urinary excretion of purine derivatives and prediction of rumen microbial outflow in goats

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

U rinary excretion of purine derivatives and prediction of rumenmicrobial outflow in goatsa b a , c*˜A. Belenguer , D. Yanez , J. Balcells , N.H. Ozdemir Baber ,

aM. Gonzalez Ronquilloa ´Departamento de Produccion Animal y Ciencia de los Alimentos. Facultad de Veterinaria. C / Miguel Servet 177, Zaragoza, Spain

b ´ ´Unidad Nutricion Animal. Estacion Experimental Zaidin. C.S.I.C. Camino del Jueves s /n- 18100 Armilla, Granada, SpaincAnkara Nuclear Agriculture and Animal Science Research Center. 06983Saray (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 theirderivatives (i.e., Allantoin, uric acid, xanthine and hypoxanthine) in selected milk goats. Three adult Granadina goats fittedwith a T-shaped cannula in the abomasum were used to determine the endogenous contribution to renal excretion of purinederivatives and urinary recovery of abomasaly infused purine bases as yeast-RNA. Animals were fed alfalfa at maintenance

0.75level. The endogenous contribution of purine derivatives was determined at fasting (11.34 mg N/W or 202.20.75

mmol /W ) and it was similar to that obtained in sheep but lower than that reported in cattle. Urinary PD excretionresponded 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 andin duodenum 0.0009 (S.E. 0.00026) i.u. /g. Again, activities were lower than those detected in cows but close to valuesdetermined 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 efficiently absorbed and the majority of their deriva-tives excreted via the kidney. The ratio of PD in

Urinary purine derivatives (PD) excretion is used urine closely reflects, and therefore may be used toto predict rumen microbial protein synthesis in predict, microbial protein flow.ruminant livestock. The principle is that duodenal However, the response model between urinary PDpurine bases (PB), as a microbial marker, are and duodenal flow of PB differs among species.

Significant variations between cattle and sheep havebeen described (Chen et al., 1990b), but also within*Corresponding author. Tel.:134-976-761-660; fax:134-976-cattle species [European-Bos Taurus vs. Zebu Cattle-761-590.

E-mail address: [email protected](J. Balcells). Bos Indicus; (Liang et al., 1994)]. It is desirable to

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

mailto:[email protected]

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

extend this technique to other species of economic 2 .1.4. Urinary recovery of abomasal PBsignificance, such as goat (capra hircus), to develop Animals were given four doses of nucleic acidsspecific models for the prediction of microbial (NA) that were infused continuously (12 h per day)protein production in the rumen (Stangassinger et al., over four successive 5-day periods. RNA yeast1995). (Torula yeast Sigma Co., St. Louis, USA) was used

The aim of this work was to establish the response as a NA source and the doses were, respectively 0,0.75model in Murciano–Granadina goats by quantifying 0.63, 0.84 and 1.05 (mmol PB/kg W ), and were

the basal or endogenous contribution to urinary PD supplied following a 334 complete crossover de-excretion after minimizing physiological rumen flow sign. These doses correspond to the amounts ofof microbial purine bases by fasting and by relating microbial NA (or PB) likely to be produced whenthe abomasal input and urinary output after abomasal animals are normally fed between zero and threeinfusion of different doses of nucleic acids (NA). times the maintenance energy requirements.The activity of the enzyme xanthine-oxidase (XO; RNA (1.1 mmol PB/g) solution was prepared byEC 1.2.3.2.) in different tissues as a key step for diluting the yeast doses in 0.5 M NaOH. Once thepurine metabolism was also determined. yeast was diluted, the pH was brought to 3.5–4.5

using 10% HCl. The RNA-solution was continuouslyinfused at a flow rate of 0.7 ml /min using aperistaltic pump (Minipuls-2 HP 18 Gilson).

2 . Material and methods

2 .1.5. Sample collection2 .1. Experiment 1: urinary excretion of purine In both trials, total urine excretion was collectedderivatives in relation to abomasal supply of daily over 10% sulphuric acid (final pH of urine waspurine bases kept below 3). Urine was weighed, its density

measured and urine samples (100 ml) were frozen2 .1.1. Animals and diets immediately at2 20 8C until analysis.

