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Clinical Science and Molecular Medicine (1975) 48, 379-390. Efficiency of utilization of urea nitrogen for albumin synthesis by chronically uraemic and normal man R. VARCOE, D. HALLIDAY, E. R. CARSON,· P. RICHARDSt AND A. S. TAVILL MRC Clinical Research Centre, Harrow, Middlesex, • Department of Systems Science, City University, London, and. tSt George's Hospital, London (Received 7 October 1974) Summary 1. The relation between .endogenous urea metabolism and albumin synthesis has been studied in ten patients with chronic renal failure and in four normal subjects, after single intravenous injections of [14C]urea, [15N]urea and 125I-Iabelled albumin. 2. The rate of urea synthesis was determined from the dynamics of plasma [14C]urea specific radioactivity and the rate of urea metabolism was estimated from the relative rates of urea synthesis and urea appearance in urine and body water. Deconvolution analysis of plasma [15N]albumin enrichment and 125I-Iabelled albumin radioactivity yielded the cumulative incorporation of 15N into total exchangeable albumin and enabled calculation of the absolute rate of urea nitrogen utilization for albumin synthesis. 3. Although the mean absolute rate of urea degradation in uraemic patients (3'7 mmol/h) was higher than in normal subjects (2'3 mmol/h) there was no significant positive correlation between urea degradation and plasma urea concentration. 4. In uraemic subjects, there was a significant positive correlation between urea synthetic rate and urea degradation rate. 5. The rate of utilization of urea nitrogen for albumin synthesis was low, but was very much higher in uraemic subjects (mean 83'8 pmol/h) compared with normal subjects (mean 6'4 pmol/h), as was the provision by urea of the nitrogen required for albumin synthesis in uraemic subjects (2'37%) compared with normal subjects (0,13%). 6. The efficiency of utilization of urea nitrogen for albumin synthesis was higher in the uraemic patients (1'3 %) than the normal subjects (0'2 %), and was higher in those patients with chronic renal failure who received a 30 g protein diet than those on 70 g of protein. A significant negative correlation was noted between efficiency of urea nitrogen utilization and the rate of synthesis of albumin. 7. These studies suggest the presence of a mechanism for the conservation of urea nitrogen in chronic renal failure which is unrelated to the extent of urea degradation, and which can only be partly explained by the higher proportion of intraluminal gut nitrogen derived from urea. Key words: albumin synthesis, deconvolution analysis, intestinal hydrolysis, nitrogen reutilization, uraemia, urea nitrogen. Introduction Since the original demonstration by Walser & Bodenlos (1959) that approximately 20% of the urea synthesized by normal man is degraded in the gastrointestinal tract, there have been a number of studies suggesting that released ammonia nitrogen may be utilized for protein synthesis. For example. urea was used to maintain a normal growth rate in protein-deprived infants, and 15Nfrom this orally administered p 5N]urea was found in haemoglobin and plasma protein (Snyderman, Holt, Dancis, Roitman, Boyer & Balis, 1962). Similarly, urea played a role in the maintenance of nitrogen balance in uraemic patients receiving a diet containing Correspondence: Dr A. S. Tavill, MRC Clinical Research Centre, Harrow, Middlesex HAl 3UJ. 379 380 R. Varcoe et al. 2 g of essential amino acid nitrogen (Giordano, 1963). Quantitative assessment of the extent of urea nitrogen utilization has been based upon isotopic studies using orally administered (l5N]ammonium chloride or [15N]urea (Snyderman et al., 1962; Richards, Metcalfe-Gibson, Ward, Wrong & Houghton, 1967; Giordano, De Pascale, Balestrieri, Cittadini & Crescenzi, 1968; Read, McLaren, Tchalian & Nassar, 1969), or intravenously injected [15N]urea (Read et al., 1969). However, evaluation of the efficiency of utilization of endogenous urea nitrogen has been limited by the effects of unrepresentative precursor ammonia nitrogen enrichment in portal venous blood after oral isotope administration and the failure to define simultaneously the true dynamics of urea metabolism and protein turnover by means of non-recycling isotopic preparations. Recently, it has been possible by means of a combined intravenous [15N]urea, [14C]urea and 125I-labelled albumin technique to demonstrate that there was an increase in the rate of urea hydrolysis and an enhanced utilization of urea nitrogen for albumin synthesis in a patient with the gastrointestinal stagnant-loop syndrome (Varcoe, Halliday & Tavill, 1974). Since uraemic patients on a lowprotein diet rich in essential amino acids may have a relative deficiency in exogenous non-essential amino acids and an increased endogenous supply of ammonia nitrogen derived from hydrolysed urea (Giordano, 1963; Richards et al., 1967), it is relevant to evaluate the efficiency of their hepatic reutilization mechanisms. We have therefore carried out studies in patients with chronic renal failure and normal volunteers on normal and low protein diets, and have measured the contribution of endogenous urea nitrogen to the synthesis of a specific liver-produced protein, namely albumin. Materials and methods Subjects The full nature and purpose of the investigation was explained to patients and