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*
Department of Internal Medicine, Division of Nephrology, University of
Genoa, Genoa, Italy.
Nephrology Department, G. Gaslini Institute, Genoa, Italy.
Correspondence to Dr. Giacomo Garibotto, Department of Internal Medicine, Nephrology Division, University of Genoa, Viale Benedetto, XV,6,16132 Genoa, Italy. Phone: 39-010-3538989; Fax: 39-010-3538959; E-mail: gari{at}unige.it
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Insulin is also an important anabolic hormone (9,10). In fasting PD patients, the whole-body antiproteolytic response to insulin has been shown to be unaltered (11) and hyperinsulinemia could contribute to preservation of muscle mass in these patients. Despite substantial glucose absorption, loss of lean body mass, with preservation or increase in fat stores, may be even more prevalent in PD than in hemodialysis patients (8,12). On one hand, the amount of glucose absorbed through the peritoneum, with its attendant hyperinsulinemia, is thought to have favorable effects on energy balance. On the other hand, catabolism induced by PD may stem from the amino acids (AA) and protein losses via the peritoneal route. Despite their potential repercussions, the effects of PD on muscle protein turnover have been unexplored so far. It has been shown that PD patients who ingest low amounts of proteins experience decreased rates of whole body synthesis and degradation, as estimated by 14C-leucine kinetics (13). Recently, it was demonstrated that increasing AA availability by giving AA intraperitoneally is followed by a stimulation of whole-body protein synthesis (PS) and that this effect is enhanced by the inhibition of protein breakdown (PB) induced by a meal (14).
Both insulin and AA are widely considered as necessary for the stimulation of muscle protein anabolism. However, neither the effects of hyperinsulinemia nor the quantitative importance of AA availability on muscle protein metabolism has been established during PD. To provide such data, we measured the kinetics of 3H-phenylalanine as well as the exchange of AA across the forearm (which is mainly made of muscle) in the basal postabsorptive state and during PD with dialysates that contained dextrose (during which systemic hyperinsulinemia associated with decreased AA availability occur) or PD performed with a combination of dextrose and AA (during which systemic hyperinsulinemia and increased AA availability take place) in two crossover studies in continuous ambulatory PD (CAPD) patients.
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Patients had been free of peritonitis for at least 2 mo before the study. All patients received vitamin supplements that contained B vitamins and folic acid. None of the patients were receiving insulin, androgenic steroids, or lipid-lowering agents. Before the patients' participation, the nature, purpose, and risks of the study were reviewed with all of the patients and their voluntary consent was obtained. The protocols described here were approved by the Ethical Committee of the Department of Internal Medicine of the University of Genoa. Procedures were in accordance with the Helsinki declaration.
Experimental Protocols
Two different protocols were used. Both consisted in crossover studies with
patients serving as their own controls. Protocol 1 was designed to study the
acute effects of dialysates that contain dextrose alone on muscle protein
turnover. In this protocol, the same patients were studied twice (in the
postabsorptive state and during PD with dextrose). Protocol 2 was designed to
study the acute effects of dialysates that contain dextrose alone or
dextrose+AA on muscle protein turnover. In this protocol, the same patients
were studied twice (when they were receiving dextrose alone and during
dextrose + AA). Five patients in protocol 1 and five in protocol 2 were
treated with the same dextrose concentration (2.27% dextrose). Because both
studies were long and fasting could influence protein turnover, a group of
patients who received only saline (time controls) were studied as a control
group for a period of time similar to that used in protocols 1 and 2.
In both protocols as well as in time control studies, the study of muscle protein turnover was performed in the postabsorptive, overnight fasted state. At 7.00 a.m., a forearm vein was cannulated with a 18-gauge polyethylene catheter and used for a primed-continuous infusion of L-(ring 2,6)-3H-phenylalanine (approximately 32 µC, 0.35 µCi/min). The tracer infusion was maintained until the end of the study. Catheters were inserted into a brachial artery and in a retrograde manner into the ipsilateral, deep forearm vein. Blood samples were taken every 30 min after the start of PD to assess the achievement of steady-state conditions of plasma substrate concentrations and specific activities (data not shown). Both phenylalanine and other AA were fairly stable in blood (coefficient of variation < 5%) during the study periods. During dextrose+AA administration, AA levels increased acutely in blood after 1 h, then progressively declined to a plateau (plateau was defined by the absence of significant slope) assessed by linear regression analysis. For determination of steady-state forearm fluxes, arterial and deep venous samples were obtained at 10-min intervals over a 30-min period in the basal state and the study periods.