Three adult Murciano–Granadina goats (41.6 kgLW; S.E. 4.53), were fitted with a T-shaped abomas- 2 .2. Experiment 2: xanthine oxidase activity inal cannula a month before the experimental pro- plasma, liver or intestinal mucosacedure began. Animals were kept in metabolic cageswith free access to drinking water. They were fed Samples of plasma, liver and intestine were takenalfalfa hay at a maintenance level (443 kJ ME/ from the local slaughterhouse (samples of tissues0.75W ; Prieto et al., 1990). were taken from the same three animals at slaughter,

and plasma was taken just before slaughter). Threeanimals, different from those used in Experiment 1,2 .1.2. Experimental procedureswere chosen at random regardless of age, sex orThe experiment was divided into two phases:previous nutrition.initially the basal excretion was determined (20

days), and after that (30 days) when animals were2 .2.1. Preparation of tissue extractsrecovered, the relationship between abomasal input

Blood samples were collected into heparinizedand urinary output was established (20 days).tubes and centrifuged at 30003 g for 15 min. Plasmasamples were analysed within 6 h. Procedures for

2 .1.3. Fasting trial extraction of liver samples and the extraction of theAnimals were penned individually in metabolic intestinal mucosa layer were adapted from those

cages and fed at a maintenance level for 10 days described by Furth-Walker and Amy (1987) and(adaptation period). After that, feed intake was Reeds et al. (1997), respectively.restricted every 2 days from 100 to 60 and 30% Liver and kidney were washed in cold 0.15 M(prior, mid and early fasting) of the previous mainte- KCl, and 1 g of tissue was homogenised in 9 ml 0.5nance feeding level. After that, a 6-day fasting period mM EDTA in 0.05 M K HPO (pH 7.5) and2 4

was commenced. centrifuged at 35 0003 g for 30 min at 48C. Super-

A. Belenguer et al. / Livestock Production Science 77 (2002) 127–135 129

natant fraction was dialysed 24 h against the same procedure after acid hydrolysis as described by´ ´EDTA–KH PO buffer for 24 h and centrifuged Martın Orue et al. (1995).2 4

again at 35 0003 g for 30 min at 48C. The superna-tant fraction was used for the assay. Intestinal 2 .4. Statistical analysissamples were taken from the duodenum and lumenand washed with cold 0.15 M KCl. After that, Analyses of variance and the simple linear regres-samples were frozen immediately in liquid nitrogen sion were performed following the procedures de-and defrosted within 2 h at 48C. Then lumen was scribed by Steel and Torrie (1980).washed with 0.05 MN-2-hydroxyethylpiperazine-N-2-ethanosulphonic acid (HEPES) buffer (pH 7.5)containing 0.25 mM EDTA and 0.25 mM phenyl-

3 . Resultsmethylsulphonyl fluoride (PMSF). One gram ofintestinal mucosa was removed by finger pressure

Animals remained in good health throughout thealong the portion of intestine, and the mucosa layerexperiment and they recovered properly and fullycells were collected at the bottom in 9 ml HEPES–consumed the experimental diets 1 week after theEDTA–PMSF buffer. The extract containing thesurgery.enzyme XO was then purified as liver samples but

using HEPES–EDTA–PMSF buffer.3 .1. Urinary excretion of purine derivative andcreatinine during fasting2 .2.2. Xanthine oxidase activity

Activity of xanthine oxidase was measured as theThe individual values for daily excretion of purinerate of uric acid produced when xanthine was

derivatives and mean values are shown in Fig. 1 andincubated with tissue extracts, as described by ChenTable 1, respectively. Allantoin excretion (mmol /et al. (1996). 0.75W ) decreased significantly with food restriction,reaching the minimum value during fasting (from

2 .2.3. Relationship between PB and N in rumen 620.9 to 128.8). After that, it remained fairly con-microorganisms extract stant. Basal excretion (fasting excretion) averaged

Samples for isolation of microbial extract were 128.8 (S.E. 9.24) ranging from 144.2 to 90.9.also taken from the same three animals at the Between animal and day-to-day variations contribu-slaughterhouse. Rumen samples (200 ml) were ted to the total variance by 22 and 19%, respectively.

0.75squeezed through four layers of surgical gauze and Uric acid excretion (mmol /W ) was not sig-the bacterial fraction was isolated by differential nificantly affected by food restriction, although dur-centrifugation (5003 g for 10 min). It was used to ing early and mid-fasting it was apparently de-precipitate the particulate material and resulting pressed. The average excretion during the fastingsupernatant was centrifuged at 20 0003 g for 20 min period was 18.5 (S.E. 2.32). Hypoxanthine andat 48C to deposit the bacterial fraction. This deposit xanthine excretion were also independent of thewas then resuspended in physiological saline solution experimental treatment except during the fastingand again centrifuged at 20 0003 g for 20 min at period, where there was a significant increase in the4 8C. The washed microbial pellet was freeze-dried urinary excretion of hypoxanthine (P ,0.05). Aver-for subsequent analyses. age basal excretion for hypoxanthine and xanthine

were 50.3 (S.E. 7.68) and 4.7 (S.E. 0.6), respective-2 .3. Analytical procedure ly.