volunteers and their consent obtained before the study. Studies were performed on six uraemic patients (group A), who had been on a 30 g protein diet, high in essential amino acids (Giovanetti & Maggiore, 1964) for 4-18 months before study, and on four uraemic patients (group B), who were eating a 70 g protein diet. Studies were also carried out on four normal volunteers (group C), eating a 70 g protein diet, two of whom were subsequently studied again after they had been eating a 30 g protein diet, rich in essential amino acids, for 9 days. The food for all subjects during the study was provided and supervised by a dietitian, and sufficient energy, usually 10500 kJ/day, was provided to maintain weight. All patients were in hospital for the study and the normal volunteers were given meals of six equal protein portions spaced throughout the day to avoid large surges in urea synthesis. Isotopic materials [14C]Urea (specific radioactivity 58·1 mCi/mmol, The Radiochemical Centre, Amersham, Bucks., U.K.) was prepared as a sterile solution of 10 /lCi in 10 ml of 154 mmol/I sodium chloride (physiological saline). Approximately 4, 8 or 16 mmol of [15N]urea (99'6 atoms % enriched, synthesized free of other labelled nitrogen by Prochem Division of British Oxygen Company) was weighed and dissolved in the [14C]urea solution (approximately 10 /lCi) and filtered through Millipore filters immediately before reweighing and injection. Purified human serum albumin was labelled with 1251 by the iodine monochloride method of McFarlane (1956, 1958). Protocol of investigation Uptake of radioactive iodine by the thyroid was blocked with oral potassium iodide. At zero time approximately 10 /lCi of 125I-labelled albumin was weighed and injected intravenously and, immediately after a heparin-treated blood sample was taken 10 min later a weighed amount of the solution of combined (l4C]urea and [15N]urea was injected intravenously and an aliquot saved for preparation of standards. Accurately timed heparin-treated venous blood samples were taken at 2 h intervals for 12 h, and then daily for 10 days from normal subjects, and approximately fifteen times in 10 days in patients with chronic renal failure. In normal subjects urine was collected at measured intervals of approximately 2 h throughout the first 12 h and thereafter as pooled 24 h collections. Patients with chronic renal failure collected 24 h urine samples for 7 days. Utilization of urea nitrogen for albumin synthesis Measurement of urea and albumin concentrations Plasma and urine urea concentrations were measured by the standard diacetyl monoxime method on a Technicon Autoanalyser II. Plasma albumin concentrations were measured by an automated Bromocresol Green dye-binding method (Doumas, Watson & Biggs, 1971). Measurement of radioactivity 1251-labelled albumin. Trichloroacetic acid-precipitable radioactivity in 2 ml aliquots of plasma was measured in a Wallac Decem series auto gamma spectrometer. [14C]Urea. Because of overlap in the radiation spectrum of 14C and 1251, protein-bound iodine was precipitated from 2 ml plasma samples with an equal volume of 20% (w/v) trichloroacetic acid and the free iodine in the supernatant was removed by exposure to LR.A. 400 (BDH Ltd) resin. The equivalent of 1 ml of plasma was adjusted to pH 7·0 with sodium hydroxide (1 mol/l), and then made to a volume of 5 ml with distilled water before being dissolved in 10 ml of Unisolve (Koch-Light Laboratories Ltd), and cooled to 17°C before counting in a LKB liquid-scintillation spectrometer. Aliquots of 1 ml of urine were treated similarly except for the omission of the deproteinizing step when there was no proteinuria. Correction for quenching was made by the external standard channels-ratio method, which was validated for 14C by use of an internal standard technique. Measurement of 15 N enrichment in albumin' Albumin samples, prepared from heparin-treated plasma by the method of Korner & Debro (1956), were dialysed against many changes of distilled water for 7 days to remove all labelled urea. Purity of the albumin thus obtained was confirmed by polyacrylamide disc-gel electrophoresis. After Kjeldahl digestion of the samples and distillation into sulphuric acid, nitrogen was liberated from the resulting ammonium sulphate solution with lithium hypobromite and analysed for its 15N content on an AEI MS.20 mass spectrometer. The 15N enrichment was expressed as atoms % excess compared with a sample obtained from each patient before administration of [15N]urea. The reproducibility of the mass spectrometry was checked on a standard D 381 enriched sample of ammonium sulphate (SD ± 0'21 %, n = 13)over a 14 months period, and also on separate purified samples of enriched human albumin (SD ±002%, n = 13). Calculations Plasma and urine [14C]urea specific radioactivity. This was obtained by dividing the plasma or urine 14C radioactivity (d.p.m.jml) by the urea concentration (mmol/ml). This method of obtaining 14C specific radioactivity was validated by injecting [14C]urea and [15N]urea simultaneously in a normal individual and comparing the decay curves of plasma 14C specific radioactivity and the [15N]urea enrichment based upon measurement of mle 28 and 30 ion currents derived from purified urea extracted from plasma (Walser, George & Bodenlos, 1954). These gave almost identical slopes (0 0143 and 0'141). Likewise, plasma samples