Study Protocol 1: Effects of PD with Dialysate that Contained Dextrose Alone. Eight male CRF patients (age 57 ± 5; range, 40 to 73 yr; creatinine clearance, 3 ± 2 ml/min) treated with CAPD for 19 mo (range, 7 to 48 mo) were studied. Their mean percentage of ideal body weight, based on medium-framed individuals (15), was 107 ± 2, body mass index = 26 ± 0.5. On the basis of their peritoneal membrane characteristics, two of them were classified as average-high and six as average-low transporters (16). Their dialysis dose, expressed as Kt/V, was 2 ± 0.1, and their protein intake, as expressed as nPCR (6), was 1.2 ± 0.1 g/kg per d. Blood urea nitrogen (BUN) was 71 ± 5 mg/dl, and albumin was 3.8 ± 0.2 g/dl.
Patients had the abdomen left dry and consumed their last meal at approximately 10 p.m. on the day before the study. After obtaining baseline samples (150 to 180 min from the start of tracer infusion), PD with a dextrose-containing dialysate was started. PD was performed with four 1-h, 2-L exchanges (dwell time, 30 min the first three, 60 min the fourth). Dialysis fluid contained 132 mmol/L sodium, 3.5 mmol/L calcium, 1.5 mmol/L magnesium, 102 mmol/L chloride, and 35 mmol/L lactate. To explore the effects of different amounts of dextrose administration on muscle protein turnover, solutions that contained different dextrose concentrations (13.6 g/L [three patients] or 22.7 g/L [five patients]) were used.
The forearm protein turnover study was repeated after approximately 200 min, after approximately 20 min from the beginning of the fourth exchange, i.e., at approximately 380 to 420 min from the beginning of the infusion. Patients were not allowed to eat during the study.
Study Protocol 2: Effects of PD with Dialysate that Contained Dextrose + AA Versus Dextrose Alone. Five CRF patients (one woman/4 men; age, 55 ± 8 yr; range, 27 to 73 yr; body mass index, 24 ± 2; creatinine clearance, 2 ± 1 ml/min; nPCR, 1.2 ± 0.1; Kt/V, 2 ± 0.1; BUN, 73 ± 7 mg/dl; albumin, 3.8 ± 0.15 g/dl; K+, 4.2 ± 0.3, HCO3-, 24 ± 1 mmol/L) treated with CAPD for 18 mo (range, 12 to 30 mo) were studied. On the basis of their peritoneal membrane characteristics, four patients were classified as average-low and one as average-high transporters. Patients had the abdomen left dry from and had consumed their last meal at approximately 10 p.m. of the previous night. On the day of the study, from approximately 6 to 10 a.m., patients received four 1-h, 2-L PD exchanges (dextrose 2.27%; dwell time, 30 min) by a cycler (Baxter HomeChoice, McGaw Park, IL). Subsequently, their dialysate was substituted for one that resulted from the combination of one 1.1% Baxter 2-L Nutrineal Æ bag (AA 1.1%) to one 5-L 2.27% and one 2-L 3.86% dextrose solution. The final dialysate dextrose and AA concentrations were 2.12 and 0.24%, respectively. The composition of the AA solution was as follows: 0.3 of tyrosine, 0.27 g of tryptophan, 0.57 g of phenylalanine, 0.64 g of threonine, 0.51 g of serine, 0.595 g of proline, 0.51 g of glycine, 0.95 g of alanine, 1.393 g of valine, 0.85 g of methionine, 0.85 g of isoleucine, 1.02 g of leucine, 0.955 g of lysine, 0.714 g of histidine, 1.071 g/L arginine; 0.184 g of calcium chloride (2H20), 0.0508 g of MgCl 2 (x6H20), 4.48 g of sodium lactate, and 5.38 g/L NaCl. Four 1-h cycler-assisted PD exchanges were performed from 10 a.m. to 2 p.m., i.e., from 240 to 480 min from the beginning of the study. The forearm protein turnover study was performed from 210 to 240 min from the start of dialysis and during the last 30 min of the dextrose+AA period, i.e., from 450 to 480 min from the beginning of the tracer infusion.
