Experimental Physiology (1991), 76, 59-65 Printed in Great Britain
RENAL CLEARANCE OF PLASMA ALLANTOIN IN SHEEP X. B. CHEN, D. J. KYLE, E. R. 0RSKOV AND F. D. DEB. HOVELL Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland (MANUSCRIPT RECEIVED 12 MARCH 1990, ACCEPTED 24 JULY 1990)
SUMMARY
The recovery in urine of an intrajugular infusion of physiological amounts of allantoin was measured in four sheep nourished by an intragastric infusion of volatile fatty acids and casein (to eliminate rumen fermentation). The recovery was 72 % (S.E.M. 7) and the remainder was presumed to have been lost by diffusion into the gut and degradation by gut microflora. Measured in two sheep, allantoin was removed from the blood at a fractional rate of 0 30 h-1, and excreted in urine at 0 23 h- . Calculation based on creatinine excretion showed glomerular filtration rate and tubular reabsorption of allantoin to be unchanged by the intravenous infusion. Maximal tubular reabsorption at 1-28 mmol day-' was saturated by the load of endogenous allantoin alone. In a second experiment with seven normally fed sheep (28-50 kg live weight, all given I kg feed), urinary excretion and plasma concentration of allantoin were linearly related. However, the errors were such that plasma allantoin concentration would be of little value as a predictor of urinary excretion. There was a nearly twofold range in allantoin excretion (the larger animals excreting less), which implied that the supply of microbial biomass to the host animal per unit of feed ingested could be profoundly affected by feeding level. INTRODUCTION
Allantoin is quantitatively the most important of the purine derivatives allantoin, uric acid, xanthine and hypoxanthine excreted in the urine of ruminants. The purine derivatives originate mainly from the degradation of absorbed microbial nucleic acids, and their urinary excretion has the potential to be used as an estimator of the rumen microbial biomass available to the ruminant (Topps & Elliott, 1965; Rys, Antoniewicz & Maciejewicz, 1975). Previous work in this laboratory has shown in sheep (Chen, Hovell, Orskov & Brown, 1990b) and in cattle (Verbic, Chen, MacLeod & 0rskov, 1990), that excretion of purine derivatives responds rapidly (within 24 h) to changes in the supply of microbial nucleic acids. In that work, known amounts of microbial nucleic acids were infused into the abomasum, and purine derivative recovery in the urine measured. The models derived to describe the input-output relationship of purines indicated that a relatively constant proportion (84% (S.E.M. 0 7) from six lambs, and 85 % from two steers) of the purine derivatives which entered the blood was excreted in the urine. Other work has shown that possibly as much as 1O % of the allantoin and uric acid produced can be recycled via saliva to the reticulo-rumen, and completely degraded therein (Chen, Hovell & 0rskov, 1990a). The objective of the present study was to examine the renal clearance of allantoin by sheep and its implication on the use of purine derivative excretion as a measure of microbial biomass supply to the ruminant.
60
X. B. CHEN AND OTHERS
METHODS
Experiment I Animals and management. Four Finn x Dorset wether sheep (53-56 kg live weight (W)) fitted with a rumen cannula and an abomasal catheter were used. They were housed in metabolism crates and were entirely nourished by the continuous intragastric infusion of all nutrients as volatile fatty acids (VFA) (with macrominerals) into the rumen, and casein (with trace minerals and vitamins) into the abomasum (Orskov, Grubb, Wenham & Corrigall, 1979; Hovell, Orskov, Kyle & MacLeod, 1987). The VFA (065 acetate: 025 propionate: 010 butyrate as molar proportions) plus casein provided 585 kJ (kg W075)-1 metabolizable energy (ME) and the casein 560 mg N (kg W075)-1 daily. These levels were equivalent to about 1 3 times the maintenance requirements for both energy and protein (Agricultural Research Council, 1984). Animals nourished by this method have no microbial fermentation in the rumen and hence no source of exogenous purines. The sheep were also fitted with a catheter in each external jugular vein; these were of polyvinylchloride (100 mm i.d.; Portex Ltd, Hythe, Kent) for allantoin infusion and of silastic (076 mm i.d.; Dow Corning Corporation, Midland, MI, USA) for blood withdrawal. Experimental design and execution. Two days after venous catheterization, the lambs were given a sterile continuous intravenous infusion of either saline (9 g NaCl 1-1) at 14 ml h-1 or allantoin solution (23 1 mmol/l-1 in saline) at 7 or 14 ml h-1 by mean of a peristaltic pump (LKB-Produkter Ltd, Sweden). In two sheep (I and J), the infusion treatments during three consecutive periods of 5, 7 and 7 days respectively were: (i) saline, (ii) allantoin at 14 ml h-1 and (iii) saline. In the other two sheep (D and E), each period was 3 days and the infusion treatments were: (i) saline, (ii) allantoin at 7 ml h-1 and (iii) allantoin at 14 ml h- . The amount of allantoin infused (up to 7 87 mmol day-1) was within the range of daily allantoin excretion (4-12 mmol day-1) by sheep given normal feeds. Daily (24 h) collections of urine were made into 1 M-sulphuric acid (final pH below 3), sampled and stored at 4 'C. Heparinized blood samples from sheep I and J were taken twice daily at 11.00 and 16.00 hours. Plasma was stored at -20 'C until analysed. On day 1 of period 3, additional samples of urine and blood were taken from sheep I and J at 0, 2, 4, 6, 8, 13 and 24 h after the termination of the allantoin infusion. The changes with time in plasma concentration and urinary excretion of allantoin were described by monoexponential equations using the maximum likelihood program of Ross (1987). With sheep D and E, only daily urine collections were made. Allantoin in urine and plasma was analysed as described by Chen, Mathieson, Hovell & Reeds (1990 c) and creatinine by the automated method of Technicon Instruments Ltd (1965). The plasma clearance of creatinine was calculated as described by Smith (1962) and taken as a measure of glomerular filtration rate (GFR).
