Ammonia (NH3+NH4+) is a toxic metabolite with deleterious effects on the central nervous system(Reference Wilkinson, Smeeton and Watt1). Exercise can be used as a model to study ammonia metabolism in an intensity-dependent way(Reference Felipo and Butterworth2–Reference Roeykens, Magnus and Rogers5). During prolonged exercise, ammonia is mainly produced by the catabolism of amino acids(Reference Snow, Carey and Stathis6). On the other hand, during high-intensity exercise, the largest source of ammonia production is from AMP deamination(Reference Graham and MacLean7). Ammonia levels are related to the appearance of both central and peripheral fatigue(Reference Hellsten, Richter and Kiens8). Therefore, controlling increases in ammonia is an important strategy in ameliorating the metabolic response to exercise and in improving athletic performance(Reference Nybo, Dalsgaard and Steensberg9, Reference Hirai, Minatogawa and Hassan10).
The combination of keto analogues with amino acids has been used to treat patients with chronic kidney disease (CKD), portal systemic encephalopathy and hyperammonaemia(Reference Meneguello, Mendonça and Lancha11, Reference Walser12). Free amino acids can be used as substrates for ATP synthesis, which produces ammonia as a side product(Reference Savica, Santoro and Ciolino13). In an opposite manner, the use of keto analogues associated with amino acids (KAAA) has been proposed as a way to synthesise amino acids whilst decreasing free ammonaemia(Reference Wu14, Reference Walser15). During metabolism, amino acids are deaminated or transaminated to form keto acids via release of the amino group(Reference Furst16). These reactions are reversible, and the use of keto analogues could reduce the blood ammonia concentration, resulting in the production of amino acids(Reference Walser17). Thus, keto analogues may serve as nutritional supplements to synthesise amino acids of high biological value, especially in CKD patients. Furthermore, it is acknowledged that resistance and aerobic exercise programmes may serve important roles in the approach to the treatment, prevention and slowed progression of CKD(Reference Johansen18).
Although KAAA supplementation is effective in the treatment of CKD, particularly for postponing the necessity for dialysis, the use of KAAA is not popular due to its cost and the requirement for a low protein intake(Reference Burns, Cresswell and Ell19). Thus, there has been a lack of interest in studies involving KAAA due to the lack of cost effectiveness in its use as a therapeutic agent. This lack of interest has limited the number of new papers published on the mechanism of action of KAAA. In the present study, we evaluated the effect of KAAA supplementation on ammonia production and blood urea levels during resistance exercise, showing metabolic effects that can enhance performance and post-exercise recovery.
Materials and methods
Male Wistar rats (12 weeks of age and body mass ranging from 280 to 350 g) were divided into four groups of twelve animals each. The group that received only KAAA (KA group) and the group that received KAAA and exercise (KAEx group) received 0·1 g Ketosteril® (Fresenius Kabi, Bad Homburg, Germany) in 0·5 ml water (0·3 g/kg). The composition of the KAAA mixture per tablet was as follows: α-keto analogues of isoleucine, 335 mg; leucine, 505 mg; phenylalanine, 430 mg; valine, 340 mg; α-hydroxy analogue of methionine, 295 mg; l-lysine acetate, 75 mg; l-threonine, 265 mg; l-tryptophan, 115 mg; l-histidine, 190 mg; l-tyrosine, 150 mg. The group that received neither KAAA nor exercise (control (Ctl) group) and the group that received only exercise (Ex group) received 0·5 ml of 0·9 % NaCl, 1 h before exercise by oral administration. The animals were maintained in collective cages (four per cage) at 22 ± 2°C with a photoperiod of 12 h and fed ad libitum (diet and water). The study was approved by the Ethics Committee in Research of the University of Tiradentes, and followed the Guiding Principles for Research Involving Animals and Human Beings.
Resistance exercise was performed according to a previous study(Reference Tamaki, Shuichi and Shoichi20) after familiarisation and determination of the load that was to be applied according to the one repetition maximum (1RM) test. Familiarisation consisted of attaching the animal to the exercise device daily without stimulating the animals to exercise, starting 6 d before the experiment. The 1RM test was performed 1 d before resistance exercise and determined the heaviest weight that could be lifted. On the day of the experiment, fifty repetitions were performed with a load equal to 75 % of 1RM. The animals were stimulated to perform the repetitions through sticker electrodes (Axelgaard ValuTrode CF3200; Axelgaard Manufacturing Co. Ltd, Fallbrook, CA, USA) placed in the tail and connected to an electrostimulator (4 mA to 15 mA at 1 Hz for 1 s; Quark Dualpex 961; Quark Medical Products, São Paulo, Brazil).
