo L, Mller K, Pedersen B, Nielsen B & Secher NH (2003). Association between fatigue and failure to preserve cerebral energy turnover during prolonged exercise. Acta Physiol Scand 179, 67–74.
Nybo L, Mller K, Volianitis S, Nielsen B & Secher NH (2002). Effects of hyperthermia on cerebral blood flow and metabolism during prolonged exercise in humans. J Appl Physiol 93, 58–64.
Nybo L & Secher NH (2004). Cerebral perturbations provoked by prolonged exercise. Prog Neurobiol 72, 223–261.
, 百拇医药
Ott P & Larsen F (2004). Blood–brain barrier permeability to ammonia in liver failure: a critical reappraisal. Neurochem Int 44, 185–198.
Siggaard-Andersen O, Wimberley PD, Fogh-Andersen N & Gthgen IH (1988). Measured and derived quantities with modern pH and blood gas equipment: calculation algorithms with 54 equations. Scand J Clin Laboratory Invest 48, 7–15.
Snow R, Carey M, Stathis C, Febbraio M & Hargreaves M (2000). Effect of carbohydrate ingestion on ammonia metabolism during exercise in humans. J Appl Physiol 88, 1576–1580.
, 百拇医药
Suarez I, Bodega G & Fernandez B (2002). Glutamine synthetase in brain: effect of ammonia. Neurochem Int 41, 123–142.
Tsacopoulos M, Poitry-Yamate C & Poitry S (1997). Ammonium and glutamate released by neurons are signals regulating the nutritive function of a glial cell. J Neurosci 17, 2383–2390.
Tyce G, Ogg J & Owen CA (1981). Metabolism of acetate to amino acids in brains of rats after complete hepatectomy. J Neurochem 36, 640–650.
Vossler D, Wilensky A, Cawthon D, Kraemer D, Ojemann L, Caylor L & Morgan J (2002). Serum and CSF glutamine levels in valproate-related hyperammonemic encephalopathy. Epilepsia 43, 154–159., http://www.100md.com(Lars Nybo, Mads K Dalsgaa)
3 Infectious Diseases, The Copenhagen Muscle Research Centre, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Abstract
We evaluated whether peripheral ammonia production during prolonged exercise enhances the uptake and subsequent accumulation of ammonia within the brain. Two studies determined the cerebral uptake of ammonia (arterial and jugular venous blood sampling combined with Kety–Schmidt-determined cerebral blood flow; n = 5) and the ammonia concentration in the cerebrospinal fluid (CSF; n = 8) at rest and immediately following prolonged exercise either with or without glucose supplementation. There was a net balance of ammonia across the brain at rest and at 30 min of exercise, whereas 3 h of exercise elicited an uptake of 3.7 ± 1.3 μmol min–1 (mean ± S.E.M.) in the placebo trial and 2.5 ± 1.0 μmol min–1 in the glucose trial (P < 0.05 compared to rest, not different across trials). At rest, CSF ammonia was below the detection limit of 2 μM in all subjects, but it increased to 5.3 ± 1.1 μM following exercise with glucose, and further to 16.1 ± 3.3 μM after the placebo trial (P < 0.05). Correlations were established between both the cerebral uptake (r2 = 0.87; P < 0.05) and the CSF concentration (r2 = 0.72; P < 0.05) and the arterial ammonia level and, in addition, a weaker correlation (r2 = 0.37; P < 0.05) was established between perceived exertion and CSF ammonia at the end of exercise. The results let us suggest that during prolonged exercise the cerebral uptake and accumulation of ammonia may provoke fatigue, e.g. by affecting neurotransmitter metabolism.
, http://www.100md.com
Introduction
During prolonged exercise the circulating levels of ammonia may become markedly elevated (Eriksson et al. 1985; Snow et al. 2000). The enhanced ammonia production seems to be a combined effect of purine nucleotide deamination and amino acid catabolism within the active myofibrils (Katz et al. 1986; Hellsten et al. 1999) and, although some ammonia remains in the skeletal muscles, the majority is released to the circulation. Since ammonia penetrates the blood–brain barrier readily (Bachmann, 2002), high levels of systemic ammonia may eventually cause accumulation in both intra- and extracellular spaces of the brain with potential detrimental effects on neurotransmission, cerebral metabolism and circulation (Banister & Cameron, 1990).
