Effect of acute acidosis on protein and amino acid metabolism in rats

ARTICLE IN PRESS Clinical Nutrition (2003) 22(5): 437–443 r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0261-5614(03)00041-4

ORIGINAL ARTICLE

Effect of acute acidosis on protein and amino acid metabolism in rats R. SAFRAŁNEK,n M. HOLECEK,n J. KADLCIŁ KOVAŁ,y L. SPRONGL,z C. MISLANOVAŁ,y M. KUKAN,y J. CHLAŁDEKz *Department of Physiology, Charles University, Hradec KraŁloveŁ, Czech Republic, yDepartment of Pharmacology andToxicology, Faculty of Pharmacy, Charles University, Hradec KraŁloveŁ, Czech Republic, zDepartment of Biochemistry, Hospital Motol, Prague, Czech Republic, yLaboratory of Perfused Organs, Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, and z Department of Pharmacology, Faculty of Medicine, Charles University, Hradec KraŁloveŁ, Czech Republic (Correspondence to: RS+ Department of Physiology, Faculty of Medicine of Charles University, S+imkova 870, P.O.BOX 38, 500 38, Hradec KraŁloveŁ, Czech, Republic).

Abstract5Background & aims: Metabolic acidosis is a common ¢nding in critical illness.The aim of the present study was to evaluate acute acidosis as a signal that induces changes in protein metabolism. Methods: In the ¢rst study, Wistar rats were infused for 6 h with HCl or saline resulting in blood pH 7.3070.03 and 7.4670.02, respectively. The whole body protein metabolism was evaluated using l-[1-14C]leucine. In the second study, soleus and extensor digitorum longus muscles from normal rats were incubated in medium, pH 7.4, 7.3 or 7.0. Protein metabolism was evaluated using l-[1-14C]leucine and tyrosine release. Results: In the in vivo study we observed increased protein turnover^protein synthesis, proteolysis and leucine oxidation and more negative protein balance in rats with acidosis.There was no change in protein synthesis in gastrocnemius muscle.We observed an increase in plasma levels of most amino acids including branched-chain amino acids and a decrease in intracellular amino acid pool in skeletal muscle. In vitro decrease in pH of 0.1had no e¡ect on protein metabolism, decrease of 0.4 decreased protein turnover and leucine oxidation. Conclusion: Acute metabolic acidosis is a protein wasting condition. Direct e¡ect of acidosis on skeletal muscle is under condition in vivo modi¢ed by neurohumoral regulations. r 2003 Elsevier Science Ltd. All rights reserved.

a-keto acid dehydrogenase) further increases negativity of nitrogen balance (7). Acidosis also decreases albumin synthesis in the liver (3) and enhances renal ammonia genesis (8). Furthermore, acidosis is associated with derangements in endocrine functions and calcium metabolism with its clinical complications. Abnormal protein metabolism caused partly by acidosis contributes to the higher morbidity and mortality in patients. Unfortunately, accelerated protein breakdown cannot be suppressed effectively by provision of exogenous nutritional substrates (9). The pathogenesis of excessive catabolism of proteins in critical illness is still not clear and a better understanding of the role of acidosis as a cause of protein catabolism can help optimise treatment options and identify new therapeutic strategies. As there is very limited number of papers dealing with effects of acidosis that is not complicated by any underlying disease on protein metabolism, especially protein synthesis, we directed the present experiments at evaluating metabolic acidosis as a signal that induces changes in protein metabolism. Using a combination of in vivo and in vitro methods we evaluated the effect of short-term acidosis on parameters of protein and amino acid metabolism in the whole body and in skeletal muscle.