analysed directly for [14C]ureaspecificradioactivity by the urease-vacuum line method (Tavill, Craigie & Rosenoer, 1968) gave similar results. Urea space. This was calculated as the 14C radioactivity administered (d.p.m.), divided by the intercept on the ordinate of the semi-logarithmic decay curve of plasma 14Cradioactivity (d.p.m.jml). Robson (1964) showed that in renal failure the amount of labelled urea excreted during the period of equilibration between plasma and urea space in patients with chronic renal failure is very small. We have confirmed this and 'excess excretion' has therefore been ignored in these patients. However, Walser & Bodenlos (1959) calculated that excess excretion in normal subjects varied from o to 19%. Excess excretion is the amount of radioactive [14C]urea appearing in the urine during the equilibration period which is the result of the high, post-injection intravascular urea specific radioactivity relative to that of urea in the extravascular body water. If equilibration of the isotopic urea throughout total body water had been instantaneous, the kinetics of urinary [14C]ureaspecific radioactivity would have been exponential from zero time. Excess urinary excretion can be determined graphically for each collection period up to equilibration by multiplying the difference between the extrapolated urinary [14C]urea specific radioactivity and the observed specific radioactivity at the midpoint of the collection by the total urea excreted during that period (Fig. 1). This was sub- R. Varcoe et al. 382 u o ~ ::> 20 '0 Observed minus expected specific radiaactivity a E ..!:. 10 ~ ~ ~ :2:u 7 6 o 5 ~ 4 .2 3 2 I I I I --..I I Urine delay time 2345678 Time (hI FIG. I. Dynamics of plasma (0) and urinary (6) [14C]urea specific radioactivity in a normal subject (A.L.) receiving a 70 g protein intake, after a single intravenous injection of approximately 10 /lCi of [l4C]urea. The graphical determination of excess urinary excretion and of urinary delay times are illustrated (see the Materials and methods section). tracted where appropriate from the actual injected dose in order to give the effective injected dose. Initial urea pool. This amount (mmol) is the urea space (I) multiplied by the initial plasma urea concentration (mmol/l). Urea synthetic rate (mmol/h). Distribution of injected [14C]urea occurred very rapidly and was followed by exponential decay of plasma urea carbon specific radioactivity. Linear regression analysis by the least-mean-square method of the semi-logarithmic plot of specific radioactivity over 12 h in normal subjects and 7 days in patients gives a slope (k), which when multiplied by the urea pool size gives the urea synthetic rate. This method for measurement of urea synthesis makes the assumption that the synthetic rate remains constant for the duration of the experiment. Urea half-life (t i) is calculated from the decay constant of the slope (k) from the formula tt = Q·693/k. Urea excretion rate (mmol/h). This is calculated from the amount of urea excreted in the urine over the period considered in calculating k. Ideally, subjects should be in a completely steady state as evidenced by a constant plasma urea concentration. However, despite run-in periods and constant protein diets plasma urea concentrations did change, usually decreasing in uraemic patients. When calculating the amount of urea metabolized in the intestine, allowance was made for changes in body urea pool mass. Thus the rate of change of urea pool (P, mmol/h) was calculated from the rate of change of plasma concentration of urea over the course of the experiment. Urea metabolized in the gut (M, mmol/h). This was calculated as the difference between urea synthesized (8) and excreted into urine (U) and accumulating in or disappearing from the pool (P), M = 8 - U - P. This calculation assumes semi-steadystate conditions, the rates of synthesis, metabolism and excretion being constant, with the urea pool changing as a result of the constant difference between synthesis and the sum of excretion and metabolism. Urine delay time. This is the interval between times at which [14C]urea specific radioactivities were the same in plasma and urine (Regoeczi, Irons, Koj & McFarlane, 1965) and is determined graphically as illustrated in Fig. 1. Plasma volume (I). This was derived from the dilution of the injected 125I-Iabelled albumin. Intravascularalbumin(g). This was calculated from the product of plasma volume and plasma albumin concentration (g/l). Fractional turnover rate of albumin. This, as ~ ~ gu o~ ~~::> .~- 'ij CZ>- 6 ~o 0.0 en E o E CZ>"'" ::;E ,......,<1. u~ s: '---' >< 5 4 3 2 'i' '2 1 0 FIG. 2. Dynamics of plasma (0) and urinary (6) [14C]urea specific radioactivity in a uraemic patient (E.O.) receiving a 30 g protein intake, after a single intravenous injection of approximately 10 /lCi of [l4C]urea. The long half-life and long urinary delay time are illustrated. Utilization of urea nitrogen for albumin synthesis ylf) mit) Rate of change of newly synthesized plasma [~]albumin 12SI-labelled albumin distribution and degradation dynanics Observed plasma ]albumln enrichment rSN FIG. 3. Model for deconvolution of experimentally observed albumin dynamics. percentage of intravascular albumin degraded per h, was calculated by resolution of the plasma 12sI_ labelled albumin radioactivity curves by the method of Matthews (1957). Under