, 百拇医药
The central nervous system (CNS) has no effective urea cycle, and the brain depends on the synthesis of glutamine from glutamate and ammonia for removal of excess ammonia (Suarez et al. 2002). However, elimination of ammonia may not only reduce the level of the excitatory neurotransmitter glutamate, but also that of -aminobutyric acid (GABA) for which glutamate is a precursor. Such disturbance of the cerebral neurotransmitter homeostasis combined with ammonia-induced perturbations of intracellular metabolism seem to be main factors underlying cerebral dysfunction during hyperammonaemia in acute liver failure (Butterworth et al. 1988; Ott & Larsen, 2004). In agreement, during prolonged exercise, increased levels of circulating ammonia may provoke cerebral perturbation that could influence central fatigue (Banister & Cameron, 1990; Davis & Bailey, 1997).
, http://www.100md.com
In support of this notion, cerebral accumulation of ammonia and exercise-induced changes in the levels of glutamate, glutamine and GABA are observed in rats (Guezennec et al. 1998). Conversely, in humans a cerebral ammonia uptake is reported during short-lasting maximal exercise without accumulation in the cerebrospinal fluid (CSF; Dalsgaard et al. 2004b), suggesting that the capacity of the brain for elimination of ammonia is sufficient during brief exercise. However, during prolonged exercise, a continuous uptake by the brain may eventually exhaust the cerebral capacity for ammonia removal. Therefore, two studies were conducted to quantify the cerebral uptake of ammonia during prolonged exercise, and to determine whether such uptake is associated with accumulation in the CSF. Glucose supplementation attenuates perceived exertion and the systemic ammonia response to prolonged exercise (Snow et al. 2000; Nybo, 2003), and trials were conducted both with and without glucose ingestion to evaluate whether there is an association between the perception of fatigue and the cerebral accumulation of ammonia.
, 百拇医药
Methods
Subjects
Study A: five endurance-trained male subjects had a mean age of 26 ± 2 years (±S.E.M.), weight of 74 ± 3 kg, height of 1.82 ± 0.03 m, and a maximal oxygen uptake of 4.9 ± 0.2 l min–1. Study B: 24 male subjects were randomly divided into three groups (rest and exercise with (CHO trial) or without glucose supplementation (placebo trial)). These subjects had an age of 24 ± 0.4 years, weight of 77 ± 2 kg, height of 185 ± 0.01 m and of 4.2 ± 0.1 l min–1.
, http://www.100md.com
Informed written consent was obtained from the subjects, and both studies were carried out in accordance with the Declaration of Helsinki as approved by the Ethical Committee of Copenhagen and Frederiksberg (KF 01-135/00).
In both studies, the subject arrived at the laboratory in the morning after an overnight (12 h) fast, and after abstaining from caffeine-containing items for 24 h. While the subjects were in a supine position, catheters were inserted in an antecubital vein, in the right internal jugular vein, and in the radial artery of the nondominant arm. Study A involved 2 days of experiments for each subject, encompassing resting measurements of cerebral blood flow (CBF), and paired arterial and jugular venous blood samples for determination of ammonia, glucose, haemoglobin and blood gases 0.5 h after catheterisation. The subject then exercised for 3 h on a cycle ergometer (Monark 829E, Sweden) at a power output (210 ± 13 W) corresponding to 60% of , either with (CHO trial) or without (placebo trial) glucose supplementation. The two trials were performed in a randomised order, and separated by 5–7 days. During the CHO trial, the arterial blood glucose level was maintained by ingestion of 250 ml of a 6% maltodextrin solution every 15th minute, while exercise with ingestion of a similar volume of a noncaloric artificially sweetened placebo resulted in progressive hypoglycaemia. Blood samples were obtained after 30 and 180 min of exercise in both trials, and CBF was measured after 180 min of exercise. Furthermore, the subject expressed his rating of perceived exertion (RPE) on the Borg scale (Borg, 1975) every 30th minute during exercise.