Patients were not allowed to eat during the study.
Control Subjects. Five CAPD patients (age, 51 ± 5 yr; range, 39 to 69 yr; 4 men, 1 woman; creatinine clearance, 4 ± 3 ml/min;, body weight, 102 ± 3% of their ideal body weight; nPCR, 1.2 ± 0.4; BUN, 76 ± 4 mg/dl; K+, 4.3 ± 0.2 mEq/L; albumin, 3.8 ± 0.4 g/dl; HCO3-, 25 ± 1 mmol/L) served as time controls. After a 180-min basal period, normal saline (0.23 ml/min) was infused for 300 min. Arterial and deep venous samples were obtained at the baseline, i.e., from 150 to 180 min from the start of the tracer infusion, and at 210 to 220, 220 to 240, and 450 to 480 min.
Blood Flow Measurement
Blood flow across the forearm was determined immediately after each
arteriovenous sampling by a indium-gallium strain gauge plethysmograph
(Angiomed Instruments, Padua, Italy). The arm that contained the deep vein was
raised approximately 20°, and a small supporting pillow was placed under
the wrist to avoid interference with venous return. The strain gauge was
wrapped around the forearm at the level of maximum circumference. The relative
change in the volume of the forearm segment under the strain gauge was
registered and was used to calculate plethysmographic blood flow. An occlusion
cuff was placed around the arm. The pressure was 60 mmHg. Each set of
measurements involved 10 separate measurements (coefficient of variation =
4%). For 1 min before and during withdrawal of each blood sample, blood flow
to the hand was interrupted by means of a sphygmomanometer cuff inflated
around the wrist to 200 mmHg. Flow was expressed per 100 ml of forearm volume.
Room temperature was 20 to 22°C.
Assays
AA were determined in whole blood and in the peritoneal effluent.
Perchloric acid (0.75 mol/L) was used for protein precipitation. An aliquot of
the supernatant was neutralized with a buffered solution, stored at -25°C,
and used for the assay of glutamine and glutamate. Another aliquot was stored
at -25°C and used for the determination of other AA. Blood samples were
processed immediately after withdrawal. AA were measured in triplicate by an
AA analyzer (Mod. 3A30, Fisons Instruments, Milan, Italy), with the use of
lithium buffers. Phenylalanine-specific activity was determined in blood, as
described previously
(17,18).
Plasma insulin was determined by RIA (Diagnostic Products, Los Angeles, CA). Arterial blood pH and P CO2 were estimated at 37°C with an ABL 505 apparatus (Radiometer Co., Copenhagen, Denmark). Blood bicarbonate concentrations were calculated by using the Henderson-Hasselbalch equation.
Hematocrit was measured by a microcapillary procedure. Albumin levels in serum and peritoneal fluid were determined by bromocresol purple dye (Sigma Diagnostics, St. Louis, MO). All other serum chemical measurements were determined by routine clinical chemistry laboratory procedures.
Calculations
The net forearm balance for AA was calculated according to the Fick
principle. Forearm phenylalanine kinetics were calculated using the A-V model
described previously
(17,18).
Briefly, the net forearm balance (NB) for substrates was calculated as
follows:
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Muscle phenylalanine rate of appearance (an expression of PB) was
calculated as follows (17):
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Muscle disposal of unlabeled phenylalanine (an expression of muscle PS) was
calculated as follows:
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The peritoneal glucose and AA absorption was calculated as the difference between the amounts initially present in the peritoneal fluid bags and the amounts finally recovered. The volume of fluid delivered to the peritoneal cavity was obtained by weighing the dialysis bag before and after it was emptied. The AA losses were calculated by multiplying the AA concentration in final fluid by the volume of fluid recovered.