steady-state albumin metabolic conditions, as defined by a constant plasma albumin concentration, the absolute synthetic rate (mgfh) could be calculated from the product of the fractional catabolic rate and the intravascular albumin pool. Urinary albumin losses in all patients were very small ( < 0·7 g/day). Deconvolution analysis of the p SNialbumin curve The observed P sN]albumin enrichments are based upon a pulse input of pSN]urea as influenced by the urea dynamics, which include distribution, hydrolysis and recycling and the distribution and degradation of newly synthesized albumin. If y(t) is the value of the observed p sN]albumin enrichment at time t and g(t) is the value at time t of the 12sI_ labelled albumin curve, which defines the albumin dynamics of distribution and degradation, these curves are related by the following convolution integral (Vitek, Bianchi & Donato, 1966): f t y(t) = _1_ g(O) m(t-'r) . g(-r) • dr o where m(t) is the rate of change of newly synthesized plasma pSN]albumin enrichment (see Fig. 3). This convolution integral can be solved for m(t) by numerical inversion (Jones, Craigie, Tavill, Simon & Rosenoer, 1968). Integrating m(t) with respect to time enables u(t) to be derived: t u(t) = f m(t)· dt o where u(t) is the plasma p SN]albumin enrichment 383 which would have been obtained had all the newly synthesized albumin remained intravascularly and undegraded. Therefore the incorporation of 1sN into albumin was obtained from the product of the maximum albumin enrichment resulting from this deconvolution and the intravascular albumin pool. Since this can be expressed as a percentage of the injected dose of pSN]urea, the utilization of endogenous urea can be obtained from the product of the 1SN incorporated and the initial urea nitrogen pool divided by the time taken to reach maximum labelling. In chronic renal failure, there was delay in completion of albumin labelling. After the period of linear labelling which followed the pulse p sN]urea input, there was a small but continuing rise in albumin enrichment which probably represents the effects of recycling of nitrogen from several sources other than solely urea. In the uraemic patients maximum albumin enrichment is defined as the time at which only these effects remain, namely the end of linear incorporation. The rate of incorporation of 1sN was obtained from the linear portion of the deconvolution curve; in practice this was calculated from the enrichment achieved between the time of intercept on the abscissa and the time at which the deconvolution curve starts to reach a plateau (Table 2; Fig. 4). Results Urea metabolism (Table 1) The selection of patients able to tolerate a normal protein intake was reflected by the generally higher rate of urea clearance in patients of group B. Only one (S.S.) with a low urea clearance and a high plasma urea concentration had later to discontinue this diet. Each of the normal volunteers had a lower plasma urea concentration at the start of the second study after being on a low-protein diet, and body urea pools were proportionately decreased. The urea space, as percentage of body weight (Table 1, column 4) calculated from the extrapolated radioactivity regression line, was similar in uraemic (mean 65'3%) and normal subjects (mean 64'8%). Excess of excretion of [14C]urea was zero in chronic renal failure, indicating the rapidity of distribution compared with renal excretion. In normal subjects, excess excretion was less than 2 % of the injected dose. The linear regression analysis of the p4C]urea R. Varcoe et al. 384 .~1i 100 "0 Cl) 98 ~ ~ 0._ 8 ~~.s ~8 i! e ~ 50 ,g.~ ~ 40 ::> 0 ~~ 30 30 20 o 60 C ~ 50 :gc: '" e 40 ~ ~ "0 ~ .0 '" 30 .---.'" if '0 20 ;fl c:....", -S E £ oS > c: 10 8 o'" 40 60 80 100 120 Time (hI 140 160 180 200 220 "o" FIG. 4. Semilogarithmic plot of the dynamics of exogenous 12sI-labelled albumin (6) and biosynthetically labelled [lsN]albumin (0) after the simultaneous intravenous administration of 12sI-labelled albumin and [lsNlurea to a uraemic patient (E.M.) The deconvoluted [lsNlalbumin enrichment (--) is plotted on a linear scale and represents the dynamics that would have been observed in the absence of distribution and degradation of the newly labelled albumin. The rate of incorporation of lsN in this patient was calculated from the enrichment achieved during the period to (i.e. the time of intercept on the abscissa) to the time at which the deconvolution curve deviates from linearity (- - -). specific radioactivity curves showed that decay constants in uraemic patients (Table 1, column 9) were very much lower than in normal subjects, yielding higher values for urea half-life in patients (groups A+B,meantt65'5h±9'7hsEM,n = 10)as compared with normal subjects (group C, mean tt 8·3 h±0'9 h SEM, n = 6). However, when converted into urea synthetic rate (Table 1, column 10) by multiplying by the urea pool, it was apparent that uraemic patients on a 30 g protein diet synthesized urea at a rate similar to that of normal subjects on this intake, whereas on a 70 g protein intake urea synthesis was higher but again similar in patients and normal subjects. Virtually all the patients in groups A and B showed a fall in body urea pool over the course of the study, in contrast to those in group C who showed a rise or no change over the much shorter time-course of the [14C]urea study. This may be