Kidney International, Vol. 58 (2000), pp. 236–241

Alterations of protein metabolism by metabolic acidosisin children with chronic renal failure

YVES BOIRIE, MICHEL BROYER, MARIE FRANCE GAGNADOUX, PATRICK NIAUDET,and JEAN-LOUIS BRESSON

Laboratoire de Nutrition Humaine, Universite d’Auvergne, Clermont-Ferrand; Service de Nephrologie Pediatrique; andCentre d’Investigation Clinique, Hopital Necker-Enfants Malades, Paris, France

Alterations of protein metabolism by metabolic acidosis in intakes, since recent data from the Growth Failure inchildren with chronic renal failure. Renal Disease Study have documented the intake of

Background. Several reports suggest that metabolic acidosis protein in children to be 153% of the recommendedmay induce significant alterations in protein metabolism anddaily allowance (RDA) [2]. Thus, protein metabolismthat its outbreak may even result in growth retardation inalterations could limit growth velocity either by a lowerchildren with chronic renal failure (CRF). However, the effects

of metabolic acidosis on protein metabolism kinetics have response to anabolic factors such as nutrients andnever been investigated in these settings. hormones [such as growth hormone (GH), insulin-like

Methods. Postabsorptive leucine metabolism, a marker of growth factor-1 (IGF-1), or insulin resistance, changes inwhole-body protein metabolism, was measured by using aIGF-binding protein distribution, circulating inhibitingprimed, continuous intravenous infusion of L-[1-13C]leucine infactors] [3–6] or by an increased action of catabolic10 CRF children who were one to four years old.

Results. Interindividual values of whole-body protein turn- events. Among the potential wasting factors, metabolicover exhibited a very large range, which was mainly accounted acidosis might play an important role [7]. Indeed, growthfor by acidotic status (plasma HCO3

2) and body compositionretardation was reported to be associated with the out-[fat-free mass (FFM)]. After correction for differences in FFM,break of chronic acidosis in children with CRF [8]. Con-plasma HCO3

2 was highly correlated with protein breakdown(R2 5 0.65, P , 0.001), so that CRF children were divided in versely, a sustained alkali therapy normalizes growthtwo groups according to their acid-base status: Group A had rates in children suffering distal tubular acidosis [9], al-a mean plasma HCO3

2 level of 15.8 6 1.5 mmol · L21 (mean 6 though this treatment alone does not seem to be efficientSD, N 5 5), whereas group B had near-normal values (HCO3

2,in CRF. Moreover, acidosis induces significant alter-22.6 6 3.0 mmol · L21, N 5 5). The leucine rate of appearanceations in protein metabolism [7] and might be involvedfrom protein breakdown was markedly higher in group A than

in group B (4.15 6 1.43 vs. 2.46 6 0.47 mmol · kg21 · min21, in the resistance to the anabolic action of hormones,respectively, P , 0.05), and the net leucine balance tended to such as GH [10].be more negative in group A (20.73 6 0.34 vs. 20.44 6 0.26 From a pathophysiological point of view, the mecha-mmol · kg21 · min21, respectively).

nism of an impaired protein deposition in CRF childrenConclusions. Metabolic acidosis in children with CRF re-necessarily involves either a reduced protein synthesissults in an excessive catabolic state, suggesting that acidosis-

related protein wasting could contribute to growth retardation. rate or an increased protein breakdown or both phenom-ena. In CRF rat models, metabolic acidosis has beenreported to be responsible for amino acids and protein

Growth retardation and protein malnutrition are com- metabolism alterations at the whole-body [11, 12] andmon clinical features in children with chronic renal fail- the skeletal muscle levels [13, 14]. This acidosis-inducedure (CRF) [1], and point toward an imbalance between muscle protein degradation is mediated by an activationprotein intake and body protein losses. Actually, this of the ubiquitin adenosine 59-triphosphate (ATP)-depen-situation is unlikely to be related only to altered protein dent pathway [15], which has also been involved in other

stress situations, such as head trauma patients [16]. Thespecific action of glucocorticoids on this proteolytic path-Key words: amino acids, kidney metabolism, muscle wasting, growth

retardation, malnutrition. way is also required to concomitantly increase the ex-pression of the proteasome mRNAs [17]. In rats, thisReceived for publication July 27, 1999acidosis-induced protein degradation is associated withand in revised form January 11, 2000

Accepted for publication February 11, 2000 an increase in the amino acid oxidation rate, particularlybranched chain amino acids, as a result of an increase 2000 by the International Society of Nephrology

