U rinary excretion of purine derivatives and prediction of rumen ...

Livestock Production Science 77 (2002) 127–135 www.elsevier.com / locate / livprodsci

Urinary excretion of purine derivatives and prediction of rumen microbial outflow in goats a ˜ b , J. Balcells a , *, N.H. Ozdemir Baber c , A. Belenguer , D. Yanez M. Gonzalez Ronquillo a a

´ Animal y Ciencia de los Alimentos. Facultad de Veterinaria. C / Miguel Servet 177, Zaragoza, Spain Departamento de Produccion b ´ Animal. Estacion ´ Experimental Zaidin. C.S.I.C. Camino del Jueves s /n- 18100 Armilla, Granada, Spain Unidad Nutricion c Ankara Nuclear Agriculture and Animal Science Research Center. 06983 Saray ( Istambul road 30 km) Ankara, Turkey Received 10 August 2001; received in revised form 12 March 2002; accepted 25 March 2002

Abstract The present study examined the relationship between duodenal flow of purine bases and urinary excretion of their derivatives (i.e., Allantoin, uric acid, xanthine and hypoxanthine) in selected milk goats. Three adult Granadina goats fitted with a T-shaped cannula in the abomasum were used to determine the endogenous contribution to renal excretion of purine derivatives and urinary recovery of abomasaly infused purine bases as yeast-RNA. Animals were fed alfalfa at maintenance level. The endogenous contribution of purine derivatives was determined at fasting (11.34 mg N / W 0.75 or 202.2 mmol / W 0.75 ) and it was similar to that obtained in sheep but lower than that reported in cattle. Urinary PD excretion responded linearly to incremental supply of purine bases throughout the abomasal cannula, these recovery (%) averaged 76. Xanthine oxidase activities in goat tissues were, in plasma 0.001 (S.E. 0.0001) i.u. / ml, in liver 0.12 (S.E. 0.021) i.u. / g and in duodenum 0.0009 (S.E. 0.00026) i.u. / g. Again, activities were lower than those detected in cows but close to values determined in sheep. A similar response model between both species (sheep and goat) is suggested.  2002 Elsevier Science B.V. All rights reserved. Keywords: Goat; Feeding and nutrition; Purine derivatives; Allantoin; Xanthine oxidase

1. Introduction Urinary purine derivatives (PD) excretion is used to predict rumen microbial protein synthesis in ruminant livestock. The principle is that duodenal purine bases (PB), as a microbial marker, are *Corresponding author. Tel.: 134-976-761-660; fax: 134-976761-590. E-mail address: [email protected] (J. Balcells).

efficiently absorbed and the majority of their derivatives excreted via the kidney. The ratio of PD in urine closely reflects, and therefore may be used to predict, microbial protein flow. However, the response model between urinary PD and duodenal flow of PB differs among species. Significant variations between cattle and sheep have been described (Chen et al., 1990b), but also within cattle species [European-Bos Taurus vs. Zebu CattleBos Indicus; (Liang et al., 1994)]. It is desirable to

0301-6226 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 02 )00081-7

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A. Belenguer et al. / Livestock Production Science 77 (2002) 127–135

extend this technique to other species of economic significance, such as goat (capra hircus), to develop specific models for the prediction of microbial protein production in the rumen (Stangassinger et al., 1995). The aim of this work was to establish the response model in Murciano–Granadina goats by quantifying the basal or endogenous contribution to urinary PD excretion after minimizing physiological rumen flow of microbial purine bases by fasting and by relating the abomasal input and urinary output after abomasal infusion of different doses of nucleic acids (NA). The activity of the enzyme xanthine-oxidase (XO; EC 1.2.3.2.) in different tissues as a key step for purine metabolism was also determined.

2. Material and methods

2.1. Experiment 1: urinary excretion of purine derivatives in relation to abomasal supply of purine bases 2.1.1. Animals and diets Three adult Murciano–Granadina goats (41.6 kg LW; S.E. 4.53), were fitted with a T-shaped abomasal cannula a month before the experimental procedure began. Animals were kept in metabolic cages with free access to drinking water. They were fed alfalfa hay at a maintenance level (443 kJ ME / W 0.75 ; Prieto et al., 1990). 2.1.2. Experimental procedures The experiment was divided into two phases: initially the basal excretion was determined (20 days), and after that (30 days) when animals were recovered, the relationship between abomasal input and urinary output was established (20 days). 2.1.3. Fasting trial Animals were penned individually in metabolic cages and fed at a maintenance level for 10 days (adaptation period). After that, feed intake was restricted every 2 days from 100 to 60 and 30% (prior, mid and early fasting) of the previous maintenance feeding level. After that, a 6-day fasting period was commenced.