Experiment 2 Animals and management, experimental design and execution. Seven sheep of 28-50 kg live weight were given 1 kg day-1 (air-dry basis) as two equal meals at 08.30 and 16.30 h, of either a standard diet containing 50 % hay, 30 % rolled barley, 10 % fishmeal and 10 % molasses with minerals and vitamins (general-purpose diet, GPD), or a pelleted dried grass (PG). The objective of this experiment was to examine the relationship between the daily urinary excretion and plasma concentration of allantoin under normal feeding conditions. Urine was collected daily (24 h), and a 10 ml jugular blood sample taken by venipuncture from the exterior jugular vein at about 1 1.00 h, from each animal daily for 15 days. Samples were analysed as in Expt 1. RESULTS
Plasma allantoin Experiment 1. Plasma allantoin concentrations averaged about 20 ,umol 1-1 during the control periods when there was no exogenous allantoin (Table 1). Concentrations increased rapidly with infusion of allantoin (49 0 and 45 9 ,umol 1I1 at hour 3, and 70 3 and 53 0,amol 1I1 at hour 5 for sheep I and J respectively). During the subsequent 7 days, plasma allantoin fluctuated between 60 and 70 ,tmol 1-1 in sheep I and 50 and 70 ,umol 1-1 in sheep J. When the allantoin infusion was stopped (period 3), plasma allantoin
61
RENAL CLEARANCE OF ALLANTOIN IN SHEEP
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concentrations declined exponentially with time with fractional clearance rates of 0-28 and 0-32 h-1 in sheep I and J respectively, (Fig. I A), and reached levels similar to those of period 1 within IO h.
Experiment 2. Plasma allantoin, which varied considerably between animals with a twofold range (69 142 #amol l'1, Table 2), was negatively correlated with animal size.
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RENAL CLEARANCE OF PLASMA ALLANTOIN IN SHEEP X. B. CHEN, D. J. KYLE, E. R. 0RSKOV AND F. D. DEB. HOVELL Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland (MANUSCRIPT RECEIVED 12 MARCH 1990, ACCEPTED 24 JULY 1990)
SUMMARY
The recovery in urine of an intrajugular infusion of physiological amounts of allantoin was measured in four sheep nourished by an intragastric infusion of volatile fatty acids and casein (to eliminate rumen fermentation). The recovery was 72 % (S.E.M. 7) and the remainder was presumed to have been lost by diffusion into the gut and degradation by gut microflora. Measured in two sheep, allantoin was removed from the blood at a fractional rate of 0 30 h-1, and excreted in urine at 0 23 h- . Calculation based on creatinine excretion showed glomerular filtration rate and tubular reabsorption of allantoin to be unchanged by the intravenous infusion. Maximal tubular reabsorption at 1-28 mmol day-' was saturated by the load of endogenous allantoin alone. In a second experiment with seven normally fed sheep (28-50 kg live weight, all given I kg feed), urinary excretion and plasma concentration of allantoin were linearly related. However, the errors were such that plasma allantoin concentration would be of little value as a predictor of urinary excretion. There was a nearly twofold range in allantoin excretion (the larger animals excreting less), which implied that the supply of microbial biomass to the host animal per unit of feed ingested could be profoundly affected by feeding level. INTRODUCTION
Allantoin is quantitatively the most important of the purine derivatives allantoin, uric acid, xanthine and hypoxanthine excreted in the urine of ruminants. The purine derivatives originate mainly from the degradation of absorbed microbial nucleic acids, and their urinary excretion has the potential to be used as an estimator of the rumen microbial biomass available to the ruminant (Topps & Elliott, 1965; Rys, Antoniewicz & Maciejewicz, 1975). Previous work in this laboratory has shown in sheep (Chen, Hovell, Orskov & Brown, 1990b) and in cattle (Verbic, Chen, MacLeod & 0rskov, 1990), that excretion of purine derivatives responds rapidly (within 24 h) to changes in the supply of microbial nucleic acids. In that work, known amounts of microbial nucleic acids were infused into the abomasum, and purine derivative recovery in the urine measured. The models derived to describe the input-output relationship of purines indicated that a relatively constant proportion (84% (S.E.M. 0 7) from six lambs, and 85 % from two steers) of the purine derivatives which entered the blood was excreted in the urine. Other work has shown that possibly as much as 1O % of the allantoin and uric acid produced can be recycled via saliva to the reticulo-rumen, and completely degraded therein (Chen, Hovell & 0rskov, 1990a). The objective of the present study was to examine the renal clearance of allantoin by sheep and its implication on the use of purine derivative excretion as a measure of microbial biomass supply to the ruminant.