236

Boirie et al: Protein metabolism in children with CRF 237

in the activity of the branched chain ketoacid dehydroge- of Necker-Enfants Malades Hospital, and the purposeof the study was explained to the parents before theynase [18, 19]. The consequences of metabolic acidosis

on protein synthesis are more controversial in vivo, since gave their written informed consent.protein synthesis tends to increase with increasing pro-

Anthropometric assessmenttein breakdown rates [12]. However, changes in proteinsynthesis are smaller than those in protein breakdown Skinfold thickness was measured at four sites (biceps,

triceps, subscapular, and suprailiac sites), and the meanso that the difference, expressed as net protein synthesis,is smaller in CRF patients than in controls [12]. result of three measurements for each site was recorded

for the lean body mass estimation (Table 1). The percent-In adults with CRF, bicarbonate supplementation hasrecently been shown to limit the accelerated rate of pro- age of body fat was also calculated from the equation

of Brooke [20].tein breakdown in the postabsorptive state. However,the consequences of metabolic acidosis on the regulation

Protocol designof protein metabolism have never been investigated inchildren with CRF [9]. Therefore, our study measured Prior to the study, protein and energy intakes were

assessed by a dietitian on a one-week history. All testsprotein metabolism in CRF children with various degreesof acid-base status, and the relationships between the were performed in the postabsorptive state, that is, the

last meal was taken between 7 and 8 p.m. before the daymagnitude of metabolic acidosis and metabolic distur-bances were analyzed to assess the sensitivity of protein of the isotopic study. The tracer infusion began the next

day between 7 and 8 a.m., thus following a 12-hour over-metabolism to various levels of metabolic acidosis in thispopulation. Changes in acid-base status have a great night fast. One catheter was inserted into an antecubital

vein for the tracer infusion and a second catheter in theimpact on protein metabolism in the postabsorptivestate, suggesting that the postabsorptive state has to be contralateral hand vein for blood sampling. Acid-base

status was assessed immediately before starting thelimited in combination with bicarbonate treatment toavoid the associated catabolic effects of fasting and aci- tracer infusion. Isotopic equilibration was obtained two

hours after a primed-continuous infusion of 13C leucinedosis in children.(4.2 mmol · kg21 bolus, followed by 0.07 mmol · kg21 ·min21). Samples were drawn at the beginning (0 min)

METHODSand every 30 minutes between 120 and 240 minutes for

Material a-ketoisocaproate (KIC) isotopic enrichments.For the determination of the natural abundance andL-[1-13C]leucine [99 mole percentage excess (MPE)]

and sodium [13C]bicarbonate (99 MPE) were supplied 13C enrichments in CO2, 10-minute expired air sampleswere collected before and during the study by using aby Cambridge Isotope Laboratories (Cambridge, MA,

USA). Isotopic purity of 13C leucine was checked by gas face mask connected to a Rudolph valve and a Douglasbag and were analyzed, as already described [21]. Mean-chromatography-mass spectrometry (GCMS). Solutions

of tracer were prepared before each study and were while oxygen consumption and CO2 production werecontinuously measured by open-circuit indirect calorim-tested for sterility and pyrogens. During each experi-

ment, priming and infusion were membrane filtered etry as previously described [22]. A transparent plasticventilated hood system was placed over the head of thethrough 0.22 mm filters.children and made air tight around the neck. Air output

Subjects was accurately estimated by a mass flowmeter, and theCO2 content of expired air was continuously measuredTen young children, aged one to four years, who at-

tended the nephrology clinics at the Necker-Enfants Ma- by an infrared CO2 analyzer. Hence, total 13CO2 excretedper minute was determined by multiplying CO2 produc-lades Hospital, were selected for the study. All subjects

had CRF caused by renal hypoplasia (N 5 5), renal tion by 13CO2 enrichment, divided by a correction factorof 0.8 for incomplete recovery of CO2 in breath.anoxia (N 5 4), or ureteral atresia (N 5 1). Each child’s

body height for age and body weight for height wereAnalytical methods and kinetics calculationscompared with the Sempe and Pedron French references

for growth. Actual height is expressed as a percentage Leucine fluxes were calculated according to the iso-topic dilution method in which labeled leucine is con-of expected height for age and actual weight as a percent-

age of expected weight for height. Clinical and anthropo- tinuously infused and plasma leucine enrichments aremeasured at plateau. However, intracellular leucine en-metric characteristics are presented in Table 1. None had

end-stage renal failure (creatinine clearance . 15 mL 3 richments may be lower than leucine enrichments inplasma, as intracellular labeled leucine is diluted by leu-min21) or infectious disease or were treated with gluco-

corticoids before or during the study period. The experi- cine released by protein breakdown [23]. For this reason,enrichment of KIC, the intracellular transaminationmental protocol was approved by the Ethical Committee