2.1.4. Urinary recovery of abomasal PB Animals were given four doses of nucleic acids (NA) that were infused continuously (12 h per day) over four successive 5-day periods. RNA yeast (Torula yeast Sigma Co., St. Louis, USA) was used as a NA source and the doses were, respectively 0, 0.63, 0.84 and 1.05 (mmol PB / kg W 0.75 ), and were supplied following a 3 3 4 complete crossover design. These doses correspond to the amounts of microbial NA (or PB) likely to be produced when animals are normally fed between zero and three times the maintenance energy requirements. RNA (1.1 mmol PB / g) solution was prepared by diluting the yeast doses in 0.5 M NaOH. Once the yeast was diluted, the pH was brought to 3.5–4.5 using 10% HCl. The RNA-solution was continuously infused at a flow rate of 0.7 ml / min using a peristaltic pump (Minipuls-2 HP 18 Gilson). 2.1.5. Sample collection In both trials, total urine excretion was collected daily over 10% sulphuric acid (final pH of urine was kept below 3). Urine was weighed, its density measured and urine samples (100 ml) were frozen immediately at 2 20 8C until analysis. 2.2. Experiment 2: xanthine oxidase activity in plasma, liver or intestinal mucosa Samples of plasma, liver and intestine were taken from the local slaughterhouse (samples of tissues were taken from the same three animals at slaughter, and plasma was taken just before slaughter). Three animals, different from those used in Experiment 1, were chosen at random regardless of age, sex or previous nutrition.

2.2.1. Preparation of tissue extracts Blood samples were collected into heparinized tubes and centrifuged at 3000 3 g for 15 min. Plasma samples were analysed within 6 h. Procedures for extraction of liver samples and the extraction of the intestinal mucosa layer were adapted from those described by Furth-Walker and Amy (1987) and Reeds et al. (1997), respectively. Liver and kidney were washed in cold 0.15 M KCl, and 1 g of tissue was homogenised in 9 ml 0.5 mM EDTA in 0.05 M K 2 HPO 4 (pH 7.5) and centrifuged at 35 000 3 g for 30 min at 4 8C. Super-


natant fraction was dialysed 24 h against the same EDTA–KH 2 PO 4 buffer for 24 h and centrifuged again at 35 000 3 g for 30 min at 4 8C. The supernatant fraction was used for the assay. Intestinal samples were taken from the duodenum and lumen and washed with cold 0.15 M KCl. After that, samples were frozen immediately in liquid nitrogen and defrosted within 2 h at 4 8C. Then lumen was washed with 0.05 M N-2-hydroxyethylpiperazine-N2-ethanosulphonic acid (HEPES) buffer (pH 7.5) containing 0.25 mM EDTA and 0.25 mM phenylmethylsulphonyl fluoride (PMSF). One gram of intestinal mucosa was removed by finger pressure along the portion of intestine, and the mucosa layer cells were collected at the bottom in 9 ml HEPES– EDTA–PMSF buffer. The extract containing the enzyme XO was then purified as liver samples but using HEPES–EDTA–PMSF buffer.

2.2.2. Xanthine oxidase activity Activity of xanthine oxidase was measured as the rate of uric acid produced when xanthine was incubated with tissue extracts, as described by Chen et al. (1996). 2.2.3. Relationship between PB and N in rumen microorganisms extract Samples for isolation of microbial extract were also taken from the same three animals at the slaughterhouse. Rumen samples (200 ml) were squeezed through four layers of surgical gauze and the bacterial fraction was isolated by differential centrifugation (500 3 g for 10 min). It was used to precipitate the particulate material and resulting supernatant was centrifuged at 20 000 3 g for 20 min at 4 8C to deposit the bacterial fraction. This deposit was then resuspended in physiological saline solution and again centrifuged at 20 000 3 g for 20 min at 4 8C. The washed microbial pellet was freeze-dried for subsequent analyses. 2.3. Analytical procedure Urine samples were centrifuged, diluted (1:100), filtered (0.2 mm Millipore Bedford MS) and analysed for PD and creatinine following the procedure described by Balcells et al. (1992). PB in RNA-yeast and rumen bacteria were analysed with the same

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procedure after acid hydrolysis as described by ´ Orue ´ et al. (1995). Martın

2.4. Statistical analysis Analyses of variance and the simple linear regression were performed following the procedures described by Steel and Torrie (1980).