Key words: acidosis; muscle; leucine; protein metabolism

Introduction Loss of lean body mass, negative nitrogen balance, increased catabolic rate, compromised wound healing, and immunosuppression are common in patients in critical illness resulting from sepsis, severe trauma, surgery or renal failure. There is growing evidence that metabolic acidosis is one of the causative factors that initiate the above-mentioned changes in protein metabolism. Earlier works demonstrated that the stunned growth of children with renal tubular acidosis is sharply improved by supplementation of sodium bicarbonate to their diet (1). Several papers demonstrated increased protein breakdown and catabolic rate in acidosis in humans (2, 3) and in animal models (4–5). Activation of ubiquitine-proteasome dependent pathway is mainly responsible for this increase in proteolysis (6). However, few data exist regarding effects of acute metabolic acidosis on protein synthesis. Concerning amino acid metabolism, increased activity of the key enzyme of branched-chain amino acid catabolism (branched-chain 437

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Materials and methods Animals Wistar rats (BioTest, Konarovice, Czech Republic) were housed in standardized cages in quarters with controlled temperature and a 12-h light–dark cycle. The rats were given free access to M2 lab chow and water. All procedures involving animals were performed according to guidelines set by the Institutional Animal Use and Care Committee of Charles University. Materials 14

l-[1- C]leucine was purchased from Amersham (Buckinghamshire, UK), [14C]bicarbonate was obtained from ICN Biomedicals (CA, USA). Amino acids, cycloheximid, Folin-Ciocalteu’s phenol reagent and albumin were purchased from Sigma Chemical (MO, USA), hydroxide of hyamine from Packard Instruments (CT, USA), Aminoplasmal 15 from B. Braun Medicals (Germany). Other chemicals were purchased from Sigma Chemicals (MO, USA) and Lachema (Brno, Czech Republic). Experimental design We carried out two separate studies. The first study was designed to assess the effects of acidosis in vivo. In the second study we measured parameters of protein and amino acid metabolism in isolated skeletal muscle. Study I Male Wistar rats (230–270 g) were used for this experiment. A polyethylene cannula was inserted into the jugular vein 24 h before the beginning of the study. Conscious unrestrained rats were placed in a glass metabolic cage and infused with either 0.2 M HCl or saline for 2 h at a rate 0.40 ml/h. The dose and the rate of infusion were estimated on the basis of our preliminary studies and literature (10, 11). Then l-[1-14C]leucine (1.9 mCi/ml) was added to the infused solutions. A priming dose of 0.7 ml infused within 4 min 35 s (i.e., 1.33 mCi of l-[1-14C]leucine) was followed by a constant infusion at a rate of 0.40 ml/h for 200 min. The rats were killed by exsanguination via the abdominal aorta exactly at the 321st minute from the beginning of the infusion. Afterwards the gastrocnemius muscle was quickly removed and immediately frozen in liquid nitrogen. The parameters of the whole body leucine metabolism were evaluated at steady-state conditions by the procedure described in detail previously (12). The expired CO2 was trapped at 10-min intervals between the 125th and 185th minute of infusion by monoethanolamine. The average value of six measurements of 14 CO2 radioactivity in the expired air at steady-state condition was used for calculations of the leucine oxidation rate. The 14CO2 recovery factor (FR) esti-

mated by infusion of [14C] bicarbonate was about 99% for HCl infused rats and 91% for control animals. Leucine specific activity (SALeu in disintegrations per minute per micromole (dpm/mmol)), turnover (QLeu), clearance (CLeu), and decarboxylation (DLeu) rates were calculated using the following formulas: SALeu ðdpm=mmolÞ ¼ QLeu ðmmol=hÞ ¼ CLeu ðml=hÞ ¼