60
X. B. CHEN AND OTHERS
METHODS
Experiment I Animals and management. Four Finn x Dorset wether sheep (53-56 kg live weight (W)) fitted with a rumen cannula and an abomasal catheter were used. They were housed in metabolism crates and were entirely nourished by the continuous intragastric infusion of all nutrients as volatile fatty acids (VFA) (with macrominerals) into the rumen, and casein (with trace minerals and vitamins) into the abomasum (Orskov, Grubb, Wenham & Corrigall, 1979; Hovell, Orskov, Kyle & MacLeod, 1987). The VFA (065 acetate: 025 propionate: 010 butyrate as molar proportions) plus casein provided 585 kJ (kg W075)-1 metabolizable energy (ME) and the casein 560 mg N (kg W075)-1 daily. These levels were equivalent to about 1 3 times the maintenance requirements for both energy and protein (Agricultural Research Council, 1984). Animals nourished by this method have no microbial fermentation in the rumen and hence no source of exogenous purines. The sheep were also fitted with a catheter in each external jugular vein; these were of polyvinylchloride (100 mm i.d.; Portex Ltd, Hythe, Kent) for allantoin infusion and of silastic (076 mm i.d.; Dow Corning Corporation, Midland, MI, USA) for blood withdrawal. Experimental design and execution. Two days after venous catheterization, the lambs were given a sterile continuous intravenous infusion of either saline (9 g NaCl 1-1) at 14 ml h-1 or allantoin solution (23 1 mmol/l-1 in saline) at 7 or 14 ml h-1 by mean of a peristaltic pump (LKB-Produkter Ltd, Sweden). In two sheep (I and J), the infusion treatments during three consecutive periods of 5, 7 and 7 days respectively were: (i) saline, (ii) allantoin at 14 ml h-1 and (iii) saline. In the other two sheep (D and E), each period was 3 days and the infusion treatments were: (i) saline, (ii) allantoin at 7 ml h-1 and (iii) allantoin at 14 ml h- . The amount of allantoin infused (up to 7 87 mmol day-1) was within the range of daily allantoin excretion (4-12 mmol day-1) by sheep given normal feeds. Daily (24 h) collections of urine were made into 1 M-sulphuric acid (final pH below 3), sampled and stored at 4 'C. Heparinized blood samples from sheep I and J were taken twice daily at 11.00 and 16.00 hours. Plasma was stored at -20 'C until analysed. On day 1 of period 3, additional samples of urine and blood were taken from sheep I and J at 0, 2, 4, 6, 8, 13 and 24 h after the termination of the allantoin infusion. The changes with time in plasma concentration and urinary excretion of allantoin were described by monoexponential equations using the maximum likelihood program of Ross (1987). With sheep D and E, only daily urine collections were made. Allantoin in urine and plasma was analysed as described by Chen, Mathieson, Hovell & Reeds (1990 c) and creatinine by the automated method of Technicon Instruments Ltd (1965). The plasma clearance of creatinine was calculated as described by Smith (1962) and taken as a measure of glomerular filtration rate (GFR).
Experiment 2 Animals and management, experimental design and execution. Seven sheep of 28-50 kg live weight were given 1 kg day-1 (air-dry basis) as two equal meals at 08.30 and 16.30 h, of either a standard diet containing 50 % hay, 30 % rolled barley, 10 % fishmeal and 10 % molasses with minerals and vitamins (general-purpose diet, GPD), or a pelleted dried grass (PG). The objective of this experiment was to examine the relationship between the daily urinary excretion and plasma concentration of allantoin under normal feeding conditions. Urine was collected daily (24 h), and a 10 ml jugular blood sample taken by venipuncture from the exterior jugular vein at about 1 1.00 h, from each animal daily for 15 days. Samples were analysed as in Expt 1. RESULTS
Plasma allantoin Experiment 1. Plasma allantoin concentrations averaged about 20 ,umol 1-1 during the control periods when there was no exogenous allantoin (Table 1). Concentrations increased rapidly with infusion of allantoin (49 0 and 45 9 ,umol 1I1 at hour 3, and 70 3 and 53 0,amol 1I1 at hour 5 for sheep I and J respectively). During the subsequent 7 days, plasma allantoin fluctuated between 60 and 70 ,tmol 1-1 in sheep I and 50 and 70 ,umol 1-1 in sheep J. When the allantoin infusion was stopped (period 3), plasma allantoin
61
RENAL CLEARANCE OF ALLANTOIN IN SHEEP
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concentrations declined exponentially with time with fractional clearance rates of 0-28 and 0-32 h-1 in sheep I and J respectively, (Fig. I A), and reached levels similar to those of period 1 within IO h.
Experiment 2. Plasma allantoin, which varied considerably between animals with a twofold range (69 142 #amol l'1, Table 2), was negatively correlated with animal size.
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