3. Results Animals remained in good health throughout the experiment and they recovered properly and fully consumed the experimental diets 1 week after the surgery.

3.1. Urinary excretion of purine derivative and creatinine during fasting The individual values for daily excretion of purine derivatives and mean values are shown in Fig. 1 and Table 1, respectively. Allantoin excretion (mmol / W 0.75 ) decreased significantly with food restriction, reaching the minimum value during fasting (from 620.9 to 128.8). After that, it remained fairly constant. Basal excretion (fasting excretion) averaged 128.8 (S.E. 9.24) ranging from 144.2 to 90.9. Between animal and day-to-day variations contributed to the total variance by 22 and 19%, respectively. Uric acid excretion (mmol / W 0.75 ) was not significantly affected by food restriction, although during early and mid-fasting it was apparently depressed. The average excretion during the fasting period was 18.5 (S.E. 2.32). Hypoxanthine and xanthine excretion were also independent of the experimental treatment except during the fasting period, where there was a significant increase in the urinary excretion of hypoxanthine (P , 0.05). Average basal excretion for hypoxanthine and xanthine were 50.3 (S.E. 7.68) and 4.7 (S.E. 0.6), respectively. Basal excretion of total PD during fasting was 202 (S.E. 12.97) mmol / W 0.75 constituting the allantoin the main proportion, 63.7%, followed by hypoxanthine 24.9%, uric acid 9.1% and xanthine 2.3%. Creatinine excretion averaged 266.8 (S.E. 20.88) mmol / W 0.75 and was not affected by food restriction.

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Fig. 1. Daily excretion (mmol / W 0.75 ) of allantoin (d), uric acid ( ), xanthine (j) plus hypoxanthine ( ) in milk selected goats during prefasting and fasting periods. See Section 3.1 for details.

Table 1 Urinary excretion of purine derivatives (mmol / W 0.75 ) in milk selected goats prior to fasting, early fasting (60% restriction), mid-fasting (30% restriction) and fasting

Prior fasting Early fasting Mid-fasting Fasting

Allantoin

Uric acid

Hypoxanthine

Xanthine

Total PD

620.9658.82 435.7631.69 244.6622.74 128.869.24

37.765.89 8.762.88 11.163.31 18.562.32

19.665.72 31.165.82 16.564.21 50.367.68

26.8610.18 11.063.97 7.061.93 4.760.60

700.8656.51 486.4631.19 279.1624.34 202.2612.97

Therefore, PD to creatinine ratio in urine samples decreased significantly from 2.94 to 0.81 mol / mol.

3.2. Urinary recovery of abomasal purine bases supply The continuous infusion of RNA solution was well accepted for the three animals and no apparent

changes in food behaviour was observed, even though goats are very sensitive to any change in the environment and they could change their daily behaviour without apparent experimental reason. Indeed, such changes that happen at random would affect the basal (dietary) flow of abomasal purine bases and it would be reflected in an increase in the residual variation.


RNA-infusion protocol was designed attending to the previous experience in relation to the reduced interval between urinary output and duodenal PB input observed in sheep (Balcells et al., 1991) and cows (Orellana Boero et al., 2001). Considering the 5-day measurement period, the first two were discarded and the mean of the last 3 days of measurements have been used to represent the urinary excretion for the corresponding level of purine infusion. Allantoin and total PD excretion responded rapidly to changes in level of RNA infusion and urinary recovery of abomasaly infused purines is presented in Table 2. For the three levels of RNA infusion recoveries were respectively 0.87, 0.63 and 0.80, averaging 0.76 (S.E. 0.062), that was taken as the reference value. Most of the registered variations in PD excretion were as allantoin, the principal PD, that represented between 82 and 90% of total excreted PD independently of the experimental treatment. No significant changes were observed in uric acid, although its proportion decreased apparently (6.42, 4.74, 4.16 and 3.61% for the basal flow and three levels of PB infusion). The remaining PD (between 2 and 5%) were excreted as salvageable compounds (xanthine plus hypoxanthine) and were also not affected by exogenous PB availability.

3.3. Xanthine oxidase activity in liver, intestinal mucosa and plasma Fig. 2 shows the production of uric acid when xanthine was incubated with plasma or tissues extracts from goats, as an indication of XO activities. The calculated XO activities are presented in Table 3. Liver (0.12 units / g) showed a much higher

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activity than plasma (0.001 units / ml) and scarce activity was found in gut mucosa (0.0009 units / g).

