Comparison of amino acid oxidation and urea metabolism in haemodialysis patients during fasting and meal intake
Jorden M. Veeneman1,3,
Hermi A. Kingma2,
Frans Stellaard2,3,
Paul E. de Jong1,3,
Dirk-Jan Reijngoud2,3 and
Roel M. Huisman1,3
1Department of Internal Medicine, Division of Nephrology, 2Department of Pediatrics, University Hospital Groningen and 3Groningen University Institute of Drug Exploration, GUIDE, Groningen, The Netherlands
Correspondence and offprint requests to: Roel M. Huisman, University Hospital Groningen, Internal Medicine, Section Nephrology, PO Box 30.001, 9700 RB Groningen, The Netherlands. Email: R.M.Huisman{at}int.azg.nl
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Abstract
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Background. The PNA (protein equivalent of nitrogen appearance) is used to calculate protein intake from urea kinetics. One of the essential assumptions in the calculation of PNA is that urea accumulation in haemodialysis (HD) patients is equivalent to amino acid oxidation. However, urea is hydrolysed in the intestine and the resulting ammonia could be used metabolically. The magnitude and dependence on protein intake of this process are unknown in HD patients.
Methods. Seven HD patients were studied twice, 1 week apart, on a similar protocol. After an overnight fast, patients fasted in the morning and received meals in the afternoon. On one day, amino acid oxidation was measured by infusion of L-[1-13C]valine. Urea production, measured from the dilution of [13C]urea, and urea accumulation, calculated from the increase in plasma urea concentration multiplied by the urea dilution volume, were measured during the other day. PNA was calculated using standard equations.
Results. Amino acid oxidation and urea production were not significantly different during fasting. Urea accumulation during fasting was significantly lower than both amino acid oxidation and urea production. Urea accumulation during feeding remained significantly lower than amino acid oxidation. PNA was equal to the average of the urea accumulation values during fasting and feeding.
Conclusion. We conclude that during fasting, urea accumulation is not associated with amino acid oxidation or urea production. During meal intake, amino acid oxidation, urea production and urea accumulation show acutely an almost identical increase. PNA represents the average of fasting and fed urea accumulation and is lower than average amino acid oxidation or urea production.
Keywords: PNA; stable isotope; urea dilution volume; urea kinetics
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Introduction
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It is well known that the clinically used protein equivalent of nitrogen appearance (PNA) represents only the amount of protein that is irreversibly oxidized. It is measured as the difference in urea concentration in blood of haemodialysis (HD) patients between two dialysis sessions. The older term protein catabolic rate has been discarded, since a substantial portion of metabolized protein is simply broken down into amino acids, which are used subsequently to synthesize new proteins. The question is whether nitrogen appearance, i.e. the urea appearing in the urine or in the rising urea concentration, of HD patients between their HD sessions is only influenced by amino acid oxidation. Urea production is the main route by which the body discards excess nitrogen. Not all urea is excreted as such: some nitrogen of urea can be released again as ammonia through the metabolic activity of the colonic microflora [1,2]. Estimates of this process range from 14 to 60% of the urea production, depending on experimental conditions [35]. This newly formed ammonia could then be reutilized in amino acid synthesis and incorporated into newly formed proteins either by intestinal bacteria [6] or by the liver [7], as has been observed in healthy humans. These observations indicate that the supposedly linear sequence of events of amino acid oxidation, urea production, amino acid hydrolysis and urea accumulation seems to be branched in the way depicted in Figure 1. Intestinal metabolism of urea leads to intestinal ammonia formation which re-enters whole body ammonia metabolism at various stages and to various extents, depending on the nutritional status of the studied subjects. It is known that this process takes place under diverse conditions both in healthy subjects and in chronic renal failure patients [8,9], but the extent is not well defined. In renal failure patients, studies measuring nitrogen balance during fasting did not include data on the anabolic response to a meal. In normal men, two groups looked at [13C]leucine oxidation and urea metabolism both during fasting and feeding [10,11]. Urea metabolism was measured using nitrogen balance studies in one study [11] while in the other, [15N15N]urea was used to estimate nitrogen flux [10]. The authors concluded that urea hydrolysis and ammonia recycling occurred especially during fasting, but that there was no salvage of nitrogen from the colonic microflora in normal man during a feeding period. Thus, depending on the source of protein, the relative contribution of the different pathways resulting in urea accumulation, as depicted in Figure 1, can vary considerably. The question arises of how urea accumulation, urea production and amino acid oxidation are related during fasting when body proteins are being oxidized and during protein intake when nutritional proteins are oxidized. We conducted the present studies in order to (i) determine the extent of amino acid oxidation, urea production and urea accumulation during a fasting and a feeding period separately; and (ii) evaluate the relationship between PNA, urea kinetics [12] and amino acid oxidation. Amino acid oxidation was estimated from measurements of 13CO2 appearance in expired air using 13C-labelled valine. Urea production and the urea dilution volume (UDV) were measured using [13C]urea dilution [13]. Urea accumulation was calculated from the measured increase of urea concentration in plasma during fasting and feeding separately, multiplied by the UDV measured from the [13C]urea dilution method.