Basal excretion of total PD during fasting was 2020.75Urine samples were centrifuged, diluted (1:100), (S.E. 12.97)mmol /W constituting the allantoin

filtered (0.2 mm Millipore Bedford MS) and ana- the main proportion, 63.7%, followed by hypoxan-lysed for PD and creatinine following the procedure thine 24.9%, uric acid 9.1% and xanthine 2.3%.described by Balcells et al. (1992). PB in RNA-yeast Creatinine excretion averaged 266.8 (S.E. 20.88)

0.75and rumen bacteria were analysed with the same mmol /W and was not affected by food restriction.


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

Table 10.75Urinary excretion of purine derivatives (mmol /W ) in milk selected goats prior to fasting, early fasting (60% restriction), mid-fasting

(30% restriction) and fasting

Allantoin Uric acid Hypoxanthine Xanthine Total PD

Prior fasting 620.9658.82 37.765.89 19.665.72 26.8610.18 700.8656.51Early fasting 435.7631.69 8.762.88 31.165.82 11.063.97 486.4631.19Mid-fasting 244.6622.74 11.163.31 16.564.21 7.061.93 279.1624.34Fasting 128.869.24 18.562.32 50.367.68 4.760.60 202.2612.97

Therefore, PD to creatinine ratio in urine samples changes in food behaviour was observed, evendecreased significantly from 2.94 to 0.81 mol /mol. though goats are very sensitive to any change in the

environment and they could change their daily3 .2. Urinary recovery of abomasal purine bases behaviour without apparent experimental reason.supply Indeed, such changes that happen at random would

affect the basal (dietary) flow of abomasal purineThe continuous infusion of RNA solution was well bases and it would be reflected in an increase in the

accepted for the three animals and no apparent residual variation.


RNA-infusion protocol was designed attending to activity than plasma (0.001 units /ml) and scarcethe previous experience in relation to the reduced activity was found in gut mucosa (0.0009 units /g).interval between urinary output and duodenal PBinput observed in sheep (Balcells et al., 1991) and 3 .4. Chemical composition of microbes extractedcows (Orellana Boero et al., 2001). Considering the from rumen goat5-day measurement period, the first two were dis-carded and the mean of the last 3 days of measure- Protein concentration (CP) in rumen bacteriaments have been used to represent the urinary extracted from the rumen liquid phase was 52.43%excretion for the corresponding level of purine (S.E. 0.1675), being the concentration (mmol /g DM)infusion. Allantoin and total PD excretion responded of adenine 68.64 (S.E. 1.9) and guanine 96.19 (S.E.rapidly to changes in level of RNA infusion and 2.18).urinary recovery of abomasaly infused purines ispresented in Table 2. For the three levels of RNAinfusion recoveries were respectively 0.87, 0.63 and 4 . Discussion0.80, averaging 0.76 (S.E. 0.062), that was taken asthe reference value. 4 .1. Xanthine oxidase activity in liver, intestinal

Most of the registered variations in PD excretion mucosa and plasmawere as allantoin, the principal PD, that representedbetween 82 and 90% of total excreted PD indepen- The authors are unaware of data of XO activity indently of the experimental treatment. No significant tissues in adult goats, although Al-Khalidi andchanges were observed in uric acid, although its Chaglassian (1965) were not able to detect XOproportion decreased apparently (6.42, 4.74, 4.16 and activity in goat blood. In relation to other species,3.61% for the basal flow and three levels of PB profile of XO activities was similar to that describedinfusion). The remaining PD (between 2 and 5%) in sheep, low activity in plasma and gut, medium inwere excreted as salvageable compounds (xanthine liver (Chen et al., 1990a), whereas those activitiesplus hypoxanthine) and were also not affected by described in cows tissues (Chen et al., 1990b), zebuexogenous PB availability. cattle (Ojeda and Parra, 2000) or buffalo (Chen et

al., 1996) were much higher.3 .3. Xanthine oxidase activity in liver, intestinal A low XO activity in goats intestine, plasma andmucosa and plasma liver would suggest that exogenous (digestive) purine

bases can pass through the gastrointestinal tractFig. 2 shows the production of uric acid when being thus available for direct incorporation into

xanthine was incubated with plasma or tissues tissue nucleotide by purine salvage pathway. Sheepextracts from goats, as an indication of XO activities. also has a low activity in intestinal mucosa, and theThe calculated XO activities are presented in Table presence in significant amounts of salvageable purine3. Liver (0.12 units /g) showed a much higher derivatives has been demonstrated in portal and