3.4. Chemical composition of microbes extracted from rumen goat Protein concentration (CP) in rumen bacteria extracted from the rumen liquid phase was 52.43% (S.E. 0.1675), being the concentration (mmol / g DM) of adenine 68.64 (S.E. 1.9) and guanine 96.19 (S.E. 2.18).

4. Discussion

4.1. Xanthine oxidase activity in liver, intestinal mucosa and plasma The authors are unaware of data of XO activity in tissues in adult goats, although Al-Khalidi and Chaglassian (1965) were not able to detect XO activity in goat blood. In relation to other species, profile of XO activities was similar to that described in sheep, low activity in plasma and gut, medium in liver (Chen et al., 1990a), whereas those activities described in cows tissues (Chen et al., 1990b), zebu cattle (Ojeda and Parra, 2000) or buffalo (Chen et al., 1996) were much higher. A low XO activity in goats intestine, plasma and liver would suggest that exogenous (digestive) purine bases can pass through the gastrointestinal tract being thus available for direct incorporation into tissue nucleotide by purine salvage pathway. Sheep also has a low activity in intestinal mucosa, and the presence in significant amounts of salvageable purine derivatives has been demonstrated in portal and

Table 2 Daily infusion of microbial nucleic acid (RNA, mmol / day) and urinary excretion of purine derivatives (PD, mmol / day) in three adult Murciano–Granadina goats feed at maintenance level PB-yeast RNA

0 10.3 13.7 17.1 †

Recovery †

mmol / day PD excretion Goat 1

Goat 2

Goat 3

10.7161.674 18.8563.869 20.4961.348 23.2862.029

13.9961.097 27.8664.963 23.9161.684 29.8661.021

9.5760.629 16.1461.396 116.4960.791 –

Increases in PD excretion / increases in duodenal PB flow.

– 0.87 0.63 0.80

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Fig. 2. Uric acid production from xanthine incubated from plasma (j), liver extracts (d) or intestinal mucosa (h) in milk-selected goats. Values are means for three animals with standard deviations indicated by vertical bars.

Table 3 Activities of xanthine oxidase (EC 1.2.3.2; XO) in plasma, liver and intestinal mucosa of goats Animals

Plasma (units / ml)

Liver (units / g)

Duodenum (units / g)

1 2 3 Mean S.E.

0.00101 0.00080 0.00118 0.001025 0.000095

0.06 0.14 0.15 0.117 0.021

0.0010 0.0013 0.0004 0.0009 0.00026

peripheral blood (Balcells et al., 1992). The urinary excretion of significant amounts of salvageable PD (xanthine, hypoxanthine) would confirm the availability of such compounds to the peripheral tissues in goats. A low XO activity in the intestinal mucosa, but also in liver and plasma, determines a low range of PD-irreversible oxidation of tissue nucleotides and therefore it would justify the low level of endogenous excretion (mmol / W 0.75 ) determined in goats (202), similar to sheep [158 (Balcells et al., 1991)], but lower than cattle [240 and 510; (Orellana Boero et al., 2001) and (Chen et al., 1990b), respectively].

4.2. Urinary excretion of purine derivative at different levels of abomasal PB supply The amount of basal losses of PD (11.34 mg-N / W 0.75 ) during fasting were consistent, but slightly higher than values reported by Fujihara et al. (1991) (8.2 mg-N / W 0.75 ). A similar trend was observed between the levels of allantoin excretion 128.8 vs. 92.79 mmol / W 0.75 . Although no changes in urinary proportion of allantoin precursors (uric acid plus hypoxanthine and xanthine) were reported by those authors with food restriction. Closer values were reported by Lindberg (1989) in milk-fed goat kids,