, 百拇医药
In study B, CSF and arterial blood samples were obtained in eight subjects at rest, and in two groups (CHO and placebo trial) of eight subjects following 2 h of exercise at 188 ± 6 W, corresponding to 60% of .
Cerebral plasma flow and blood analysis
The CBF was measured with the Kety–Schmidt technique in the desaturation mode using 133Xe as the tracer (Kety & Schmidt, 1948; Madsen et al. 1993). 133Xe dissolved in 0.9% saline was infused for 30 min via the catheter placed in the antecubital vein at 0.5 mCi min–1 at rest, and 1.5–2.5 mCi min–1 during exercise (depending on the size of the subject and his ventilation). Arterial blood samples were obtained from the radial artery, and cerebral venous blood was sampled from the internal jugular vein. To empty catheter dead space, 3 ml blood were drawn simultaneously from the catheters immediately before 1 ml of blood was drawn into syringes of known weights at exact times (t) –2, –1, 0, 0.5, 1, 2, 3, 4, 6, 8 and 10 min (t = 0 denoting the stop of infusion). After blood sampling, the syringes were re-weighed to obtain the weight of the blood sample. Subsequently, the syringes were sealed in gas-tight cylinders to avoid the escape of 133Xe, and counted for 10 min on a scintillation counter (Cobra II, Packard Instruments, Meriden, CT, USA) for determination of decay- and background-corrected 133Xe activity (counts per minute per gram) in individual samples. The CBF (millilitres per minute per gram of cerebral tissue) was calculated according to the Kety–Schmidt equation (Kety & Schmidt, 1948) with brain–blood partition coefficients for 133Xe calculated on the basis of the individual haemoglobin concentration (Hedt-Rasmussen et al. 1966). Global CBF was estimated assuming an average brain mass of 1400 g (Miller & Corsellis, 1977; Dekaban, 1978), and cerebral plasma flow was calculated on basis of the global CBF and the corresponding haematocrit. The cerebral uptake of ammonia was calculated as cerebral plasma flow multiplied by the arterial to jugular venous difference. Blood samples for determination of ammonia were drawn into glass tubes containing EDTA. These samples were immediately spun at 2200 g for 5 min at 4°C, and plasma was subsequently stored at –80°C. Plasma and CSF ammonia were analysed using spectrophotometrical determinations. Plasma glutamine and glutamate were determined by reversed-phase high performance liquid chromatography, while CSF glutamine was determined according to the methods described by Lund (1985). Arterial and jugular venous values of haemoglobin, glucose and blood gas variables were determined on an ABL 700 apparatus (Radiometer, Copenhagen, Denmark) with an accuracy of 0.1 mM, and the haematocrit and the arterial and jugular venous oxygen content were calculated on the basis of the haemoglobin concentration, the oxygen saturation and the partial pressure of oxygen (Siggaard-Andersen et al. 1988).
, http://www.100md.com
Cerebrospinal fluid
CSF was obtained following local anaesthesia of the skin with 2% lidocaine (lignocaine) through a 25 gauge pencil-point cannula (Braun, Melsungen, Germany) advanced between the third and fourth lumbar vertebrae until penetration of dura. In order to avoid the subjects developing a spinal headache, subjects rested for 1 h after the puncture while isotonic saline (1 l) was administered intravenously. The procedure was well tolerated and, except for one subject, who transiently developed headache 2 days after the experiment, there were no complications.
, http://www.100md.com
Statistical analysis
Two-way (time-by-trial) repeated measures analysis of variance (ANOVA) was performed to evaluate differences between and within trials. Following a significant F-test, pair-wise differences were identified using Tukey's significance (HSD) post hoc procedure. Furthermore, simple linear regression was used to test the strength of the association between variables. Data are presented as means ± S.E.M. unless otherwise indicated.