Blood was collected through cardiac puncture before exercise (Ctl and KA groups) or immediately after exercise (Ex and KAEx groups). The blood samples were immediately centrifuged to obtain sera, which was subsequently frozen and stored at − 70°C for future biochemical analysis. Biochemical analyses of glucose, urea, urate and creatinine concentrations were performed using commercially available spectrophotometric assays (Labtest, Minas Gerais, Brazil). Lactate and ammonia were measured using an enzymic UV method (Randox, Crumlin, Co. Antrim, UK) on a Dade Model Dimension RXL Automated Chemistry Analyzer (Dade Behring, Eschborn, Germany), and haematological parameters were analysed using a Sysmex SE-9500 Automated Hematology Analyzer (TOA Medical Electronics, Kobe, Japan). Standard curves were taken at a minimum r value of 0·98 and the experimental points were always within the calibration curve and at least 20 % above the lower limit of detection.
Statistical significance was evaluated by one-way ANOVA. Significances (P < 0·05) were confirmed using the Tukey test as a post hoc analysis. Data are reported as mean values with their standard errors.
Results
We used a weight-lifting exercise to evaluate the effect of KAAA on blood ammonia concentration after resistance exercise. There was an increase of about 40 % in ammonia in resting animals due to KAAA supplementation. Compared with unsupplemented controls, exercise resulted in a twofold increase in ammonia levels in the animals. However, the supplemented group had a much smaller increase (about 20 %) in ammonia levels after exercise (Fig. 1(A)). Since KAAA is proposed to decrease blood urea levels, we evaluated the response of blood urea to acute KAAA supplementation during exercise. We did not measure the effect of supplementation on blood urea at rest. With no supplementation, exercise significantly increased blood urea levels by 17 % compared with the levels in the Ctl group. However, with KAAA supplementation, blood urea was reduced to 75 % of the pre-exercise values (Fig. 1(B)).
Acute supplementation of keto analogues associated with amino acids (KAAA) affects ammonia (A), urea (B) and urate (C) metabolism. Ctl, control group (neither KAAA nor exercise); KA, KAAA-only group; Ex, exercise-only group; KAEx, KAAA and exercise group. Values are means, with standard errors represented by vertical bars. a,b,c,d Mean values with unlike letters were significantly different (P < 0·05). Inset of Fig. 1(A) shows the increase in ammonia levels in experimental and control groups when normalised against rest values.
To differentiate the ammonia production derived from AMP deamination from that derived from amino acid deamination, we measured urate, the end metabolite of inosine monophosphate. Blood urate levels increased 28 % in the group supplemented with KAAA, independent of exercise. This effect was enhanced in the supplemented group after exercise. There was no change in blood urate levels in the control group in response to exercise (Fig. 1(C)).
KAAA supplementation has been shown to increase creatinine clearance. Our study model did not detect a change in blood creatinine in response to exercise. However, blood creatinine decreased by 40 % in the groups supplemented with KAAA, independent of exercise (Table 1).
(Mean values with their standard errors)
Ctl, control (no keto analogues associated with amino acids or exercise); KA, keto analogues associated with amino acids only; Ex, exercise only; KAEx, keto analogues associated with amino acids and exercise.
a,b,c Mean values within a row with unlike superscript letters were significantly different (P < 0·05; ANOVA).
To understand the effect of KAAA on glucose maintenance, we measured glucose levels after exercise. Supplementation increased glucose levels in resting animals by 10 %. Exercise did not change glucose levels in either the Ctl or KAEx groups (Table 1).
Blood lactate is an indicator of glucose utilisation during exercise. Exercise promoted a 57 % increase in blood lactate in the Ctl group. The supplementation promoted a twofold exercise-induced increase in blood lactate (Table 1).
Discussion
It is widely reported that ammonia production increases during exercise and that ammonia could be a deleterious metabolite that promotes fatigue(Reference Hellsten, Richter and Kiens8, Reference Tamaki, Shuichi and Shoichi20, Reference Banister and Cameron21). The production of ammonia can lead to significantly elevated systemic ammonia levels to levels between 90 and>200 μmol/l. Patients with either liver or kidney disease also show sharp increases in ammonia levels that may range from 70 to 300 μmol/l in liver disorder patients(Reference Wilkinson, Smeeton and Watt1, Reference Olde Damink, Deutz and Dejong22). Patients with CKD have lower peak levels of ammonia during exercise, but experience ammonia increases of about 30–60 % compared with resting values(Reference Wilkinson, Smeeton and Watt1, Reference Pimentel, Brusilow and Mitch23, Reference Deferrari, Garibotto and Robaudo24). KAAA has been widely used as a supplement to treat patients with kidney failure and as a therapeutic agent for liver failure and encephalopathy(Reference Walser12). Additionally, regular physical activity and close clinical and dietary monitoring, including the use of keto analogues, should be recommended in patients with CKD(Reference Cupisti, Licitra and Chisari25).