with a urinary excretion of total PD of 242 mmol / W 0.75 , constituting allantoin the principal proportion 60% followed by hypoxanthine 26%, uric acid 13%, and not detecting xanthine in urine samples. In relation to other species, basal excretion in goats seems to show a similar behaviour than sheep; fasting values obtained simultaneously in both species were quite similar [7 mg-N / W 0.75 in sheep vs. 8.2 mg-N / W 0.75 in goats; (Fujihara et al., 1991)]. The recorded basal values (202 mmol / W 0.75 ) are similar to those obtained in adult sheep using different methodology [177–202 mmol / W 0.75 ; (Giesecke et al., 1984; Lindberg and Jacobson, 1990; Balcells et al., 1991)]. Indeed, values are much lower than those values obtained in cattle even when data obtained with the same methodology are compared (Blaxter and Wood, 1951). In ruminants, most of the PD excreted in urine comes from partial metabolism of microbial nucleic acid absorbed into the duodenum. However, a significant fraction of the urinary PD originates from the endogenous nucleic acid turnover and that fraction is defined as the urinary PD excretion when there is no duodenal flow (or absorption) of NA. During fasting evidence does not exist that duodenal flow of PB is stopped, although this would be the method of choice to reduce exogenous rumen output of PB when there is no other methodology available, and in such a way it has been recently applied to buffaloes (Chen et al., 1996), Zebu cattle (Liang et al., 1999) and Zebu crossbreed animals (Ojeda and Parra, 2000). In this sense, in sheep, endogenous values obtained in fasting animals (7.0 mg-N / W 0.75 ) were not too dissimilar from those values obtained in intragastric fed animals [9.8 mg-N / W 0.75 ; (Fujihara et al., 1991)]. Results obtained in fasting Kedah– Kelantan cattle (274 mmol / W 0.75 ; Liang et al., 1999) were also similar to those values reported by Verbic et al. (1990) and Orellana Boero et al. (2001) in intragastric fed steers (348) or by labelling exogenous PB in cows (235), respectively. Thus, with the caution previously cited, fasting excretion could be taken as a proximate estimation of endogenous losses when no other method is available. A close relationship was found between urinary excretion of purine derivatives and duodenal supply of purines, as previously reported in other species as sheep (Chen et al., 1990a; Balcells et al., 1991) or

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cattle (Orellana Boero et al., 2001). That response was mostly explained by allantoin, the contribution of allantoin precursors (uric acid, xanthine and hypoxanthine) was independent of the experimental treatment. In agreement with our results, allantoin accounts for most of the variation in PD excretion in goat kids (Lindberg, 1991; Fujihara et al., 1994), although in those papers uric acid also responded significantly to the experimental treatment. Urinary recovery of abomasaly infused purines (0.76; S.E. 0.062) was lower than those values reported by Lindberg (1991) in milk-fed goat kids (from 127 to 74% in females and from 95 to 74% in males). Probably, the age of the animals and the methodology of the approach may explain most of the existing variations. Results, however, are similar to those obtained in sheep 0.84–0.80 (Chen et al., 1990b; Balcells et al., 1991) and cows 0.77–0.73 (Verbic et al., 1990; Beckers and Thewis, 1994). The results presented in relation to the low level of XO, the appearance of salvageable PD (xanthine plus hypoxanthine) in urine samples and also the incorporation level in different tissues (intestinal mucosa, liver, muscle and spleen) obtained in lactating goats (Ozdemir Baber, unpublished results) seem to indicate a similar behaviour in the response model between goats and sheep in relation to PD metabolism. In sheep, this relationship has been shown to be curvilinear (Chen et al., 1990a; Balcells et al., 1991) and it would reflect the degree of biochemical feedback on de novo synthesis process by the salvage of absorbed exogenous purines by tissues (Nolan, 1999). However, these factors are likely to be affected when the absorption of purine compounds is low, the endogenous component being negligible (taken as zero) when the feeding level of the animals increased from sub-maintenance to maintenance level (Chen et al., 1990a). Similar conclusions were reported in sheep also by Balcells et al. (1991). If that is accepted, the amount of absorbed purines (X mmol / day) can simply be estimated as PD excretion (Y) / Incremental recovery (0.76), then: X 5 Y / 0.76 Assuming that absorbed purines (X) are equal to PD excreted and that the relation purine bases: total

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N coming from the microbial flow out from the rumen is constant and similar to the liquid population of the rumen compartment, Microbial Nitrogen (MN) could be calculated as follows: MN (g / day) 5 X /(0.92 3 1.97) where 0.92 is the true digestibility of duodenal PB (Chen et al., 1990a) and 1.97 (mmol PB / g N) the ratio between purine bases (164 mmol / g DM) and N (83.8 mg / g DM) content in microbial population extracted from rumen of goats.

5. Conclusion This work has defined the relationship between intestinal supply of purine bases and the urinary excretion of their derivatives. Urinary recovery of abomasal purine is adjusted to a factor of 0.76 accounting to non-renal losses for 0.24. Tissue activity of XO, endogenous excretion and also PD distribution in urine sample seems to suggest that purine metabolism in adult goats is similar to that described previously in sheep.

Acknowledgements This work has been supported by the projects AGL2001-0941-CO2-02 and OLI96-2162-CO2-01. The authors wish to thank M. Fondevila Camps for the critical review of this manuscript.

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