, 百拇医药
Results
In study A, the arterial ammonia concentration increased from 22 ± 2 μM at rest to 42 ± 13 μM by the end of the CHO trial, and further to 56 ± 13 μM in the placebo trial (P < 0.05), whereas in study B including less-trained subjects, the arterial ammonia level was 116 ± 18 μM by the end of the CHO trial and 190 ± 44 μM following the placebo trial (P < 0.05 between trials and P < 0.01 compared to study A and rest). At rest and after 30 min exercise, there was a net balance of ammonia across the brain, whereas 3 h of exercise elicited a cerebral ammonia uptake of 3.7 ± 1.3 μmol min–1 in the placebo trial and 2.5 ± 1.0 μmol min–1 in the glucose trial (P < 0.05 compared to rest; not different across trials). The cerebral plasma flow was similar at rest compared to both exercise conditions (416 ± 17 ml min–1 in the placebo trial and 410 ± 18 ml min–1 during the CHO trial).
, http://www.100md.com
In all subjects, CSF ammonia was below the detection limit of 2 μM at rest, but increased (P < 0.05) to 5.3 ± 1.1 μM following exercise with glucose, and to 16.1 ± 3.3 μM after the placebo trial. Moderate to high correlation coefficients were observed between the arterial ammonia level and both the cerebral uptake (Fig. 1) and the CSF level (Fig. 2), and a weaker correlation (r2 = 0.37; P < 0.05) was observed between CSF ammonia and perceived exertion during the final stage of the exercise trials (Fig. 3). Following exercise, CSF glutamine was not different between treatments (539 ± 16 μM in placebo and 529 ± 12 μM in the glucose trial), but there was a weak correlation (r2 = 0.30; P < 0.05) between CSF glutamine and CSF ammonia (Fig. 4), and CSF glutamine tended (P = 0.08) to be higher following exercise as compared to rest (495 ± 24 μM). In contrast, there was no correlation between the cerebral ammonia uptake and the cerebral arterial to jugular venous (a–v) differences of either glutamine or glutamate (Table 1).
, 百拇医药
The rather large interindividual variation in the systemic and cerebral ammonia responses to prolonged cycling may relate to differences in the subjects training. Thus, the subjects with no or only a very small increase in arterial plasma ammonia after 3 h of moderate intense cycling (<40 μmol l–1) were competitive cyclists with a maximal oxygen consumption of 70 ml min–1 kg–1 and a training volume above 20 h per week. All data points in the plot were used to derive the r2 value.
, 百拇医药
The symbols represent individual values from study B. All data points in the plot were used to derive the r2 value.
RPE values represent the individual scores during the last 15 min of the prolonged cycle trial, whereas the CSF sample was obtained immediately after the trial. All data points in the plot were used to derive the r2 value.
The symbols represent individual values from study B. All data points in the plot were used to derive the r2 value.
, 百拇医药
Discussion
The present paper demonstrates that prolonged exercise provokes a cerebral uptake of ammonia and an increased concentration in the CSF, especially when conducted without glucose supplementation. The cerebral ammonia uptake and CSF levels following exercise displayed relatively large interindividual variation, being most pronounced in subjects with high levels of circulating ammonia. These subjects also experienced the largest difficulty in completing the exercise trials, indicating that ammonia could constitute a link between muscle metabolism and central fatigue.