The total decrease in extracellular AA concentration was estimated as the product of total body water (kg) x 0.33 (extracellular fluid volume) x decrease in plasma AA levels over basal levels. Total body water was calculated according to Watson et al. (20).
Extracellular volume was assumed to be one third of total body water. The percentage of the decrease in extracellular AA accounted for by dialysis removal was calculated as the ratio of removed amounts/total decrease in extracellular fluids. These calculations assume also that changes in plasma AA concentrations reflect changes in extracellular AA concentrations (21).
Statistical Analyses
All data are presented as the mean ± SEM. Statistical analysis was
performed using the two-tailed t test to compare arterial with venous
data. When the arteriovenous difference was different from 0 (P <
0.05) or when intragroup statistical significances were to be evaluated, a
repeated-measure ANOVA was used to compare the overall changes during the
phases of the study. When ANOVA indicated statistical significance (P
< 0.05), a post hoc F-based test was performed between phases. To
establish the differences in AA concentrations between patients and controls,
we used the unpaired t test and one-way ANOVA followed by post
hoc analysis. Linear regression and correlation were used to evaluate the
relationship between two variables. Stepwise multiple regression analysis was
used to discover which independent variables were significantly related to the
dependent variables when the contribution of other independent variables was
factored out and to discover what proportion of variations in dependent
variables was accounted for by each independent variable and by the model.
Statistical analysis was performed with the Statview Statistical Package
(Abacus, Berkeley, CA).
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AA Balance across the Forearm
Protocol 1: PD with Dextrose Alone. In the postabsorptive state, the
CRF patients studied exhibited lower arterial blood levels of serine, valine,
leucine, and tyrosine and higher levels of proline, citrulline, glutamate, and
cyst(e)ine as compared with those obtained in our laboratory in 35 normal
subjects (22). As shown in
Table 2, arterial
concentrations of several AA decreased during PD. A significant decrease was
observed for valine and leucine (-21 and 23%, respectively), tyrosine (-20%),
and cyst(e)ine and threonine (11 to 18%). For some other AA, such as taurine,
lysine, citrulline, and serine, a small, not statistically significant
decrease was observed in whole blood. Alanine was the only AA that tended to
increase (+10%). Despite the increase in insulin levels, forearm AA balance
remained on the average unchanged. A tendency toward an increased release of
valine, threonine, and alanine was observed but did not achieve statistical
significance. The release in plasma was similar in both quantitative and
qualitative terms to what was observed in whole blood; therefore, it is not
reported in the table. Changes in arterial plasma AA followed a similar trend
to that observed in whole blood. Only for taurine and serine, the decrease was
greater in plasma than in whole blood, suggesting that these AA are
predominantly transported by way of plasma across the peritoneal membrane.
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As a general trend, the amount of AA removed via PD was related to their arterial concentration. According to our estimate, removal with the peritoneal fluid accounted for 50 to 70% (valine, isoleucine, leucine, tyrosine, serine) or 70 to 100% (taurine, threonine, proline, lysine) of the decrease in extracellular AA levels (Table 2).
Protocol II: PD with Dextrose + AA. During PD with dextrose + AA, a rise in arterial blood was observed for several AA as compared with treatment with dextrose alone (Table 2). Most marked increments were found for valine and isoleucine (approximately 70%), leucine (approximately 60%), threonine and arginine (approximately 40%), histidine and lysine (approximately 20 to 26%), and serine and glycine (approximately 16 to 20%). Alanine levels did not change. The relationship between the percentage increase in blood AA (excluding alanine) and the molar ratio of AA contained in the dialysate gave a correlation coefficient close to unity (r = 0.90, P < 0.0005).
As compared with the basal period in which patients received PD with dextrose alone, forearm NB from AA became less negative (from -640 ± 223 to -277 ± 206 nmol/min per 100 ml; P < 0.05), owing to the occurrence of a net uptake of valine and a decreased release of serine, glycine, alanine, lysine, histidine, and arginine (Table 2). For several AA (valine, threonine, isoleucine, glycine, leucine, serine, tyrosine, histidine), variations in their exchange across the forearm were directly related to changes in their arterial concentration (P < 0.04 to 0.01).