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Fig. 1. Whole body nitrogen metabolism is represented schematically, showing the interactions of amino acid oxidation, urea production and urea accumulation in HD patients. Solid lines represent measured values from the results; dotted lines represent possibilities mentioned in the Discussion.
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Subjects and methods
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Study subjects
Non-diabetic stable HD patients, aged <65 years, in the Dialysis Center Groningen were asked to participate in this protocol. Seven patients, two females and five males, gave their consent. The medical ethics committee of the University Hospital of Groningen approved all studies, and written informed consent was obtained from all participants. Two patients had been diagnosed as having chronic glomerulonephritis (one with hypertension), three had nephropathy due to hypertension, one had polycystic kidney disease and one quiescent Wegener's disease. All patients had been clinically stable for >3 months before the study protocol. Medications included phosphate binders, iron, multivitamins, anti-hypertensive drugs, calcitriol and, in six patients, recombinant human erythropoietin. No patients received hormone or immunosuppressive agents for 6 months before the study. Patients were dialysed on low-flux biocompatible dialysers for 3.54 h three times weekly. Blood flow ranged from 250 to 350 ml/min and dialysate flow was 500 ml/min. All patients used standard dialysate with 140 mEq Na+ and 34 mEq bicarbonate. Residual renal function was 6 ml/min in one patient and <3 ml/min in the other six patients, and urinary flow was 500 ml per 24 h for one patient, <400 ml per 24 h for two patients, and none in four patients. Three weeks prior to the study, all subjects visited the Dialysis Center Groningen for a dietary interview and instructions on dietary recording. Patients consumed their regular protein intake, which was 1.0±0.1 g/kg/day. During the study, blood pressure and hydration status were monitored, as well as body weight and serum albumin. These values were stable.
Materials
L-[1-13C]valine, NaH[13C]O3 and [13C]urea (>99% atom percent enrichment, respectively) were purchased from Cambridge Isotope Laboratories (Andover, MA). Chemical purities were confirmed before use. Pyrogen- and bacteria-free solutions were prepared in sterile saline by the hospital dispensary. Meal portions consisted of 150 g of yogurt (5.7 g of protein, 7.4 g of carbohydrate and 5.4 g of fat; Domo, The Netherlands), 20 g of cream (0.5 g of protein, 0.7 g of carbohydrate and 6.3 g of fat; Friesche vlag, Ede, The Netherlands) and 5 g of protein-enriched milk powder (1.5 g of protein, 2.4 g of carbohydrate and 0.8 g of fat; Fortify, Nutricia, The Netherlands). Consumption of a meal portion every 30 min for 3 h resulted in a dietary valine intake of 9.8 mmol/h and a fluid intake of 350 ml/h. Meals were designed to give at least 0.6 g/kg of protein and 15 kcal/kg. It was assumed that gastric emptying during the meal was not influenced by the dialysis procedure since our patients had no history of dyspeptic symptoms during the 3 months before both protocols.