Leu radioactivity ðdpm=mlÞ ; Leu concentration ðmmol=mlÞ

infusion rate ðdpm=hÞ ; SALeu in plasma ðdpm=mmolÞ

QLeu ðmmol=hÞ ; plasma Leu ðmmol=mlÞ 14

DLeu ðmmol=hÞ ¼

CO2 production rate ðdpm=hÞ SALeu in plasma ðdpm=mmolÞ  FR

The whole body leucine metabolism was considered to take place within a common metabolic pool represented by free plasma leucine. Due to the fact that exogenous leucine intake (E) was zero in our protocol, QLeu estimates the leucine released from protein, i.e. the protein breakdown (B) as described by the equation: Q=In+D=B+E. Using this formula, rates of leucine incorporation into protein (In), the oxidized fraction of leucine (OF=D  100/Q) and the fraction of leucine incorporated into protein (IF=In  100/Q) were calculated. The samples for measurement of protein synthesis in gastrocnemius muscle were processed as described elsewhere (13). To avoid contamination by ketoisocaproate, samples were treated with 30% hydrogen peroxide, which causes the carboxyl carbon of ketoisocaproate to be released as CO2 (12,14,15). The fractional protein synthesis rates were calculated using the equation derived by Garlick (16): Sb li ð1  eKs t Þ Ks ¼   ; Si ðli  Ks Þ ð1  elit Þ ðli  Ks Þ In the equation, Sb and Si are the specific activities of the protein-bound and free acid-soluble tissue leucine pools, respectively, in dpm/mmol li is the rate constant for the rate of rise of specific activity of leucine in the acid-soluble amino acid pool per day; t is the duration of l-[1-14C]leucine infusion in days, and Ks is the fraction of protein mass renewed each day, in percent per day. The value of 38/day was taken to represent li in different tissues of rats under different treatments on the basis of literature. Study II Female Wistar rats (30–50 g) were used for this study. Soleus and extensor digitorum longus muscles of both legs were dissected under anesthesia with pentobarbital (6 mg/100 g body weight, intraperitoneally) as described in detail by Maizels (17). Isolated muscles were fixed to stainless-steel clips to provide slight tension and

ARTICLE IN PRESS CLINICAL NUTRITION

immediately transferred to 2.5 ml of modified Krebs-Henseleit bicarbonate buffer with 6 mM glucose and 2 mU/ml insulin that was gassed with O2/CO2 (19:1). Muscles were preincubated for 30 min in a thermostatically controlled bath (371C) with a shaking device (70 cycles/min). After preincubation, muscles were quickly rinsed in 0.9% NaCl, blotted and transferred to a second set of vials containing fresh media identical in composition and volume. One muscle was placed in each vial, so that control and experimental incubations could be compared from the same rat. Protein synthesis and leucine oxidation were measured after 1-h incubation. A solution of amino acids — Aminoplasmal 15 (2.2 mM amino acids) and glutamine (0.5 mM) were added into the media for preincubation and incubation. [1-14C]Leucine (0.6 mCi/ml) was present in incubation medium only. At the end of the incubation, hydroxide hyamine (0.4 ml) was added to the hanging well and the reaction was stopped by the addition of 35% (v/v) perchloric acid solution (0.2 ml) to the incubation medium (18). At the end of each incubation, muscles were removed from the incubation flasks, quickly rinsed in cold 0.9% NaCl and blotted, and then dropped into liquid nitrogen and pulverized. Muscles were further homogenized and proteins precipitated in 6% (v/v) HClO4. Amount of l-[1-14C]leucine incorporated into proteins was estimated after their dissolution in 2 M NaOH. Protein synthesis was calculated using leucine specific activity and expressed as nmol of incorporated leucine/g wet weight/hour. Proteolysis was estimated after 2-h incubation. Total protein breakdown was expressed as the rate of tyrosine release into the medium. To prevent reincorporation of amino acids released during proteolysis, 0.5 mM cycloheximide was added into the medium. Tyrosine was measured fluorimetrically as described by Waalkes (19). To assess the viability and energy status of the muscles we measured ATP, ADP, AMP, calculated energy charge ([ATP+ADP/2]/[ATP+ADP+AMP]), weighed wet and dry muscles and calculated wet/dry weight ratio. No statistically significant differences at Po0.05 were found in any parameters when comparing the values before (energy charge in soleus mucle 0.8770.03, extensor digitorum longus 0.9070.02) and after (soleus muscle 0.8270.013, extensor digitorum longus 0.9070.017) incubation.

blood gas system AVL 995 (AVL, Graz, Austria). The radioactivity of the samples was measured with the liquid scintillation radioactivity counter LS 6000 (Beckman Instruments, CA, USA). ATP, ADP, and AMP were measured using high-performance liquid chromatography. Preparation of samples and analytical procedure is described in detail by Vajdova (21). Statistical analysis Results are expressed as the mean7SE. F-test, paired or unpaired t-test was used for analysis of the data. A difference was considered significant at Po0.05.