Phenylalanine Kinetics across the Forearm
Protocol I: PD with Dextrose. In the basal, postabsorptive state, as
well as during PD with dextrose, protein degradation exceeded PS (P
< 0.05 to 0.003) and the forearm muscle was in a negative protein balance
(Figure 1A). However, during
PD, muscle protein degradation decreased on the average by 17% (P
< 0.01). Protein degradation was inversely related to insulin levels
(r = -0.578; P < 0.002;
Figure 2), suggesting the
occurrence of an insulin-induced inhibition of muscle PB. However, no net
muscle anabolism ensued in response to the increase in insulin levels. Muscle
phenylalanine net balance (NB) was on the average unchanged, owing to the
occurrence of an average 26% decrease in muscle PS (P < 0.04).
Furthermore, during the PD study, the NB of phenylalanine across the forearm
was only marginally related (r = -0.28; P < 0.064) to
insulin levels.
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Changes in muscle PS during PD were related directly to arterial BCAA (r = 0.32; P < 0.04) and potassium concentrations (r = 0.682; P < 0.001). Moreover, basal arterial potassium predicted the dialysis-induced averaged changes in PS (r = 0.797; P < 0.004). In a multiple linear regression model, BCAA and potassium levels accounted for 52% of the observed variability (multiple r2 = 0.522; P < 0.001), with a greater weight for potassium (partial r2 = 0.46; P < 0.002) than for BCAA (partial r2 = 0.059; P < 0.06). There was no significant relationship between the total amount of potassium removed or changes in arterial potassium levels and PS. When two patients with the lowest basal plasma potassium levels (3.4 and 3.7 mEq/L, respectively) were excluded, BCAA were the only predictors of changes in muscle PS.
In the basal period, as much as 70 ± 6% of phenylalanine that appeared from PB was cycled back into PS. During PD with dextrose, this value declined to 63 ± 5% (P < 0.05) in the last period of the study (200 to 215 min, 66 ± 5%; 215 to 230 min, 66 ± 5%; 230 to 240 min, 63 ± 5%), owing to a significant decrease in PB not counterbalanced by a decrease in phenylalanine NB. During PD with dextrose, whole-body phenylalanine flux declined by approximately 14% (basal, 0.573 ± 0.06; PD with dextrose, 0.478 ± 0.05, 0.480 ± 0.06, 0.486 ± 0.06 µmol/kg per min; P < 0.02).
Protocol II: PD with Dextrose + AA Versus PD with Dextrose Alone. At the end of the PD with dextrose study, net phenylalanine balance across the forearm was negative, as already observed during protocol 1 (Figure 1B). This occurred even in the presence of low absolute values for protein degradation, which, however, were greater than the low absolute rates of PS. In response to PD with dextrose + AA, no change in muscle protein degradation was observed. However, the overall response to the increased AA availability was a decrease in the negative muscle protein balance observed with respect to when dextrose alone was used. This occurred because of a 20% increase in muscle PS. We could not find a significant relationship between protein degradation and insulin when dextrose + AA have been studied. This may have occurred because all patients showed a sizable increase over baseline insulin levels, which suppressed protein degradation. However, when all of the experimental conditions (baseline, dextrose, and dextrose + AA) were considered, the correlation between protein degradation and insulin levels continued to be significant (r = -0.325; P < 0.02).
Whole-body phenylalanine flux increased by approximately 9% (PD with dextrose, 0.55 ± 0.12; PD with dextrose + AA, 0.60 ± 0.11, 0.62 ± 0.12, 0.60 ± 0.12 µmol/kg per min; P < 0.05 to 0.02) during PD with dextrose and AA as compared with the baseline period when patients received dextrose alone.