Boirie et al: Protein metabolism in children with CRF238

Table 1. Clinical and biological data of the children with chronic renal failure

Weight Lean massAge Height Plasma HCO32

Subjects Gender months cm mmol/Lkg

1 M 12 66 8.0 6.2 15.02 F 2 56 3.8 3.0 16.73 M 35 81 9.1 8.2 17.34 M 42 88 11.9 10.0 16.65 F 17 66 6.0 5.8 13.66 F 48 96 12.8 12.1 20.57 F 16 68 8.1 7.0 25.48 M 22 72 7.2 6.3 26.19 M 25 76 11.0 8.9 22.010 M 26 80 9.6 8.4 19.1Mean 24 75 8.8 7.6 19.2SD 14 12 2.7 2.5 4.2

product of leucine, best predicts the isotopic enrichment Statistical analysisof intracellular leucine and permits calculation of whole- Results are expressed as mean 6 SD. Covariance anal-body protein turnover according to the reciprocal pool ysis was used to assess the contribution of clinical (age,model [23]. Therefore, KIC was isolated and converted HCO3) and anthropometric parameters (weight, height,to trimethylsilyl-quinoxalinol derivatives [24], and KIC fat free mass, fat mass) to the variance of protein kinetics.enrichments were measured by selected ion-monitoring Then analysis of variance was performed to comparechemical ionization gas chromatography as already de- the results of protein kinetics between groups (Statview;scribed [22]. This model is a one-compartmental model Abacus Concept, Berkeley, CA, USA).in which leucine appearance in the pool corresponds tothe tracer infusion rate (I) and leucine derived from

RESULTSprotein breakdown (that is, endogenous leucine rate ofSubject characteristicsappearance or Endo Ra), whereas leucine is removed

The children’s anthropometric measurements are pre-from the pool, for either irreversible oxidation to CO2

sented in Table 1. Growth retardation was present in all(Ox) or protein synthesis (that is, nonoxidative leucinechildren (89.9 6 4.8% of expected height for age), anddisposal or NOLD). At steady-state enrichment, leucinetheir weight was 90.7 6 10.8% of expected weight forflux is determined as follows:height. Mean protein (2.7 6 1.1 g · kg21) and energy

Total Ra 5 I (Ei/Ep) (mmol · kg21 · min21) intakes (116.3 6 32.2 kcal · kg21 · day21) were close tothe RDA for age.where I is the [1-13C] leucine infusion rate in mmol · kg21 ·

min21, Ei the enrichment of the infused 13C leucine (99Leucine kineticsMPE), and Ep the enrichment of plasma KIC at plateau

Isotopic data in the whole population indicated a wide(MPE). This flux includes the tracer infusion.range of distribution for whole-body protein breakdownOxidation rate is calculated from the ratio of total(15.07 to 38.10 mmol · min21, minimum to maximum13CO2 production (mmol · kg21 · min21) to the enrichmentvalue), protein synthesis (13.37 to 30.96 mmol · min21),of the immediate precursor of leucine oxidation, that is,and leucine oxidation (1.45 to 10.75 mmol · min21; TableKIC, as follows:2). To explain this large interindividual variations in pro-

Ox 5 (E13CO2 3 VCO2)/(Ep KIC 3 k) tein kinetics between the subjects, relationships betweenclinical or metabolic parameters (weight, height, bodywhere k is the correction factor for incomplete recoverycomposition, plasma HCO3

2) were tested via analysis ofof 13CO2 in breath (0.8).covariance. Results showed that neither weight, height,From the two prior equations, Endo Ra (that is, whole-or age explained the differences between individuals forbody protein breakdown) and NOLD (that is, whole-protein metabolism. Moreover, the factor explaining thebody protein synthesis) can be estimated:major part of the variance in protein breakdown in this

Endo Ra 5 Total Ra 2 I population was plasma bicarbonate concentration. Thus,the median value of the bicarbonate concentration rangeNOLD 5 Total Ra 2 Oxwas chosen to divide the children in two groups. One

HCO32 concentrations and pH values were measured in group (group A) had metabolic acidosis with plasma

HCO32 less than 18.2 mmol · L21 (N 5 5; mean plasmaarterialized blood using a blood gas analyzer.