explained by a normal diurnal rhythm in the volunteers, whereas in chronic renal failure a supervised protein intake at either level may have permitted urea loss by all routes to exceed synthesis. Urinary urea delay times estimated graphically can only be approximate. Only one normal subject (A.L.) had a measurable delay time, which was 1 h and 1·5 h in the two successive studies. In all three other volunteers the delay was virtually zero. In chronic renal failure the delay time was usually longer and the plasma and urine ['4C]urea specific radioactivity decay curves occasionally diverged, the plasma falling faster than the urine with a progressive lengthening of delay times. The rate of urea metabolism (Table 1, column 13) was greatest in the uraemic patients on a normal protein intake. However, when expressed as a percentage of the urea synthetic rate (Table 1, column 14) it was highest in uraemic patients on a lowprotein diet and progressively lower in normally fed uraemic patients, and normal subjects on a 70 g protein intake. Both normal subjects when put on a low-protein intake increased their rate of urea breakdown as a percentage of synthesis but not in absolute terms. There was a significant positive correlation between the plasma urea concentration TABLE I. Urea metabolism data in uraemic patients and normal subjects F F F F M M Group A E.M. E.O. J.G. R.C. M.R. J.S. MeanA±sEM Group C S.H. R.V. A.L. (I) P.B. (I) A.L. (2) P.B. (2) Mean C±SEM M F M M M M Group B J.A. M C.P. M S.S. M F.S. M MeanB±sEM MeanA+B±sEM Sex Patient 70 70 70 70 30 30 70 70 70 70 30 30 30 30 30 30 (I) 90'0 56'5 65'0 65'0 65'0 64'0 68'0 67'7 60'7 68'3 47'0 44'0 47'7 50'0 56'0 65'0 (2) Body weight g/24 h) (kg) Dietary &rotein 4·0 4·0 3·5 4·5 2·5 3-3 3·6±0·3 20·2 20·2 36·8 13-8 22-8±4'3 25-4± 3-3 26,5 41,5 37·8 15·2 10·7 31·3 27·2±4·6 (3) (mmol/I) (5) Urea space (% of body wt) = 53·0 59 34,7 62 48·7 74 41·4 64 46·4 71 37·5 59 43·6±2-6 64·8±2·4 52·2 76 41·5 61 41·0 67 47·0 68 45-4±2'3 68'0±2'7 38·2±2·8 65·3±2·5 29·9 60 23-1 53 34·2 72 27·4 54 43·0 76 43-1 66 33'5±3'1 63'5±3'5 (4) (I) Urea space Plasma urea concentration = 212 139 170 186 ll6 124 158±14 1054 838 1509 649 1013±160 932±ll2 792 959 1293 416 460 1349 878±149 (6) Urea pool (mmol) 2650 3025 3286 3246 3279 2150 2939±170 571 721 217 742 563±105 362±72 166 136 130 248 446 238 227±44 (7) 0·00 0·00 +2·00 +3095 +0·50 +1·03 -0,37 -0,33 -0,40 +0·30 -0,46 -0'73 -0,22 -0'38 -0,20 +0'50 (8) pool (mmol/h) in urea (ml/h) Change Urea clearance = = -0·0543±0·0027 -0,0923 ±0·0028 -0·1026±0·0240 -0·ll05±0·0286 -0·1028±0·0161 -0·07l8±0·0071 -0,0891 ±0'0081 -0'0138±0'0005 -0,0257 ±0·0009 -0,0085 ±0·0004 -0·0244±0·0035 -0,0181 ±0·0036 -0·0136±0·0022 -0·0076±0·0003 -0·0084±0·0002 -0·0056±0·0002 -0·0177±0·0015 -0,0161 ±0·0010 -0·0079±0·0003 -0·0106±0·0019 (9) 4·4 4·8 4·8 309 4,6 7·9 5·1±0·5 (ll) ll·5 10·6 \2·8 \2'1 17·4 ll·5 20·6 14·6 ll·9 8·2 8·9 7·1 1309±\06 10·7±1·0 14,5 12·2 21,5 15·1 \2·8 7·7 15·8 n-z 16·2±1·6 1\06±1·3 IH±I'5 7·7±1·2 6,0 8·1 7·2 7·4 7·4 10·7 7·8±0·6 (10) 92 95 66 71 69 80 79±5 84 70 60 71 7I±4 67±3 73 59 67 53 62 74 65±3 (12) rate 0·9 0·7 309 2·1 3·2 2-8 2-3±0'5 2·7 6·7 5·5 4·3 4'8±0'7 3·7±0·4 2·1 4·0 2·6 309 3·0 2·3 3·0±0·3 (13) 8 5 22 10 27 31 17±4 19 31 43 27 30±4 36±3 35 49 36 53 41 21 39±4 (14) rate Urea metabolized [I4C)Urea specific Urea excreted Urea radioactivity rate synthetic Xof rate (mmo /h) constant±sE %of. (mmol/h) synthetic synthetic (mmol/h) (fraction/h) = Urea clearance values are calculated as mean values of 24 h collection periods throughout the course of study except in normal subjects, where a single urea clearance was calculated over the course of the [14C]urea study. Group A: uraemic patients on a low protein diet. Group B: uraemic patients on a normal protein diet. Group C: normal subjects. Column (6) (3) x (4). Column (10) (9) x (6). Column (12) 100 x (11)/00). Column (13) (10)-(11)-(8). Column (14) 100 x (13)/(10). ~ 00 Vl V> 0:;' ~ ~ sS· :::: 5= l:l ::: ~ .... ::s ::; ~ ~ -. l:l ~ :::: .... ~ o' ;:: l:l - s-. Group C S.H. R.Y. A.L. (I) P.B. (1) A.L. (2) P.B. (2) Mean C±SEM GroupB J.A. C.P. S.S. F.S. Mean B±SEM Mean A+B±SEM Group A E.M. E.O. J.G. RC. M.R J.S. MeanA±sEM umn (9) (6) = = 70 70 70 70 30 30 70 70 70 70 30 30 30 30 30 30 (I) 45 48 47 48 46 47 47±0·4 30 32 30 40 33±2·1 35±H 38 33 33 35 38 38 36±0·9 (2) 117 106 127 106 124 106 96 83 72 104 106 69 83 77 95 125 (3) 80 80 80 50 70 50 100 90 90 90 90 80 70 60 60 100 (4) (h) Protein Concn, of Intravenous Linear albumin content plasma pool incorporation of diet albumin (g) interval (g/I) (g/24 h) Col umn m (9) 100 (13) x IoiiO = Column (14) 6'0 17-8 9.1 7'1 18'7 13'7 17-3 13'7 36·7 14'0 35·2 261'0 27·0 84·5 12'3 8·8 8·0 21·5 13·2 8·6 26·5 16·6 18·9 13·0 30·2 16·7 42-8 206'7 25-4 74·4 13-3 12-6 8·0 14'1 16'0 7·9 14·7 7'8 8'1 7·3 7·7 7-7 7·5 30'8 7·3 7·6 8·4 7·9 0·10 0'15 0·08 0·11 0·18 0·21 0·14±0·02 0·23 0·18 0·39 0·22 0·26±0·04 0·39±0·08 0·57 0·67 0·35 0·98 0·16 0'16 0'48±0'12 5·3 5'2 3·4 8·2 6'0 10·4 6·4±0·93 48·5 33·5 130·8 31-7 6H±20'4 83-8±15'7 100·3 160·6 129·3 135·9 24·5 43'2 99·0±20·2 604 235 450 403 654 472 470±56 456 468 380 364 417±23 369±29 440 281 194 294 316 496 337±41 3-6 6'8 2-8 5'2 4·6 7'5 5'4 5·4±0·6 5'2 5'4 4'3 4'2 4·8±0·3 4·2±0·3 5'6 3-8±0'5 5'0 3'2 2'2 3'4 0·08 0'19 0·07 0·18 0·08 0·19 0·13±0·02 0·94 0·62 3'04 0'75 1'34±0'49 2'37±0'60 2·01 5·02 5·88 4·00 0·68 0·77 3'06±0'83 eo 1·8 1·4 