Time Controls
In time-control subjects in the baseline period, forearm was in a negative
balance because of a PB greater than PS. There was no change over time with
regard to net phenylalanine balance or rates of phenylalanine uptake or
release across the forearm (protein breakdown: baseline, 40 ± 5; 200 to
220 min, 38 ± 8; 220 to 240, 36 ± 7; 440 to 460 min, 41 ±
6 nmol/min per 100 ml; PS baseline, 27 ± 5; 210 to 220 min, 23 ±
6; 220 to 240, 25 ± 7; 450 to 480 min, 27 ± 6 nmol/min per 100
ml; NB baseline: - 13 ± 3; 200 to 220 min, - 13 ± 4; 220 to 240,
- 13 ± 3; 440 to 460 min, - 14 ± 3 nmol/min per 100 ml;
P = NS). No change over time in either arterial or forearm exchange
of AA was observed (data not shown).
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Insulin has been shown both in vitro (9) and in vivo (10) to be a powerful anabolic agent. Insulin suppresses BCAA metabolism (23) as well as protein degradation through specific pathways in muscle (24). Whereas uremia impairs insulin sensitivity with regard to glucose (25), insulin sensitivity regarding AA and whole-body protein metabolism seems to be unaltered in patients with ESRD (11,26). It is noteworthy that human muscle is extremely sensitive to insulin's antiproteolytic effect. For this effect, a dose-response curve has been demonstrated: infusions as low as to obtain insulin concentrations of approximately 30 µU/ml maximally suppress (-40%) protein degradation (27). The effects of insulin on phenylalanine muscle balance are half-maximal at insulin concentrations of only 14 µU/ml. Also in PD patients studied here, when dextrose alone was used in the peritoneal dialysate, rates of muscle protein degradation were inversely related to arterial insulin levels (Figure 2). Similar to the normal condition (25), the best fitting of the relation shows a curvilinear relationship with no decrease in protein degradation for increases in insulin levels above 30 to 35 µU/ml. Therefore, PD, with its attendant hyperinsulinemia, theoretically could be followed by an anticatabolic effect. However, in the present study, the negative phenylalanine balance across the forearm observed in the basal, postabsorptive state remained unchanged despite a doubling of the insulin levels. In addition, the tracer data disclosed a marked decrement in phenylalanine kinetics. It is noteworthy that in previous studies in which insulin levels had been raised systemically in the high, physiologic range (60 to 90 µU/ml), a decrease in the estimate of PS (28,29) prevented the occurrence of a net anabolic effect in muscle tissue if the concurrent hypoaminoacidemia had not been avoided. In this setting, the decrease in AA availability derived from an insulin-dependent increase in AA uptake by extramuscle (29) or a decrease in the release from muscle (30) tissues. In our study, the decrease in AA levels observed with the use of dextrose was not associated with a diminished muscle release. According to our estimate, a large portion of this decrement could be accounted for by the removal by the peritoneal fluid. However, it cannot be excluded that an inhibition of AA release occurred in extramuscle tissues, such as splanchnic organs.
Patients with CRF display several abnormalities in postabsorptive blood and muscle AA profile, with a decrease in essential AA. In our study, PD with dextrose caused a further decrease in the abnormal arterial concentration of several AA. Besides leucine and valine, we observed a decrease also in arterial serine (in plasma) and tyrosine. Circulating and intracellular pools of these AA are low in uremia owing to decreased production by the kidney (31,32), and it is possible that these AA are limiting muscle PS in conditions of increased demand. We also observed a significant decrease in plasma arterial taurine levels. PD patients have a decrease in muscle intracellular taurine, which correlates with plasma taurine levels (33). Taurine does not participate in PS; however, taurine depletion may cause a number of abnormalities at the cardiovascular and neurologic levels (34). Therefore, according to our findings, PD acts to restrain the already diminished circulating pools of these AA in uremia.
We have not been able to show any relationship between changes in blood glucose and protein turnover in our patients. In previous studies in which glucose was administered intravenously, an increase in insulin levels, with a decrease in protein degradation and unchanged PS, were observed (35). However, in a study in which endogenous insulin was controlled, neither parenteral glucose infusion rates nor plasma glucose concentrations affected whole-body proteolysis (36). Our data and those from the literature suggest that it is the insulin response rather than glucose per se that affects protein turnover.