, 百拇医药
Considering a CSF volume of 150–300 ml (Lüders et al. 2002), the exercise-induced elevation of CSF ammonia by 5–16 μM corresponds to the accumulation of 1–5 μmol in the CSF. This value approximates the amount of ammonia taken up by the brain every minute during the later stages of the exercise trials and, even with the assumption that ammonia is distributed equally in all brain tissues, the cerebral accumulation accounts only for the ammonia taken up by the brain during a few minutes of exercise. Consequently, a major part of the ammonia taken up by the brain seems to be metabolised or removed from the CSF by other means. The CNS has no effective urea cycle, as it lacks carbamoyl-phosphate synthase I and ornithine transcarbamylase, and the predominant pathway for removal of excess ammonia is formation of glutamine on the basis of glutamate and ammonia (Felipo & Butterworth, 2002). The weak correlation between CSF ammonia and glutamine following exercise may support the idea that some ammonia is removed via glutamine synthesis. In accordance, during liver failure, both acute and chronic hyperammonaemia is associated with reductions of CNS glutamate (Tyce et al. 1981; Mans et al. 1994) and elevated glutamine levels (Vossler et al. 2002), and corresponding observations have been made following exercise in rats (Guezennec et al. 1998). On the other hand, the results should be interpreted with caution since there were no changes in the a–v differences of glutamate and glutamine, and CSF glutamine following exercise was not significantly different across trials.
, 百拇医药
A cerebral uptake of ammonia, but without changes in the CSF level of ammonia, has previously been reported during maximal exercise with a duration of 12 min (Dalsgaard et al. 2004b). In contrast, the cerebral uptake of ammonia during the prolonged exercise trials elevated CSF ammonia several fold, suggesting that it takes a certain time, and thereby ammonia load, before the capacity for ammonia removal is exhausted (Meeusen & De Meirleir, 1995; Guezennec et al. 1998).
, http://www.100md.com
Activation of the brain either by a mental challenge or when exercise requires a determined effort provokes a cerebral uptake of glucose out of proportion to that of oxygen (Nybo & Secher, 2004). During prolonged exercise part of this ‘surplus’ glucose could combine with ammonia in the process of amino acid synthesis explaining some of the ammonia uptake (Dalsgaard et al. 2004a,b), which seemingly is metabolised by the brain. During a comparable bout of prolonged exercise the cerebral ‘surplus’ uptake of glucose that is not accounted for by oxidation (or by lactate release from the brain) is calculated to 35 μmol min–1 (Nybo et al. 2002, 2003). If the ammonia uptake in that study equates to 1–5 μmol min–1 as in the present study, amino acid synthesis may explain some 3–14% of the ‘surplus’ glucose uptake. These calculations confirm that amino acid synthesis explains only a fraction of the ‘surplus’ carbohydrate uptake during brain activation as demonstrated with brief maximal exercise in man (Dalsgaard et al. 2004a,b). The other way around, elevated ammonia levels in the brain may also influence the cerebral oxygen to carbohydrate uptake ratio as ammonium stimulates glycolysis in glia cells (Tsacopoulos et al. 1997).
, http://www.100md.com
There is substantial evidence supporting that ammonia toxicity plays a role in the encephalopathy associated with both acute and chronic liver failure, which may elevate arterial ammonia to between 200 and 500 μM and lead to altered blood–brain permeability, cerebral oedema and impaired regulation of brain metabolism and circulation (Ott & Larsen, 2004; Butterworth et al. 1987). However, global CBF was not different across the two trials and it remained unchanged compared to rest, although both the plasma level and the cerebral uptake of ammonia were increased by exercise. It should be considered, though, that while some subjects following exercise in study B had blood and CSF ammonia levels of similar magnitude to that reported during acute liver disease (Ott & Larsen, 2004), the systemic and cerebral ammonia responses were modest in study A for which CBF was determined. However, in study A, exercise without glucose supplementation reduced the cerebral metabolic rate of glucose (see Table 1), but presumably this perturbation of the cerebral metabolism relates to inadequate substrate (glucose) availability rather than to the elevated level of ammonia (Nybo et al. 2003).