One of the problems associated with human studies has been ensuring that subjects have adhered to the recommended diet and have properly taken the supplements. Here, we used a previously described resistance exercise animal method(Reference Burns, Cresswell and Ell19) to investigate a possible ammonia-chelating effect of KAAA during exercise in rats. The production of ammonia during exercise occurs via both AMP deamination and branched-chain amino acid metabolism(Reference Wilkinson, Smeeton and Watt1).
The use of KAAA increased ammonaemia during the resting state, demonstrating that amino acid metabolism during exercise is associated with anaplerosis of Krebs cycle intermediates(Reference Gibala, Lozej and Tarnopolsky26, Reference Gibala, MacLean and Graham27). Increases in ammonia levels in response to exercise can be managed through the use of amino acids or carbohydrates that interfere with ammonia metabolism(Reference Carvalho-Peixoto, Alves and Cameron28). It is possible to propose that the amino acids in the supplement are being using either as carbon skeleton donors to obtain energy or as gluconeogenic precursors. Even with an increase in ammonia levels at rest, KAAA supplementation was able to reduce the exercise-induced increase in blood ammonia by 80 %. When compared with the non-supplemented exercise group, the absolute decrease was 20 %. Previous data in our laboratory showed that there is a habituation of basal ammonia levels in response to amino acid supplementation, since the resting ammonia level decreases with an increase in basal blood urea levels correlated to supplementation time(Reference Bassini-Cameron, Monteiro and Gomes3). On the basis of these data, we postulate that the effect of KAAA supplementation on basal ammonia levels can be diminished by chronic KAAA use.
Our exercise model increased ammonia and urea levels in animals without any changes in urate levels. It has been pointed out that excess ammonia is metabolised to urea by the liver for excretion to minimise toxicity(Reference Felipo and Butterworth2). During exercise, KAAA was able to decrease the blood urea concentration to 75 % of the resting urea level. This finding is related to the widely described therapeutic effect of KAAA (for a review, see Savica et al. (Reference Savica, Santoro and Ciolino13)). Urate appears more quickly in blood in response to exercise compared with urea(Reference Bessa, Nissenbaum and Monteiro29). KAAA supplementation increased resting urate levels. However, we detected changes in blood urate in the supplemented exercise group when compared with the non-supplemented exercise group after resistance exercise. It is known that during high-intensity exercise such as resistance exercise, the largest quantity of urate is produced when the ATP:ADP ratio decreases which leads to increases in both AMP deamination and urate synthesis(Reference Hellsten, Richter and Kiens8). It is possible that our resistance exercise model with fifty repetitions activates pathways associated with resistance and prolonged exercise. KAAA supplementation promoted an increase in creatinine clearance. This is a well-described effect of this supplement in chronically ill patients(Reference Walser12). Taking these results together, we postulate that the majority of ammonia production results from the deamination of amino acids instead of AMP(Reference Graham and MacLean7, Reference Hellsten, Richter and Kiens8, Reference Zhao, Xu and Du30).
Some studies have shown that amino acid supplementation increased the pool of Krebs cycle intermediates during exercise(Reference Roeykens, Magnus and Rogers5, Reference Bruce, Constantini-Teodosiiou and Greenhaff31). KAAA supplementation produced a 10 % increase in resting glucose levels that were maintained even after exercise. Since KAAA is a mixture of ketogenic and glucogenic keto analogues and amino acids, we postulate that KAAA provide glucose for exercise. It is important to state that the use of the amino acids from KAAA as carbon skeleton donors augments the net ammonia release. On the other hand, the anaplerosis using the keto analogues does not increase ammonia release. However, both situations increase ATP synthesis, leading to a decelerating ammonia production due to AMP deamination.
The results of the present study showed that KAAA supplementation exacerbated blood lactate levels after exercise. It is known that lactate is formed during glycolysis in active skeletal muscles and many conditions can attenuate lactate levels during exercise, such as muscle glycogen depletion(Reference Mourtzakis and Graham32). Thus, such alterations in the present study may be explained by KAAA providing glucose for exercise through gluconeogenesis. Since the central nervous system has no effective urea cycle and depends on the synthesis of glutamine for removal of the excess ammonia(Reference Nybo, Dalsgaard and Steensberg9, Reference Suárez, Bodega and Fernandez33), high levels of blood ammonia have been proposed to be related to the development of both local and central fatigue(Reference Nybo, Dalsgaard and Steensberg9, Reference Banister and Cameron21, Reference Cooper34). Here, we describe for the first time that acute supplementation of KAAA can be used to reduce the increase in ammonia levels caused by resistance exercise. The practical significance of these findings may be important for the individual exerciser and merits further research to examine the efficacy of chronic KAAA intake. Therefore, we believe that the present study contributes important data to our understanding of metabolism and that these findings could be helpful for the development of future therapies.
Acknowledgements
The present study was supported in part by Tiradentes University.
R. D. A., E. S. P. and L.-C. C. were responsible for the study design. All authors contributed to data collection and interpretation, and manuscript writing.
The authors have no conflicts of interest to declare.