Results Characteristics of experimental animals in study I are shown in Table 1. There was a significant decrease in pH, buffer base, base excess, and bicarbonate level in HCl infused rats. There were no changes in sodium and potassium levels. In HCl infused rats significant increase in blood glucose level was observed. Table 2 shows that acute acidosis induced a significant increase in most of the evaluated parameters related to the whole body leucine metabolism (i.e. leucine turnover, oxidation, and incorporation into protein). However, we did not observe significant changes in fractional rate of protein synthesis in gastrocnemius muscle. Table 3 shows that acidosis caused a significant increase in plasma levels of most amino acids, including valine, leucine, and isoleucine. Amino acid concentrations in gastrocnemius muscle were less affected—threonine and glutamine decreased while leucine and phenylalanine increased; (Table 4). In the second, in vitro, study we did not observe significant differences in protein synthesis, protein breakdown and leucine oxidation between muscles incubated in media of pH 7.3 or 7.4 (Figs. 3 and 4). However, as shown in Figs. 1 and 2, decrease in pH from 7.4 to 7.0 decreased leucine oxidation and proteolysis, both in soleus and extensor digitorum longus muscles. Furthermore, in soleus muscle incubated in medium of pH 7.0 (control pH 7.4) a significant decrease in protein synthesis was found (Fig. 1).

Table 1 Study I. Parameters of experimental animals

Other techniques Protein content was measured according to Lowry (20). Amino acid concentrations in deproteinized samples of blood plasma or skeletal muscle tissue were determined with high-performance liquid chromatography (Waters, MA, USA) after precolumn derivatization with o-phthaldialdehyde. The blood pH, HCO 3 , buffer base and base excess were analyzed using the automatic

439

Body weight (g) Glucose (mmol/l) Na+ (mmol/l) K+ (mmol/l) pH Base excess (mmol/l) Buffer base (mmol/l) HCO 3 (mmol/l) Mean7SE. *Po0.05 vs control.

Control (n=9)

Acidosis (n=10)

25279 9.070.3 14070 3.0570.08 7.4670.02 0.7170.92 4971 22.370.8

24375 11.170.4* 13971 3.2970.90 7.3070.03* 4.0171.16* 4571* 19.670.8*

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Acidosis (n=10)

127713 108711 1973 1572 852784 1.6070.19

241733* 197727* 4577* 1972 10057105 1.5870.15

FPSR; fractional rate of protein synthesis is expressed as the fraction of protein mass renewed each day (%/day). Mean7SE, *Po0.05 vs control.

Table 3 The effect of acidosis on amino acid concentrations in blood plasma

Taurine Aspartate Threonine Serine Asparagine Glutamate Glutamine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Arginine Derived values Branched-chain amino acids Total amino acids

Control (n=9)

Acidosis (n=10)

179714 571 143711 184715 3674 74710 365714 328724 318731 164713 2272 7876 152711 5174 5174 5875 1771 201719 10078

290737* 571 16678 22378* 4978* 80714 406713* 335720 455772* 250711* 2872* 9975* 238715* 6172* 6972* 4674 2072 227717 10274

393730 25397154

587730* 31657120*

Mean7SE in mmol/l, *Po0.05 vs control.

Taurine Aspartate Threonine Serine Asparagine Glutamate Glutamine Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Ornithine Lysine Arginine Derived values Branched-chain amino acids Total amino acids

Control (n=9)

Acidosis (n=10)

5.7970.23 0.3370.03 0.8170.07 0.4770.03 0.1570.02 1.6070.09 2.0370.22 2.6170.19 2.9270.20 0.2370.01 0.0270.00 0.1470.01 0.2370.01 0.1670.02 0.0770.00 0.0270.00 0.0470.00 0.2670.02 0.1070.01

4.1570.46 0.4970.07 0.5470.07* 0.5470.05 0.1570.01 1.4970.12 1.3470.11* 2.1170.19 2.6370.15 0.2970.03 0.0270.00 0.1570.01 0.2970.02* 0.1470.01 0.1070.01* 0.0270.00 0.0370.00 0.2670.02 0.1570.03

0.6070.03 17.9870.80

0.7470.06 14.9070.70*

Mean7SE in mmol/g. *Po0.05 vs control.