, 百拇医药
Glucose ingestion lessened perceived exertion at the same time as it attenuated the exercise-induced systemic and cerebral ammonia responses. This finding taken together with the relation between CSF ammonia and perceived exertion during the final stage of exercise supports the idea that cerebral ammonia accumulation may provoke central fatigue (Banister & Cameron, 1990; Guezennec et al. 1998). However, whether exercise elevates the cerebral ammonia level to an extent that influences glutamatergic neurotransmission and other CNS functions may depend on the exercise mode and individual differences. For instance, both glucose supplementation (Snow et al. 2000) and aerobic training (Baldwin et al. 2000) attenuate ammonia production during prolonged exercise. Accordingly, the highest levels of plasma and CSF ammonia were observed in the subjects with the lowest following exercise without glucose supplementation. On the other hand, some of the endurance-trained subjects had either no or negligible cerebral ammonia uptake during prolonged exercise (especially when they were supplemented with glucose; Fig. 1); yet, their perception of effort increased during the CHO trial from ‘fairly light’ at 30 min to ‘somewhat hard’ at 180 min (2 units on the RPE scale) demonstrating that other factors are also important for the development of fatigue. Therefore, we stress that fatigue should be acknowledged as a complex phenomenon influenced by both central and peripheral factors (cf. Fitts, 1994; Davis & Bailey, 1997; Nybo & Secher, 2004), and it has been noted that ammonia may also have detrimental effects on skeletal muscle metabolism (Brouns et al. 1990). However, the present data demonstrate that prolonged exercise has profound effects on the cerebral ammonia balance, supporting the notion that accumulation of ammonia in the brain may provoke fatigue, especially when untrained subjects exercise for a prolonged period without glucose supplementation.
, 百拇医药
References
Bachmann C (2002). Mechanisms of hyperammonemia. Clin Chem Lab Med 40, 653–662.
Baldwin J, Snow R & Febbraio M (2000). Effect of training status and relative exercise intensity on physiological responses in men. Med Sci Sports Exerc 32, 1648–1654.
Banister E & Cameron B (1990). Exercise-induced hyperammonemia: peripheral and central effects. Int J Sports Med 11, 129–142.
, http://www.100md.com
Borg G (1975). Simple rating for estimation of perceived exertion. In Physical Work and Effort, ed. Borg G, pp. 39–46. Pergamon, New York.
Brouns F, Beckers E, Wagenmakers A & Saris W (1990). Ammonia accumulation during highly intensive long-lasting cycling: individual observations. Int J Sports Med 11, 78–84.
Butterworth R, Giguere J, Michaud J, Lavoie J & Layrargues G (1987). Ammonia: key factor in the pathogenesis of hepatic encephalopathy. Neurochem Pathol 6, 1–12.
, http://www.100md.com
Butterworth R, Girard G & Giguere J (1988). Regional differences in the capacity for ammonia removal by brain following portocaval anastomosis. J Neurochem 51, 489–490.
Dalsgaard M, Ogoh S, Dawson E, Yoshiga C, Quistorff B & Secher NH (2004a). The cerebral carbohydrate cost of physical exertion in humans. Am J Physiol Regul Integr Comp Physiol 287, R534–540.
Dalsgaard M, Ott P, Dela F, Juul A, Pedersen B, Warberg J, Fahrenkrug J & Secher NH (2004b). The CSF and arterial to internal jugular venous hormonal differences during exercise in humans. Exp Physiol 89, 271–277.
, 百拇医药
Davis JM & Bailey SP (1997). Possible mechanisms of central nervous system fatigue during exercise. Med Sci Sports Exerc 29, 45–57.
Dekaban AS (1978). Changes in brain weights during the span of human life: relation of brain weights to body heights and body weights. Ann Neurol 4, 345–356.
Eriksson L, Broberg S, Bjorkman O & Wahren J (1985). Ammonia metabolism during exercise in man. Clin Physiol 5, 325–336.
, 百拇医药
Felipo V & Butterworth R (2002). Neurobiology of ammonia. Prog Neurobiol 67, 259–279.
Fitts RH (1994). Cellular mechanisms of muscle fatigue. Physiol Rev 74, 49–94.
Guezennec C, Abdelmalki A, Serrurier B, Merino D, Bigard X, Berthelot M, Pierard C & Peres M (1998). Effects of prolonged exercise on brain ammonia and amino acids. Int J Sports Med 19, 323–327.
Hellsten Y, Richter E, Kiens B & Bangsbo J (1999). AMP deamination and purine exchange in human skeletal muscle during and after intense exercise. J Physiol 520, 909–920.