Experimental design
In the amino acid oxidation protocol, patients were fasted overnight and were studied during a midweek day without dialysis, having been dialysed the afternoon before. This protocol has been described in earlier studies by our group [14,15] in which we discuss the fate of non-oxidized amino acids and the influence of dialysis on protein metabolism. In short, patients were admitted to the Hospital Research Unit at
7:30 a.m. A catheter was inserted into the dorsal vein of the hand of the shunt arm to collect baseline blood samples. Breath samples were taken simultaneously into 10 ml glass containers. A schematic diagram of the metabolic study day is shown in Figure 2A. The NaH13CO3 infusion was started at 8:00 a.m. During the first hour, whole body bicarbonate flux was measured using a primed constant infusion of 13C-labelled bicarbonate (5 µmol/kg bolus followed by a continuous infusion of 5 µmol/kg/h). Four breath samples were taken between 30 and 60 min at 10 min intervals after the start of the NaH13CO3 infusion. The NaH13CO3 infusion was discontinued immediately after the last breath sample was taken and the L-[1-13C]valine infusion was started with a bolus of 15 µmol/kg followed by a continuous infusion of 7.5 µmol/kg/h for 4 h. A second catheter was then inserted in the contralateral arm to collect blood samples. Blood and breath samples were taken simultaneously every 30 min for 2 h after the start of the [13C]valine infusion. During the fourth hour, blood and breath samples were taken every 15 min. At 1:00 p.m., the meal was started by consumption of the first portion of the protein- and energy-enriched meal and continued for 3 h by consumption of a portion every 30 min. The infusion of [13C]valine was continued during this time. Blood and breath samples were taken every 30 min for 2 h after the start of the meal, while during the last hour samples were taken every 15 min. The study day ended at 4:00 p.m.; all catheters were removed and patients were observed until stable and then discharged after
30 min. After 1 week, the same seven subjects were studied according to a 7 h tracer protocol, partly similar to the first day (Figure 2B). Patients received a single bolus injection of 10 µmol [13C]urea/kg in
1 min at 08:00 a.m. Blood samples were taken during the whole study day for the measurement of urea concentrations and urea enrichment at the same time points described above. Meal intake started at 12:00 p.m. and continued for 3 h. The study day ended at 3:00 p.m.; all catheters were removed and patients were observed until stable and then discharged after
30 min. The two study periods were randomized.

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Fig. 2. Schematic representations of both study days. (A) The protocol used to study whole body protein metabolism in chronic HD patients during a non-dialysis day. After an overnight fast, whole body amino acid oxidation was measured in the morning while fasting. This was followed in the afternoon by the measurement of whole body amino acid oxidation in patients consuming a protein- and energy-enriched meal in six portions. (B) The protocol used to study whole body urea production and urea accumulation in chronic HD patients. A bolus of [13C]urea was given at 8 a.m. and steady state was reached within 120 min. After the overnight fast, urea production and urea accumulation were measured in the morning during fasting. Fed urea production and accumulation were measured in the afternoon during intake of six small meals.
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Analytical methods
A 4 ml aliquot of blood was drawn for each sample in liquid heparinized vacuum tubes and centrifuged at 3000 r.p.m. Plasma was extracted and stored at 20°C until analysis. Breath samples were collected in gas collection tubes using a straw, as described previously [15]. Subjects exhaled normally through a straw into the glass container. After exhalation was completed, tubes were closed immediately and stored at room temperature until analysis. Urine samples, when produced, were collected during the study day and their volume and urea concentrations (micro Kjeldahl analysis) were measured to calculate total excreted urea. These values were then added to the urea accumulation numbers. Albumin concentrations were determined by standard clinical chemistry methods. Determination of valine oxidation requires 13C enrichment measurements for breath CO2, plasma valine and plasma ketoisovaleric acid (KIVA), an intracellular metabolite of valine. Amino acids were isolated from plasma using a cation exchange column (SCX-100, 209800, Alltech, Deerfield, IL) and converted to the N(O)-methoxycarbonyl methyl ester (MCF) derivative. The determination of [1-13C]KIVA isotopic enrichment was done as described previously [15].