Protein synthesis 30 25

( 7)

*

20 15 10

Proteolysis

Leucine oxidation

350

120

300 (10) 250 200 150 100

5

50

0

0

7.4 7.0

*

nmol Try/g wet wt./h

Proteolysis (mmol Leu/kg/h) Protein synthesis (mmol Leu/kg/h) Leucine oxidation (mmol/kg/h) Leucine oxidized fraction (%) Plasma clearance of leucine (ml/kg/h) FPSR in muscle (%/day)

Control (n=9)

Table 4 Study I. The effect of acidosis on amino acid concentrations in gastrocnemius muscle

nmol Try/g wet wt./h

Table 2 The effect of acidosis on the whole body leucine metabolism and protein synthesis in muscle

nmol Leu/g wet wt./h

440

100

( 7)

*

80 60 40 20

7.4 7.0

0

7.4 7.0 pH

Fig. 1 The effect of severe acidosis on protein synthesis, proteolysis and leucine oxidation in isolated soleus muscle. Paired t-test. Mean7SE. *Po0.05; pH of incubation media 7.4 (&) or 7.0 (’), respectively.

Discussion We investigated the influence of acute metabolic acidosis on the whole body and skeletal muscle protein and amino acid metabolism. To date, almost all experimental and clinical studies investigated the effect of acidosis on protein metabolism concerned with chronic metabolic acidosis. We used a short period of acidosis to determine what are its acute effects. Results of in vivo experiment clearly show that acute acidosis results in negative protein balance with increased protein turnover (i.e. increased protein breakdown, protein synthesis and leucine oxidation). Our results are in agreement with several studies including experiments with HCl infused dogs (22) and ovine fetal (23). In the latter study, acidosis resulted in increased leucine oxidation and protein breakdown, but protein synthesis did not change. Increased protein turnover was also observed in chronic acidosis

that was induced by addition of ammonium chloride to the diet, both in rats (4) and in humans (2). However, it should be noted that in contrast to other studies Jeevanandam et al. (24) using a pulse dose of l-[15N] alanine and [13C] urea observed decreased protein turnover in HCl induced acidosis in dogs. We found no change in the fractional rate of protein synthesis in gastrocnemius muscle in rats with acidosis. This suggests that in vivo skeletal muscle is not responsible for increased whole body protein synthesis in acute acidosis. This is in agreement with data obtained in humans, where muscle protein synthesis decreased after 48 h of acidosis induced by administration of ammonium chloride (25). In addition, changes in protein synthesis in skeletal muscle were not observed even in chronic acidosis (26, 27).

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20 15 10 5

(10)

*

250 200 150 100 50

0

0 7.4 7.0

100 (10)

*

80 60 40 20

7.4 7.0

7.4

Proteolysis

7.0 pH

15 10 5

7.4 7.3

250 200 150 100 50 0

(9) nmol Leu/g wet wt./h

nmol Tyr/g wet wt./h

nmol Leu/g wet wt./h

20

200

300 (9)

25 (9)

0

Leucine oxidation

350

30

20 15 10 5

0

Fig. 2 The effect of severe acidosis on protein synthesis, proteolysis and leucine oxidation in isolated extensor digitorum lonus muscle. Paired t-test. Mean7SE. *Po0.05; pH of incubation media 7.4 (&) or 7.0 (’), respectively.

Protein synthesis

25 (8)

7.4 7.3

150

100

50

0

7.4 7.3 pH

Fig. 3 The effect of mild acidosis on protein synthesis, proteolysis and leucine oxidation in isolated soleus muscle. Paired t-test. Mean7SE. *Po0.05; pH of incubation media 7.4 (&) or 7.3 (’), respectively.