, 百拇医药
Hedt-Rasmussen K, Sveinsdottir E & Lassen NA (1966). Regional cerebral blood flow in man determined by intra-arterial injection of radioactive inert gas. Circ Res 18, 237–247.
Katz A, Broberg S, Sahlin K & Wahren J (1986). Muscle ammonia and amino acid metabolism during dynamic exercise in man. Clin Physiol 6, 365–379.
Kety SS & Schmidt CF (1948). The nitrous oxide method for the quantitative determination of cerebral blood flow in man: theory, procedure and normal values. J Clin Invest 27, 476–483.
, http://www.100md.com
Lüders E, Steinmetz H & Jancke L (2002). Brain size and grey matter volume in the healthy human brain. Neuroreport 13, 2371–2374.
Lund P (1985). UV-method with glutaminase and glutamate dehydrogenase. In Methods of Enzymatic Analysis, 3rd edn, chap. 3, ed. Bergmeyer, HU, pp. 357–363. Verlag Chemie, Weinheim.
Madsen PL, Sperling BK, Warming T, Schmidt JF, Secher NH, Wildschidtz G, Holm S & Lassen NA (1993). Middle cerebral artery blood velocity and cerebral blood flow and O2 uptake during dynamic exercise. J Appl Physiol 74, 245–250.
, 百拇医药
Mans AM, DeJoseph MR & Hawkins R (1994). Metabolic abnormalities and grade of encephalopathy in acute hepatic failure. J Neurochem 63, 1829–1838.
Meeusen R & De Meirleir K (1995). Exercise and brain neurotransmission. Sports Med 20, 160–188.
Miller AK & Corsellis JA (1977). Evidence for a secular increase in human brain weight during the past century. Ann Human Biol 4, 253–257.
Nybo L (2003). CNS fatigue and prolonged exercise – effect of glucose supplementation. Med Sci Sports Exerc 35, 589–594.
, 百拇医药
Nybo L, Mller K, Pedersen B, Nielsen B & Secher NH (2003). Association between fatigue and failure to preserve cerebral energy turnover during prolonged exercise. Acta Physiol Scand 179, 67–74.
Nybo L, Mller K, Volianitis S, Nielsen B & Secher NH (2002). Effects of hyperthermia on cerebral blood flow and metabolism during prolonged exercise in humans. J Appl Physiol 93, 58–64.
Nybo L & Secher NH (2004). Cerebral perturbations provoked by prolonged exercise. Prog Neurobiol 72, 223–261.
, 百拇医药
Ott P & Larsen F (2004). Blood–brain barrier permeability to ammonia in liver failure: a critical reappraisal. Neurochem Int 44, 185–198.
Siggaard-Andersen O, Wimberley PD, Fogh-Andersen N & Gthgen IH (1988). Measured and derived quantities with modern pH and blood gas equipment: calculation algorithms with 54 equations. Scand J Clin Laboratory Invest 48, 7–15.
Snow R, Carey M, Stathis C, Febbraio M & Hargreaves M (2000). Effect of carbohydrate ingestion on ammonia metabolism during exercise in humans. J Appl Physiol 88, 1576–1580.
, 百拇医药
Suarez I, Bodega G & Fernandez B (2002). Glutamine synthetase in brain: effect of ammonia. Neurochem Int 41, 123–142.
Tsacopoulos M, Poitry-Yamate C & Poitry S (1997). Ammonium and glutamate released by neurons are signals regulating the nutritive function of a glial cell. J Neurosci 17, 2383–2390.
Tyce G, Ogg J & Owen CA (1981). Metabolism of acetate to amino acids in brains of rats after complete hepatectomy. J Neurochem 36, 640–650.
Vossler D, Wilensky A, Cawthon D, Kraemer D, Ojemann L, Caylor L & Morgan J (2002). Serum and CSF glutamine levels in valproate-related hyperammonemic encephalopathy. Epilepsia 43, 154–159., http://www.100md.com(Lars Nybo, Mads K Dalsgaa)