Calculations
The rate of appearance of intracellular valine R(a) was calculated at isotopic steady state using the reciprocal pool model as described by Matthews et al. [16] for leucine kinetics. In our isotopic model, enrichment of plasma KIVA is assumed to provide an estimate of intracellular enrichment of valine as described previously [15]. R(a) in µmol valine/kg/h was calculated according to the following equation:
 | (1) |
where MPEi(V) is the isotopic enrichment of the valine in the infusate in mol percent excess, MPE(KIVA) is the isotopic enrichment of KIVA in plasma in mol percent excess, and i(V) is the infusion rate of [1-13C]valine in µmol/kg/h. The rate of oxidation of valine was calculated applying the approach described previously [15]. Whole body bicarbonate flux is estimated using a short-term primed continuous infusion of NaH13CO3 prior to infusion of [13C]valine. In this way, it is possible to obtain a two-point calibration in which one point is the background 13CO2 enrichment at no infusion of NaH13CO3 and the second point is the measured value of enriched CO2 at the applied continuous infusion rate of NaH13CO3. The [13C]bicarbonate flux originating from the oxidation of [13C]valine was calculated by linear interpolation of the measured 13CO2 enrichment in expired air at steady state during [13C]valine infusion between the two points of the calibration:
 | (2) |
in which ibic(V) is the [13C]bicarbonate production during valine infusion, Zco2(B) is the tracer-to-tracee ratio of expired air at isotopic steady state during the NaH13CO3 infusion, Zco2(V) is the tracer to-tracee ratio of expired air at isotopic steady state during the [13C]valine infusion, and i(b) is the NaH13CO3 infusion rate in µmol/kg/h. Using the KIVA tracer-to-tracee ratio in plasma, we calculated the amount of valine that needed to be oxidized to sustain this production of [13C]bicarbonate as follows:
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where 5 is the number of carbon atoms in valine. This means that one can calculate the oxidation rate of an amino acid without measuring Vco2.
During fasting:
 | (4) |
During feeding, an increase in recovery of labelled CO2 has been observed in comparison with fasting from 0.74±7 to 0.84±8, thus by
13%. This value has only been validated in control subjects. We adopted it for both the control subjects and the HD patients. Correction of the rate of oxidation of valine during feeding for this increased recovery is necessary since we obtained the two-point calibration by measuring 13CO2 enrichment while the subject was fasting. O(fed) was calculated according to:
 | (5) |
Amino acid oxidation values were converted to urea production values. For this purpose, we assumed that valine content of protein is 5.5 g per 100 g of muscle protein. This 5.5 g corresponds to 47 mmol valine. We also assumed that 100 g of protein contains 16 g of nitrogen, which corresponds to 1.14 mol N. Oxidation of an amount of protein containing 1 mol of valine produces 1140/47 = 24 mol of nitrogen, which corresponds to 12 mol of urea assuming that initially all nitrogen is converted to urea. The oxidation of 1 mol of valine produces 1 mol of NH3, and rates of amino acid oxidation, calculated as µmol valine/kg/h, were converted into µmol urea equivalents/kg/h by multiplying by a factor of 12.
Urea production was measured by isotope dilution of [13C]urea as described previously [13]. Briefly, urea enrichment of the plasma samples of each subject was calculated using the slope and intercept of the calibration lines prepared in plasma samples obtained before the bolus injection of [13C]urea. The fasting state was defined as the third hour after infusion, while the fed state was defined as the second and third hour after the start of ingestion of the meal. For the calculations, UDV, plasma urea concentration and the synthetic rate of urea during fasting and during the last 2 h of feeding were measured and a steady state during these periods was assumed. The calculations of UDV and urea production were performed as described previously [13]. Nitrogen losses via the sweat were assumed to be negligible [17], as were faecal losses [18].
Amino acid concentrations in plasma were measured by the AccQ Tag method using HPLC according to the manufacturer's protocols (Waters, Breda, The Netherlands) during the last hour of the fasting and the last hour of the feeding period.
Urea accumulation, measured during the non-dialysis study days, was calculated by the increase of the plasma urea concentration per hour, multiplied by the UDV calculated by standard equations [12,13] and expressed per kg body weight. Urea hydrolysis was defined as urea production minus urea accumulation.