7.8 4·2 6·4 5·6 4·5±1·1 5·4 13·4 11·0 8·6 9·6±1·5 7·4±0·9 4·2 8·0 5·2 7-8 6·0 4·6 6·0±0·6 0'29 0'37 0·04 0·20 0·09 0'19 0·20±0·05 0·90 0·25 1·19 0·37 0·68±0·19 1·27±0·25 2·39 2'01 2'49 1·74 0·41 0·94 l-66±0'31 104 X Total ISN Injected Proport on Rate of Albumin N required Proportion N available Efficiency of Albumin incorporated dose of ofi njected urea N synthetic for of total N from urea utilization enrichment (Jlmol) ISN ISN utilization rate synthesis required degradation of available achieved (mmol) incorporated for albumin (mgfh) (mmol/h) provided by (mmol/h) N (atoms % into albumin synthesis urea N co excess) (%) (pmol/h) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) Utilization of urea N for albumin synthesis 2xcolumn (13) in Table I (9) (11) x 10 = = (10) = 6'25 x 14 Column (13) Column (12) Column (11) Deconvoluted [lsN]albumin data (8) urea N pool x 1000 100 x (4) (3) X (5) 6'25x14 (6) Column (8) = x 100 Col Calculations Group A: uraemic patients on a low-protein diet. Group B: uraemic patients on a normal protein diet. Group C: normal subjects. 2. Albumin metabolism data in uraemic patients and normal subjects: utilization of urea nitrogen for albumin synthesis based upon incorporation of lSN after intravenous administration of [ 1 SN lurea Patient TABLE w r- '"...'" l::l <;) ~ ~ ~ 00 0\ Utilization of urea nitrogen for albumin synthesis (Table 1, column 3) and the urea metabolized as a percentage of the urea synthetic rate (Table 1, column 14) (r = 0'56, P<0'05, n = 16). However, the absolute amount of urea metabolized (Table 1, column 13) failed to show a significant positive correlation with the plasma urea concentration (Table 1, column 3) (r = 0'33, P>O·I, n = 16). Finally, the only other significant correlation noted was in chronic renal failure between urea synthetic rate (Table 1, column 10) and urea metabolic rate (Table 1, column 13)(r = 0'76, P < 0,05, n = 10). Albumin metabolism (Table 2) Plasma albumin concentration was lower in both groups of uraemic patients than in the normal subjects (Table 2, column 2). Since all subjects were in a steady albumin metabolic state, the rate of 12'I-Iabelled albumin degradation could be equated with synthesis, which was generally lower in uraemic patients, but not invariably so even on a reduced protein intake (Table 2, column 10). It was apparent from the sequential measurements of [1 'N]albumin enrichment (Fig. 4) that biosynthetically labelled albumin could be detected in plasma as early as 2 h in normal subjects and 3 h in chronic renal failure. In all groups there were examplesoflatent periods of 3-4 h, and in one patient (J.A.) there was a long latent period of 23 h before [1'N]albumin was detected (samples at 4 and 8 h being below the limits of detection, i.e, < 0·0001 atoms % excess). As indicated in the Materials and methods section, both the dynamics and magnitude of l'N incorporation into albumin are derived by deconvolution of the [1'N]albumin enrichment curve, thereby permitting direct comparison between the rate of release of urea nitrogen by metabolism and the rate of utilization of this nitrogen for albumin synthesis. The cumulative albumin enrichment achieved over the linear period of labelling is shown in column 5 of Table 2. Since deconvolution provides the plasma albumin enrichment that would have been achieved had there been no extravascular distribution of labelled albumin, the total incorporation of l'N within this period (Table 2, column 6) can be obtained from the product of enrichment (column 5) and the plasma albumin pool (column 3). Since the injected dose of l'N is known (Table 2, column 7), the percentage of this dose which appeared in albumin can be derived (column 8). On the assump- 387 tion that after distribution through body water the injected [1'N]urea behaves identically with endogenous urea, the absolute rate of urea nitrogen utilization for albumin synthesis can be calculated (Table 2, column 9). This can then be expressed as the percentage of the total nitrogen requirements for the measured rate of albumin synthesis (Table 2, column 12) and as a percentage of the rate of urea nitrogen released by metabolism, the latter being an indication of the efficiency of utilization of available urea nitrogen by the liver (column 14). In spite of the greatly expanded urea pool in groups A and B, with its resultant dilution of enrichment of precursor [1'N]urea, the percentage of the injected dose incorporated was greater in most cases than in group C (Table 2, column 8). When this factor is taken into account in calculation of the rate of urea nitrogen utilization (Table 2, column 9) the differences between patients with chronic renal failure and normal subjects become more apparent, with uraemic patients using very much more (mean 83·8 pmol/h±sEM 15'7, n = 10) than normal subjects (mean 6·4 Jlffiol/h ± SEM 0,93, n = 6). On average, in uraemic subjects a lowprotein diet seemed to promote utilization of urea nitrogen. In both normal subjects there was a higher utilization on the 30 g than on the 70 g protein diet. The contribution of urea nitrogen to the nitrogen required for albumin synthesis (Table 2, column 12) in normal subjects was small. However, urea nitrogen forms a consistently higher fraction of albumin synthetic requirements in uraemic patients (up to 80-fold higher), the highest values being seen in uraemic patients on