Boirie et al: Protein metabolism in children with CRF 239

Table 2. Leucine kinetics in children with chronic renal failure ascalculated from the reciprocal pool model

Total Ra Oxidation NOLD Endo Ra Balance

Subject lmol/kg · min

1 3.34 0.49 2.84 3.27 20.432 4.96 0.62 4.34 4.83 20.493 3.36 1.06 2.30 3.29 20.994 3.07 0.59 2.48 3.00 20.535 6.42 1.26 5.16 6.35 21.196 2.98 0.84 2.14 2.92 20.787 1.92 0.28 1.65 1.86 20.218 2.20 0.20 2.00 2.14 20.149 2.50 0.61 1.89 2.44 20.5410 2.99 0.59 2.40 2.92 20.52Mean 3.38 0.65 2.72 3.30 20.58SD 1.35 0.32 1.14 1.34 0.32

Abbreviations are: Total Ra, whole body leucine flux; NOLD, protein synthe-sis; Endo Ra, protein breakdown.

Fig. 1. Relationship between plasma bicarbonate and endogenous leu-cine rate of appearance, i.e., whole-body protein breakdown, in a popu-lation comprised of children with chronic renal failure (r 2 5 0.65; P ,0.01).

HCO32 15.8 6 1.5 mmol · L21 and mean pH 7.33 6 0.06;

patient numbers 1 to 5). The other group (group B) hadno acidosis, as most of them received bicarbon-

plasma bicarbonate concentration (Fig. 1). Noticeably,ate treatment (N 5 5; mean plasma HCO32 22.6 6

the leucine oxidation rate was also strongly related to3.0 mmol · L21 and mean pH 7.42 6 0.11; patient numbersplasma bicarbonate (r 2 5 0.57, P , 0.01).6 to 10). Height and weight did not significantly differ

between the two groups (89.9 6 6.0 vs. 89.9 6 4.0%,groups A vs. B, P 5 NS, and 91.2 6 11.4 vs. 90.2 6 DISCUSSION11.5%, A vs. B; P 5 NS, respectively). To further sub-

Protein malnutrition is a frequently observed situationstantiate the results, we then compared the kinetic datain uremic patients, even encountered before the begin-with respect to groups.ning of dialysis therapy [3]. Whole-body protein synthe-

As shown in Table 2, a higher protein breakdownsis and breakdown have already been investigated in

(Endo Ra) was found in group A than in group B (4.15 6CRF adult patients [12, 15]. From these studies, meta-

1.43 vs. 2.46 6 0.47 mmol · kg21 · min21, groups A vs. B, bolic acidosis appears to be one of the major determi-P , 0.05). This difference was even larger when protein nants of protein wasting that can be related to an activa-breakdown data were corrected for differences in fat- tion of the ubiquitin proteolytic pathway [15]. However,free mass (FFM; 4.84 6 1.43 vs. 2.81 6 0.49 mmol · kg21

when CRF appears early during life, as in children suffer-FFM · min21, A vs. B, P , 0.02). This high whole-body ing from renal disease, growth can be hampered. Theproteolysis rate in the acidotic group was also associated magnitude of acidosis-mediated protein wasting and itswith a 58% higher, but not statistically different, leucine specific effects on protein kinetics to our knowledge hasoxidation rate per kg body weight (0.80 6 0.33 vs. 0.50 6 never been investigated in CRF children [9]. Thus, we0.26 mmol · kg21 · min21, A vs. B) or per kg fat free mass studied 10 CRF children with various degrees of meta-(0.92 6 0.30 vs. 0.57 6 0.28 mmol · kg21 FFM · min21, bolic acidosis and using a stable isotopic approach basedA vs. B). The increased protein breakdown in group A on 13C-leucine dilution. Among the kinetic parameters,was associated with an increased nonoxidative leucine whole-body protein breakdown appeared to be mark-disposal or NOLD (index of whole body protein synthe- edly increased as the intensity of metabolic acidosissis; 3.42 6 1.26 vs. 2.02 6 0.28 mmol · kg21 · min21, groups increased, with a tight correlation between plasma bicar-A vs. B, P 5 0.04). Finally, leucine balance was more bonate and protein breakdown. This observation suggestsnegative in group A, as compared with group B, but that protein breakdown is very sensitive to acid-basenot significantly (20.73 6 0.34 vs. 20.44 6 0.26 mmol · status, as in the adult CRF patients and that bicarbonatekg21 · min21, A vs. B, P 5 0.11). concentration lower than 18 mmol/L could be considered