PNA was determined from the rise in plasma urea concentration during the interdialytic interval and the measured urea distribution volume. Calculations were performed as described previously [12]. PNA was normalized to the actual post-dialysis dry weight of the patient.
Statistics
All values are given as means±SD. To compare the influence of the protein meal, the fasted and fed states were compared using paired Student's t-test. Differences between methods were compared using the same test. Statistical significance was assumed when p<0.05. Correlations between the methods were tested using the Pearson's correlation method in SPSS version 10 (SPSS Inc. Chicago, IL). The average slope was calculated from the individual slopes and was tested as a one-sample t-test vs the line of identity or a slope of 1. The average intercept was calculated from the individual intercepts and was tested as a one-sample t-test vs 0.
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Results
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Table 1 shows demographic and clinical data of the patients studied. Body weight remained stable during the 3 weeks before the experiments. Patients were clinically stable, and albumin concentrations and body weight did not deviate from the values that are presented in Table 1 during the 3 weeks before the study. Also, C-reactive protein concentration was <2 mg/l in all patients during the 3 months before the study. During the measurement of urea and valine kinetics, a stable metabolic state was achieved. UDV was 50±8% of body weight, which was within the normal range. Valine concentrations during the urea infusion day were lower than during the valine infusion day during both fasting and meal intake, which can be explained by the [13C]valine infusion. Protein intake was 15 g protein/h which accounted for an intake of 0.2 g protein/kg/h. Conversion of this value to urea equivalents resulted in an intake of 1100 µmol urea N/kg/h. Amino acid concentrations in plasma increased, except for histidine, citrulline (significant decrease), glutamine, glutamic acid, glycine and taurine (Table 2). This increase is equivalent to 54 mmol amino acid N per 3 h, which is equal to 125 µmol urea N/kg/h.
In Figure 3a, steady-state enrichments in KIVA are shown during fasting (fifth hour) and meal intake (eighth hour). In Figure 3b, the percent change in urea concentration with time is shown compared with the average of the first 30 min. The error bar at 30 min represents the interindividual differences, while the rest of the data points represent the intra-individual differences compared with the time point at 30 min. During meal intake, these differences became somewhat larger due to the large inter-individual differences in urea accumulation. A linear increase over time was observed both during fasting (r2 = 0.86) and during feeding (r2 = 0.81). The coefficient of variance (CV) of the urea concentration measurement was 2.3%. In Figure 3C, the percentage change in urea enrichment is shown compared with the enrichment in plasma after 2 h. The error bar at 2 h represents the inter-individual difference, which was due to the differences in urea pool size. The rest of the data points represent the intra-individual differences compared with the time point at 2 h. A linear decrease over time was observed both during fasting (r2 = 0.97) and during feeding (r2 = 0.98). These data show the steady state in time (Figure 3A), while the linear increase or decrease in time due to urea production/accumulation shows that isotopic dilution was complete during the two study periods.

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Fig. 3. (A) Plasma -[1-13C]ketoisovaleric acid (KIVA) enrichment. Steady-state enrichments in KIVA are shown during fasting (fifth hour) and meal intake (eighth hour). (B) The percentile change in urea concentration over time is shown compared with the average of the first 30 min. The error bar at 30 min represents the inter-individual differences, while the rest of the data points represent the intra-individual differences compared with the time point at 30 min. (C) The percentile change in urea enrichment is shown compared with the enrichment in plasma after 2 h. The error bar at 2 h represents the inter-individual difference, which was due to the differences in urea pool size. The rest of the data points represent the intra-individual differences compared with the time point at 2 h.
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Only one patient produced a significant volume of urine. Patient 4 had a urine production of 350 ml during the whole study day during both the fasting and the meal period, with a urea concentration of 30 mmol/l. This reflects a urea excretion rate of 1.75 mmol/h, equivalent to 23 µmol urea N/kg/h. The other six patients did not produce urine during the study day.