Complementing the decrease in muscle PS and AA availability observed with dextrose alone, an increase in muscle PS with no significant variation in protein degradation occurred when arterial AA levels were raised by the use of dextrose combined with AA. This finding is in accordance with previous studies in which PS was shown to increase in the whole body (37) or in muscle (38) when AA levels were raised. The increase in AA levels during dextrose+AA was markedly dependent on their individual concentration in the dialysate. This is in keeping with previous observations obtained in children (38,39). Moreover, our data suggest that the amount of individual AA administered is a strong determinant of its arterial level and uptake by muscle. Alanine was an exception because this AA increased in blood only slightly even when it was present in sizable amounts in the dialysate. It is likely that this AA did not rise in blood because its release from the forearm decreased with the use of dextrose+AA. It is also of note that splanchnic organs are effective in removing alanine from the circulation (40). As also observed in our laboratory in previous studies in patients with CRF (40), variations in AA exchange across the forearm were related directly to changes in their arterial concentration. However, it is noteworthy that we have not been able to demonstrate a fully anabolic action by the use of dextrose+AA. Why was the stimulation of both mechanisms for muscle protein accretion, i.e., a submaximal inhibition of muscle protein degradation and a stimulation of PS, not followed by a net anabolic response? One possible explanation is that the amount or the proportions of AA administered to our patients were too low to cause a sizable and appropriate increase in arterial supply to peripheral tissue and stimulate PS. Several reports have underscored the major role of AA availability in conditioning their transport and PS in muscle, both during hyperinsulinemia and independent of insulin concentration (37,39,41). In fact, the amount of AA given intraperitoneally was only 22 g, i.e., the amount of AA provided by a small steak, and their arterial concentrations therefore were only slightly increased, in comparison to what is observed in CRF patients who received protein meals (40,42). If the total amount administered was low, then it is also possible that the formula of AA given was limited as to some AA. Available data also indicate that PS requires a full complement of essential AA (43). Studies in healthy subjects indicate that limiting the availability of one of more essential AA, including BCAA, adversely affects whole-body PS but not proteolysis (44). Moreover, it has been shown recently that BCAA, mainly leucine, influence PS by modulating the availability of protein translation factors, an effect that is independent from insulin (45). We calculated the percentage variations in AA exchange across the forearm during PD with dextrose+AA versus dextrose alone, i.e., the percentage of AA retained in muscle by the use of intraperitoneal AA, and put them in relation with the percentage of AA composition of muscle protein (Figure 3). Some AA (valine, serine, threonine, and isoleucine) were retained by muscle in large proportions relative to the muscle AA composition, whereas other AA (leucine, tyrosine, and lysine) were taken up by the forearm in more limited amounts. It is of note that the AA formula used in the present study contained relatively high amounts of valine and relatively low amounts of tyrosine and lysine. Our results show that even if incomplete, this solution may stimulate muscle PS and prevent the decrease in muscle PS induced by PD with dextrose alone. It would be of interest, however, to compare the effects with those of complete AA mixtures. It is possible that a more complete solution is more efficient in causing anabolism, therefore reducing other negative effects, such as azotemia and acidosis, and the use of AA like a caloric source, which have been observed with intraperitoneal AA (46).
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It is of note that this study was performed during an overnight fast, a condition in which peripheral tissues are in a negative nitrogen balance owing to a continuous release of AA by muscle. The negative AA balance across the forearm in patients studied therefore must be considered as a physiologic condition and is not inherent to the uremic condition or to the treatment with PD. Basal rates of muscle protein turnover in patients studied were in the lower range of values observed in the normal condition both in the literature (17,26) and in our laboratory. In addition to being reduced, basal rates of muscle protein turnover in patients studied proved to be efficient. According to the model of forearm phenylalanine kinetics studied, as much as 60 to 65% of phenylalanine deriving from proteolysis is cycled back into PS in the normal condition (17,25,47). A figure even higher than this is found in PD patients observed in the present study, in which as much as 70% of phenylalanine deriving from PB was cycled back into phenylalanine disposal. Therefore, PD patients studied in the postabsorptive state seem to be minimally catabolic. This observation is in keeping with their well-preserved nutritional conditions. Our data indicate that in fasted patients, PD with dextrose induces acutely a change in the efficiency by which AA are cycled back into PS, as expressed by the net muscle protein balance unchanged in front of a decrease in protein turnover. One could speculate that a decrease in protein turnover and of its efficiency, as demonstrated by the present study during PD with dextrose, may result in a limited potential for net skeletal muscle protein accretion in uremia or predisposes to reduced anabolism when nutrient intake is diminished or during superimposed catabolic illnesses. However, the negative effects of dextrose reported here may not be manifested in regular PD patients who eat at least three times a day. Moreover, it is possible that the PD regimen that we used, which is similar to regimens used in patients who are treated with automated devices but is uncommon for the majority of PD patients, emphasized the metabolic effect of PD.