protein restriction. However, it is noteworthy that protein restriction produced no effect on the provision of these requirements in the two normal subjects who were studied twice. The highest efficiency of utilization of available urea nitrogen was seen in group A, and although there were individual exceptions, efficiency was higher in groups A and B than group C. Of the two subjects studied twice, one (P.B.) showed no change in efficiency of utilization after a short period of protein restriction; the other (A.L.) doubled the efficiency but still showed the lowest value seen in any study. No significant positive correlation was seen between the rate of urea nitrogen utilization for albumin synthesis (Table 2, column 9) and the amount of nitrogen available from urea degradation (Table 388 R. Varcoe et al. 2, column 13) (r = 0'28, P>O'I, n = 16), in spite of the previous finding of a significant correlation between urea synthesis and urea degradation. When expressed in terms of efficiency of utilization of released nitrogen, the highest positive correlation existed between efficiency (Table 2, column 14) and the absolute rate of utilization (Table 2, column 9) (r =0'90, P < 0'001, n = 16). In contrast, negative correlations were apparent between both efficiency and the absolute rate of utilization of urea nitrogen and the total nitrogen utilized for albumin synthesis (Table 2, column 11) (r = -0,53 and -0'54 respectively, P < 0'05, n = 16). Discussion Urea metabolism Calculation of the parameters of urea metabolism from the dynamics of intravenously administered isotopically labelled urea has been well established in previous studies (Walser & Bodenlos, 1959; Jones, Smallwood, Craigie & Rosenoer, 1969; Robson, Kerr & Ashcroft, 1968; Deane, Desir & Umeda, 1968; Scholz, 1968; Walser, 1970). Calculations in the present study were based upon observations over at least one half-life of the injected urea. In all subjects the urea space was consistent with the expected total body water and with the data of Scholz (1968), who demonstrated that there was no increase in the volume of urea distribution in renal failure and that the urea space was nearly identical with the volume of total body water measured with tritiated water. As expected the ['4C]urea plasma half-life was considerably longer than normal in chronic renal failure (mean tt 65'5 h). The values in normal subjects (mean 8·3 h) were similar to the 7 h mean value observed by Walser & Bodenlos (1959) and the 6·7 hand 9·2 h values recorded by Jones et al. (1969). Likewise, the mean percentage of urea synthesized which was metabolized by uraemic patients (39%) is similar to the rate of urea degradation reported by Robson (1964) and Deane et al. (1968), but considerably less than the average of 70% for urea degradation as percentage of synthesis recently reported by Walser (1974) in uraemic patients receiving essential amino acids or keto acid analogues of five essential amino acids. The higher percentage degradation seen in patients and normal subjects receiving a low-protein intake is predominantly the result of a relative reduction in urea synthesis. Nevertheless, in absolute terms there is more urea degraded in chronic renal failure than in normal subjects regardless of the dietary protein, although the difference was not as great as that described by Jones et al. (1969), who claimed that the enterohepatic circulation of urea nitrogen was at least twice that of normal subjects. Although uraemic patients on a normal protein intake tend to degrade more urea than those on dietary protein restriction, no consistent effect was noted in the two normal subjects when placed on 30 g of protein for 9 days. This phenomenon was evident in chronic renal failure in spite of the fact that those able to tolerate a normal protein intake had on average a lower plasma urea concentration. The estimation of the quantity of urea nitrogen recycled is dependent on the assumption that synthesized urea not appearing in the urine has been hydrolysed in the gut and not lost by other routes. Although there is no detectable urea in the faeces of normal individuals it might be expected that there would be an increased faecal loss in uraemia. However, Wilson, lng, Metcalfe-Gibson & Wrong (1968) have shown that there was no detectable faecal urea in two-thirds of their patients with renal failure, with a mean faecal dialysate urea concentration of only 6% of the blood urea concentration in the whole group. Evidence that urea hydrolysis in the gut is the main extra-renal source of urea clearance has been provided by the observation that antibiotic treatment can virtually eliminate degradation (Walser & Bodenlos, 1959). Although urea diffuses readily through most body tissues, the wall of the colon is relatively impermeable (Billich & Levitan, 1969), and colonic bacterial ureases would therefore have to act upon urea delivered intraluminally from the small intestine. However, Gibson, Sladen & Dawson (1973), on the basis of studies in patients with ileostomies or excluded colonic loops, suggest that a significant proportion of urea breakdown can occur in the small bowel. Since urea is readily diffusible into the small intestine, and faecal bacterial urease activity is increased in uraemia (Brown, Hill & Richards, 1971), and as mentioned uraemic patients tend to have on average a higher rate of urea metabolism, it