The individual results of PNA, protein oxidation, urea production and urea accumulation in the seven patients are presented in Table 3 and the mean results in Figure 4A and B. During fasting (Figure 4A), urea production and amino acid oxidation yielded similar results. Urea accumulation (plus excretion) was significantly lower than the other two values. During meal intake (Figure 4B), urea production and amino acid oxidation were not significantly different, but urea accumulation remained significantly lower than amino acid oxidation. The increase in all three parameters due to the meal is shown in Figure 4C. PNA, calculated over the whole interdialytic interval, was 218±25 µmol urea N/kg/h (Table 3), equivalent to 1.1±0.1 g protein/kg/day. This was not significantly different from protein intake estimated from the dietary diary (Table 1).

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Fig. 4. (A) Amino acid oxidation, urea production and urea accumulation during fasting. Amino acid oxidation and urea production values were significantly higher than urea accumulation values. (B) Amino acid oxidation, urea production and urea accumulation during feeding. Urea accumulation was significantly lower than amino acid oxidation. (C) Increase in amino acid oxidation, urea production and urea accumulation due to meal intake. *P<0.05; NS, not significant; O1 and O7, individual outlying patient.
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Figure 5 shows that there is a systematic difference between urea accumulation and the other two parameters. All parameters were significantly correlated (oxidation and urea accumulation, r = 0.802, P<0.01, Figure 5A; production and accumulation, r = 0.771, P<0.01, Figure 5B; production and oxidation, (r = 0.538, P<0.05, Figure 5C). The intercept with the y-axis in Figure 5A and B is significantly higher than 0 (represented in the formula in each graph), and for Figure 5C was not different from 0. The slope (also represented in the formula in each graph) of all three correlations was not significantly different from 1.

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Fig. 5. Correlation graph for protein oxidation with urea accumulation (A), urea production and urea accumulation (B), and protein oxidation and urea production (C). Individual patients are characterized by two matching symbols, one during fasting and the other during feeding. In each graph, the line of identity is shown and the correlation line is shown as a dotted line for all points in each graph. Formulas in each graph represent the slope and intercept of the correlation.
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Discussion
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The main findings of this study were that during fasting, urea production and amino acid oxidation were higher than urea accumulation values, implying urea hydrolysis in HD patients up to almost 50% [10]. This indicates that especially during fasting, urea hydrolysis is of quantitative importance [3,19]. All parameters increased significantly after meal intake, with greater dispersion of values. During meal intake, urea accumulation was significantly different from amino acid oxidation but not from urea production. Long-term PNA calculations were comparable with time-averaged urea accumulation values for fasting and feeding.
We combined valine oxidation values, estimates of urea production measured with [13C]urea, and compared these with experimentally derived values of urea accumulation during a fasting and a feeding period. The oxidation rate of valine was used to calculate whole body amino acid oxidation, which we subsequently converted into values of urea metabolism. This procedure has been validated in healthy control subjects [5]. Dilution of 13C-labelled urea is a sensitive method to measure total urea production. It has been used previously in our laboratory to calculate urea distribution volume and urea kinetics [13]. By combining these values for valine oxidation and urea metabolism, we thought we might be able to specify the relative contributions of the various processes to whole body nitrogen flows, as shown in Figure 1, in more detail. During fasting, when nitrogen intake was zero, amino acid oxidation was found to be equivalent to 200 µmol urea N/kg/h. This is comparable with the value of 159 µmol urea N/kg/h (61 mg N/kg/12 h) during fasting reported in the study by el Khoury et al. [10]. Urea production in our study was 190 µmol urea N/kg/h, which is not significantly different from the amino acid oxidation. Earlier studies showed a similar agreement between amino acid oxidation and urea production in control subjects [3,20]. However, we observed that the rate of urea accumulation was only
100 µmol urea N/kg/h. This raises the question as to where this excess urea production, or non-accumulated urea, of
100 µmol urea N/kg/h has gone. As has been shown in control subjects and pre-dialysis patients, up to 50% of produced urea can be hydrolysed in the intestines [3]. Varcoe et al. [21] showed a significant recycling of urea, which was confirmed by Mitch [8] in pre-dialysis patients. Theoretically, if all ammonia arising during intestinal urea hydrolysis would result in intrahepatic urea production, this would imply a total urea production of 300 µmol urea N/kg/h, comprising urea production due to amino acid oxidation and urea hydrolysis. Since we observed a value of
200 µmol urea N/kg/h for the rate of urea production, alternative routes need to be assumed for deposition of intestinally arising ammonia (or detoxification). Ammonia, formed by intestinal hydrolysis of urea, could be incorporated into the amino acid pool, as was shown by others in control subjects [6]. We suggest that intestinal ammonia is used in the production of (non-)essential amino acids, either by the colonic microflora, or by hepatic metabolism in chronic HD patients. On the other hand, one should realize that it might also indicate that amino-N, arising during amino acid oxidation, is not directed exclusively into urea formation [2,7]. With the current data, these routes cannot be studied separately.