An estimate of the effects on muscle mass during the use of dextrose+AA in comparison with dextrose alone may be offered by the changes in forearm phenylalanine NB induced by the use of dextrose+AA. Considering that the mean fraction of forearm made up of muscle tissue is approximately 0.6, that forearm blood flow in forearm muscle is approximately 60 to 70% of total flow, that muscle is on the average 40% of body weight, and that phenylalanine content in proteins is approximately 280 µmol/g protein (18,48), the change in net phenylalanine release from the forearm during the use of dextrose+AA versus dextrose alone would account for a less negative muscle balance of approximately 8 to 9 g/d.
There is evidence that muscle protein accretion is achieved through the combined effect of an energy-induced, insulin-mediated inhibition of PB and an AA-dependent stimulation of PS. Given the action in muscle induced by PD described in the present study, we hypothesize that in fasting PD patients, muscle mass could be maintained better by preventing, through continued AA supplementation, the dialysis-induced decrease in AA availability. Findings observed in the present study may also be relevant as regard to both the understanding of the pathophysiology of chronic renal failure and its treatment and the design of nutritional regimens that include the use of insulin and AA.
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Acknowledgments |
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Portions of the data reported in this paper appeared in an abstract presented at the 31st Annual Meeting of the American Society of Nephrology, Miami Beach, November 5-8, 1999.
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  H. L. Tjiong, R. Swart, J. W. Van den Berg, and M. W. Fieren Dialysate as food as an option for automated peritoneal dialysis NDT Plus, October 1, 2008; 1(suppl_4): iv36 - iv40. [Abstract] [Full Text] [PDF]
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 H. L. Tjiong, T. Rietveld, J. L. Wattimena, J. W. van den Berg, D. Kahriman, J. van der Steen, W. C. Hop, R. Swart, and M. W. Fieren Peritoneal Dialysis with Solutions Containing Amino Acids Plus Glucose Promotes Protein Synthesis during Oral Feeding Clin. J. Am. Soc. Nephrol., January 1, 2007; 2(1): 74 - 80. [Abstract] [Full Text] [PDF]
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 H. L. Tjiong, J. W. van den Berg, J. L. Wattimena, T. Rietveld, L. J. van Dijk, A. M. van der Wiel, A. M. van Egmond, M. W. Fieren, and R. Swart Dialysate as Food: Combined Amino Acid and Glucose Dialysate Improves Protein Anabolism in Renal Failure Patients on Automated Peritoneal Dialysis J. Am. Soc. Nephrol., May 1, 2005; 16(5): 1486 - 1493. [Abstract] [Full Text] [PDF]
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 D. S. C. Raj, P. Zager, V. O. Shah, E. A. Dominic, O. Adeniyi, P. Blandon, R. Wolfe, and A. Ferrando Protein turnover and amino acid transport kinetics in end-stage renal disease Am J Physiol Endocrinol Metab, January 1, 2004; 286(1): E136 - E143. [Abstract] [Full Text]
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, the composition of
actin and myosin in skeletal muscle
(
, the percentage
of AA retained in muscle during dextrose +AA versus dextrose alone.
Some AA (valine, serine, threonine, and isoleucine) were retained by muscle in
large proportions relative to the muscle AA composition, whereas some AA
(leucine, tyrosine, and lysine) were taken up by the forearm in more limited
amounts.