Decrease in pH of the incubation media from 7.4 to 7.3 had no effect on protein synthesis, protein breakdown and leucine oxidation in muscles. This is in agreement with observation that incubation of epitrochlearis muscles in acidified media and perfusing the hindquarters of normal rats with acidified media had no effect on protein degradation (4). Furthermore, in our experiment, the decrease in pH of incubation media from 7.4 to 7.0 resulted in the decrease in protein turnover. We conclude from our data that acidosis has a direct effect on skeletal muscle, but only in severe acidosis. Our results indicate that the direct effect of acidosis on skeletal muscle is opposite to the changes in whole body protein metabolism that take place in acidosis in vivo. Therefore, direct effects of acidosis on protein and amino acid metabolism in vivo are probably of minor importance. We suggest that the physiologically important metabolic effects of acidosis must be mediated by neurohumoral changes. Several factors have already been identified but their importance and interactions are yet to be established. For example, it has been suggested that glucocorticoids are necessary for stimulation of protein degradation in skeletal muscle (27). There are also changes in insulin

0

7.4 7.3

Leucine oxidation

250

200

200 (11) nmol Leu/g wet wt./h

nmol Tyr/g wet wt./h

25 (10)

Proteolysis

30

120 nmol Leu/g wet wt./h

300

Protein synthesis

nmol Tyr/g wet wt./h

350

30 nmol Leu/g wet wt./h

Leucine oxidation

Proteolysis

nmol Leu/g wet wt./h

Protein synthesis

150 100 50 0

7.4 7.3

441

(8)

150 100 50 0

7.4 7.3 pH

Fig. 4 The effect of mild acidosis on protein synthesis, proteolysis and leucine oxidation in isolated extensor digitorum lonus muscle. Paired t-test. Mean7SE. *Po0.05; pH of incubation media 7.4 (&) or 7.3 (’), respectively.

secretion and sensitivity, in growth hormone/IGF-I axis, and thyroid hormone concentrations (10, 26, 28–31). Cytokines, well-established catabolic factors, may interact with hormonal changes and enhance protein catabolism (32, 33). These derangements and factors not yet identified seems to be of crucial importance for mediating effects of metabolic acidosis on skeletal muscle and play a role in causing negative protein balance in acidosis. We incubated slow and fast twitch skeletal muscles. In literature there are data showing that as far as proteolysis and energy metabolism are concerned, fast and slow twitch muscles react differently to different stimuli (34–37). In our study, incubation of both fast twitch (extensor digitorum longus) and slow twitch (soleus) muscles in media of pH 7.0 decreased protein turnover. However, protein synthesis was decreased only in soleus muscle. These data suggest that also regulation of protein synthesis in fast and slow muscle fibers is different. Plasma levels of amino acids in acidosis undergo timedependent changes. In our study, acute acidosis induced an increase in plasma levels of most amino acids including valine, leucine, and isoleucine. These findings concur with studies that evaluate changes in amino acid metabolism in different models (2, 22). This increase in plasma amino acids results probably from stimulated proteolysis and enhanced release of amino acids from tissues. However, after 5 days of acidosis, plasma leucine, valine, and isoleucine levels are significantly decreased and stay low in chronic acidosis (4). This is also true in patients with chronic renal failure with acidosis (38). The cause of increased blood glucose level observed in our study is not clear. In acute metabolic acidosis decreased hepatic glucose release (39) and inhibited glucose uptake due to impaired glycolysis was found (40). It should be noted, that depending on the duration of acidosis and employed model, authors find increase (41, 42), decrease (39) or no change (22) in blood glucose level.

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In conclusion, our data show that acidosis has a direct effect on skeletal muscle. Furthermore, metabolic acidosis has been proved to be a protein wasting condition even in the first hours of acidosis due to higher increase in protein breakdown than protein synthesis. From our results in acute acidosis and findings in chronic acidosis increased protein turnover seems to be a uniform reaction to metabolic acidosis both in rats and humans, without regard to the length of acidosis. Thus correction of metabolic acidosis, which has been proved to be beneficial in patients with chronic renal failure and associated metabolic acidosis (39, 40), could be also effective in critically ill patients with acute acidosis and possibly reduce morbidity and mortality. Acknowledgements This study was supported by the Grant No.305/01/0578 of the Grant Agency of the Czech Republic. We are grateful to I. Altmannova´, L. Kriesfalusyova´, and R. Rysˇ ava´ for the technical support.

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