During protein intake, amino acid concentrations increased in our patients and so did amino acid oxidation, urea production and urea accumulation. The increase in amino acid concentration represented an N retention equivalent to 125 µmol urea N/kg/h, which was equal to 11% of the total protein intake (1100 µmol urea N/kg/h). Amino acid oxidation during meal intake increased to 441 µmol/kg/h. Thus half of the ingested protein is not used immediately for protein synthesis and 40% of this half is oxidized immediately. In the urea pool, most of these oxidized amino acids are found in urea production values, i.e. 360 µmol/kg/h. Of these, 300 µmol/kg/h is retained in the urea pool in body water in the HD patient. Thus, after meal intake, amino acid oxidation, urea production and urea accumulation all increase
200 µmol/kg/h (see Figure 3C) and the differences between these values are no longer significant during meal intake. This is partly explained by the larger variability in all three parameters. Also, we suggest that intestinal bacteria could decrease their utilization of urea as a fuel and start using food-derived components. This decreases urea hydrolysis, thus reducing the difference between urea accumulation and the other two parameters. The difference between amino acid oxidation and urea production on the one hand and urea accumulation on the other is confirmed by the offset in the correlation between amino acid oxidation/urea production and urea accumulation of
150 µmol/kg/h, which represents the amount of urea that can be hydrolysed by the intestinal microflora in stable HD patients and reused for other purposes.
In summary, amino acid oxidation and urea production were both
200 µmol/kg/h, while urea accumulation was 100 µmol/kg/h (Figure 1). During meal intake, these values all increased
200 µmol/kg/h so that values of 400 µmol/kg/h were reached for amino acid oxidation and urea production, and urea accumulation became 300 µmol/kg/h. This implies that meal intake does not have a major influence on the recycling of amino-N and the intestinal metabolism of urea as depicted in Figure 1. PNA was
200 µmol/kg/h (1 g/kg/h), which was comparable with the protein intake of 0.93 g/kg/day. A net degradation of body protein stores would have resulted in a PNA higher than protein intake (Figure 1). Without bowel uptake and reuse of urea-derived ammonia, PNA and amino acid oxidation would be the same under any circumstances, even during net degradation of body proteins.
We conclude that in fasting HD patients with little urinary urea excretion, there is agreement between amino acid oxidation and total urea production, but these values do not agree with urea accumulation. Amino acid oxidation, urea production and urea accumulation show an acute response to meal intake, and the differences between the three parameters diminish with meal intake. PNA represents the time-averaged urea accumulation for fasting and meal intake, but is lower compared with the average amino acid oxidation or urea production. These findings appear to have no practical consequences at the moment, but they show that in the body of HD patients, breakdown of amino acids to urea, especially during fasting, is taking place at a higher rate than measured by urea accumulation. The excess urea (non-accumulated urea) is probably salvaged by intestinal bacteria. In future studies, attempts should be made to measure these directly in HD patients.
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Acknowledgments
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The authors would like to thank Henk Elzinga for analysis of the breath samples. The authors also appreciate the time given by the patients and staff of the Dialysis Center Groningen and the out-patient renal function ward. Part of this work was presented at the 34th annual meeting of the American Society of Nephrology, 2001. This work was supported by a grant from the Dutch Kidney Foundation (no. C 97-1694).
Conflict of interest statement. None declared.
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Received for publication: 8. 7.03
Accepted in revised form: 23. 2.04