would be expected that the latter would correlate with plasma urea concentration. The fact that our results, like those of Robson (1964) and Walser (1974), fail to show such a correlation emphasizes the conclusion that urea degradation must be in- Utilization 0/ urea nitrogen for albumin synthesis dependent of diffusion and that some component of the chronic uraemic state leads to a relative reduction in gastrointestinal urea hydrolysis. The finding of a significant correlation between plasma urea concentration and the fraction of synthesized urea which is metabolized must be interpreted with caution because of the apparent over-riding importance of dietary protein in regulating the urea synthetic rate. There is no evidence that factors exist in chronic renal failure which simultaneously increase urea breakdown and decrease urea synthesis. Urea nitrogen reutilization The extent of reutilization of ammonia nitrogen released by urea degradation is indicated by two sets of data. First, the correlation between the rate of urea synthesis and the rate of urea degradation suggests that urea synthesis may be promoted by ammonia returning to the liver in portal blood. Secondly, a direct indication of the extent of utilization of urea nitrogen for albumin synthesis is obtained by the present method for provision and analysis of [1sN]albumin incorporation data. Although 1SN recycling may be perpetuated by exchange phenomena, e.g. transamination reactions as suggested by Jones, Craigie, Koj & McFarlane, (1967), the pathways of initial fixation of ammonia nitrogen are probably those which are available for reutilization of endogenous urea nitrogen in the process of net nitrogen gain. Fixation of ammonia nitrogen into arginine, glutamic acid and glutamine and aspartic acid represents a potential net contribution to nitrogen balance. The advantages of the present approach to the quantification of the extent of urea nitrogen reutilization are twofold. First, intravenous administration of [lsN]urea together with [l4C]urea as a single injection permits equilibration with endogenous urea. Thereafter, the fate of both isotopic forms of urea is a valid indication of endogenous metabolism, and in particular of intestinal hydrolysis (Wolpert, Phillips & SummerskiII, 1971). Secondly, the simultaneous intravenous administration of 12sI-labelled albumin, followed by frequent blood sampling, permits deconvolution of an accurately defined plasma [l sN]albumin enrichment curve, thereby establishing with a precision that has not been possible previously the time-course of incorporation of 1 sN into total exchangeable albumin. In this way it has been possible to make 389 a quantitative assessment of the fate of endogenous urea which has left the circulation to be hydrolysed in the lumen or mucosa of the intestine. From measurements of the rate of turnover of albumin the extent to which recycled urea nitrogen fulfils the requirements for albumin synthesis has been determined, and from the rate of metabolism of [l4C]urea the efficiency of utilization of urea nitrogen for this purpose has been calculated. As with the extent of urea hydrolysis, the incorporation of urea nitrogen into albumin does not appear to be dependent upon the urea concentration iii. body water. Although the provision of nitrogen requirements for albumin synthesis and the efficiency of utilization of released urea nitrogen for this purpose was on average greater in patients with renal failure than normal, the reason for these observations is not immediately evident. Although all measured albumin synthetic rates (expressed per unit body weight) were within the normal range of 135-260 mg day" ' kg- 1 (Dykes, 1968), the significant inverse relationship between nitrogen requirements for albumin synthesis and the efficiency of utilization of available urea nitrogen are evidence for. a possible hepatic mechanism for adjusting the reclamation of urea nitrogen. The level of dietary protein may be a major factor in such a mechanism since in dietary protein restriction urea nitrogen will form a greater proportion of total intraluminal nitrogen. Any suggested mechanisms must take into account the fact that the patients with the lowest urea clearance were those requiring restriction of dietary nitrogen and as such were also the ones whose fractional urea turnover was the slowest. These two factors, namely prolonged protein restriction and the fact that urea has an enhanced survival time in body water, may be the key mediators of long-term adaptation in nitrogen economy in chronic renal failure. Evaluation of their relative importance will require further observations of the effects of each studied in isolation. Whichever factor is predominant it appears that in chronic uraemia under the circumstances studied the utilization of urea nitrogen for albumin synthesis is of minor nutritional significance. Because of the relatively high contribution of albumin to overall hepatic protein synthesis and the predominant enterohepatic route of ammonia nitrogen fixation, it is unlikely that the utilization of urea nitrogen for the synthesis of other hepatic or extrahepatic proteins is of any greater proportional significance. 390 R. Varcoe et al. Acknowledgments This project was approved by Northwick Park Hospital Ethical Committee (Project E. 142). 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