Regression analysis of leucine kinetics and biological as a threshold where protein breakdown is strongly stim-data revealed that the level of protein breakdown cor- ulated in children.rected for differences in lean body mass was strongly From a methodological point of view, branched-chaincorrelated to the acid-base status, as 65% of the interindi- amino acid leucine was chosen to trace the metabolic

fate of the free amino acids in CRF patients. It is a well-vidual variance in protein breakdown was explained by

Boirie et al: Protein metabolism in children with CRF240

known essential amino acid giving an accurate estimation metabolic needs for body maintenance and cellular activ-ities. This particular link between growth and proteinof whole-body proteolysis in humans. A potential error

in leucine oxidation assessment is the CO2 production metabolism has already been described in several otherspecies [27]. Thus, it appears that a bicarbonate supple-measurement. CO2 production not originating from a

metabolic source, but coming as a consequence of the mentation may be of benefit to avoid substantial proteinloss in this pathological conditions, and the question ofbuffering effect of CO2, is possible. This overproduction

would dilute the labeled CO2 so that 13CO2 excretion when and how bicarbonate should be administered arisesfrom this work.would be underestimated. Thus, stimulation of leucine

oxidation during metabolic acidosis is likely and may This study also indicates that the postabsorptive stateshould be shortened as much as possible in children withcorrespond either to a direct activation of branched-

chain-ketoacid-dehydrogenase (BCKDH) or to a greater CRF. We have shown that the protein breakdown rateis higher than the protein synthesis rate, since all theavailability of proteolysis-derived leucine. It has been

reported that metabolic acidosis stimulates the BCKDH, children were studied during the postabsorptive state, aknown condition of protein mobilization. We have nothe complex branched-chain amino acid-catabolyzing en-

zyme located in the muscle and the liver [14]. data on protein metabolism response to feeding in chil-dren, and only a few studies have investigated the re-If an error on oxidation rate would result in an overes-

timation of protein synthesis, the calculation of protein sponse to feeding in animal models or in human adultswith CRF. Recent experiments in CRF human adultsbreakdown has solid foundations. Indeed, in the postab-

sorptive state, leucine turnover rate is the result of leu- during feeding have indicated a reduced postprandialhandling of exogenous nitrogen sources [25]. These post-cine released from protein breakdown so that leucine

isotopic dilution gives an accurate estimate of whole- prandial alterations associated with a postabsorptive cat-abolic state may further alter protein balance in CRFbody proteolysis. From our study, the increased protein

turnover in CRF acidotic children is the result of an children. A new therapeutic approach may then arisefrom the modulation of dietary protein absorption byincreased protein breakdown in association with a ten-

dency to a higher protein synthesis. This pattern is in using slow or fast dietary proteins [28]. Indeed, we haveshown that it is possible to limit the postprandial rise inagreement with that reported in adult CRF patients dur-

ing metabolic acidosis [12]. It is noticeable that a correla- plasma amino acid concentrations without suppressingthe anabolic action of dietary proteins [28]. This concepttion between plasma bicarbonate level and protein

breakdown (muscle release of amino acids) was already is promising, but long-term effects of such a diet haveto be assessed in a larger population of CRF patients [29].suggested in adults CRF patients [25]. The correction of

acidosis by sodium bicarbonate clearly improves protein In conclusion, children with CRF are particularly af-fected by metabolic acidosis, since whole protein break-turnover abnormalities, but the mechanisms and the im-

plication of other catabolic factors involved in these al- down is very sensitive to acid-base status in this condi-tion. Acidosis during CRF appears to be a major riskterations remain unclear. Glucocorticoids have been re-

ported to potentialize the effect of acidosis on protein factor for protein malnutrition, hence for growth delay.breakdown in adrenalectomized rats [13]. This actioncould be exerted at the level of proteasome subunits ACKNOWLEDGMENTS[17]. Acidosis could also limit the anabolic action of We thank all of the medical staff and the dieticians of the Nephrology

Department, Dr. J. Merckx for technical help, Dr. F. Rocchicciolli forhormonal factors such as insulin, GH, or IGF-1, as al-providing GCMS analysis facilities, and Pr. A. Mariotti for providingready suggested [10, 26].IRMS determinations facilities.

There was no correlation between protein breakdownrates and the parameters of malnutrition or the magni- Reprint requests to Dr. Yves Boirie, Laboratoire de Nutrition Hu-

maine, B.P. 321-58, rue Montalembert, 63009 Clermont-Ferrand Cedextude of growth retardation in our patients. This result1, France.

was not unexpected, as body composition and height at E-mail: yves.boirie@clermont.inra.frthe time of tracer infusions reflected the interplay ofmany factors (such as origin of CRF and duration of REFERENCESCRF) over months or years, which this cross-sectional,

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