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

†PB-yeast RNA mmol /day PD excretion Recovery

Goat 1 Goat 2 Goat 3

0 10.7161.674 13.9961.097 9.5760.629 –10.3 18.8563.869 27.8664.963 16.1461.396 0.8713.7 20.4961.348 23.9161.684 116.4960.791 0.6317.1 23.2862.029 29.8661.021 – 0.80

†Increases in PD excretion/ increases in duodenal PB flow.


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 3Activities 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 0.00101 0.06 0.00102 0.00080 0.14 0.00133 0.00118 0.15 0.0004Mean 0.001025 0.117 0.0009S.E. 0.000095 0.021 0.00026

peripheral blood (Balcells et al., 1992). The urinary 4 .2. Urinary excretion of purine derivative atexcretion of significant amounts of salvageable PD different levels of abomasal PB supply(xanthine, hypoxanthine) would confirm the availa-bility of such compounds to the peripheral tissues in The amount of basal losses of PD (11.34 mg-N/

0.75goats. W ) during fasting were consistent, but slightlyA low XO activity in the intestinal mucosa, but higher than values reported by Fujihara et al. (1991)

0.75also in liver and plasma, determines a low range of (8.2 mg-N/W ). A similar trend was observedPD-irreversible oxidation of tissue nucleotides and between the levels of allantoin excretion 128.8 vs.

0.75therefore it would justify the low level of endogen- 92.79mmol /W . Although no changes in urinary0.75ous excretion (mmol /W ) determined in goats proportion of allantoin precursors (uric acid plus

(202), similar to sheep [158 (Balcells et al., 1991)], hypoxanthine and xanthine) were reported by thosebut lower than cattle [240 and 510; (Orellana Boero authors with food restriction. Closer values wereet al., 2001) and (Chen et al., 1990b), respectively]. reported by Lindberg (1989) in milk-fed goat kids,


with a urinary excretion of total PD of 242mmol / cattle (Orellana Boero et al., 2001). That response0.75W , constituting allantoin the principal proportion was mostly explained by allantoin, the contribution

60% followed by hypoxanthine 26%, uric acid 13%, of allantoin precursors (uric acid, xanthine andand not detecting xanthine in urine samples. hypoxanthine) was independent of the experimental

In relation to other species, basal excretion in treatment. In agreement with our results, allantoingoats seems to show a similar behaviour than sheep; accounts for most of the variation in PD excretion infasting values obtained simultaneously in both goat kids (Lindberg, 1991; Fujihara et al., 1994),

0.75species were quite similar [7 mg-N/W in sheep although in those papers uric acid also responded0.75vs. 8.2 mg-N/W in goats; (Fujihara et al., 1991)]. significantly to the experimental treatment.

0.75The recorded basal values (202mmol /W ) are Urinary recovery of abomasaly infused purinessimilar to those obtained in adult sheep using (0.76; S.E. 0.062) was lower than those values

0.75different methodology [177–202 mmol /W ; reported by Lindberg (1991) in milk-fed goat kids(Giesecke et al., 1984; Lindberg and Jacobson, 1990; (from 127 to 74% in females and from 95 to 74% inBalcells et al., 1991)]. Indeed, values are much lower males). Probably, the age of the animals and thethan those values obtained in cattle even when data methodology of the approach may explain most ofobtained with the same methodology are compared the existing variations. Results, however, are similar(Blaxter and Wood, 1951). to those obtained in sheep 0.84–0.80 (Chen et al.,

In ruminants, most of the PD excreted in urine 1990b; Balcells et al., 1991) and cows 0.77–0.73comes from partial metabolism of microbial nucleic (Verbic et al., 1990; Beckers and Thewis, 1994).acid absorbed into the duodenum. However, a sig- The results presented in relation to the low level ofnificant fraction of the urinary PD originates from XO, the appearance of salvageable PD (xanthine plusthe endogenous nucleic acid turnover and that frac- hypoxanthine) in urine samples and also the incorpo-tion is defined as the urinary PD excretion when ration level in different tissues (intestinal mucosa,there is no duodenal flow (or absorption) of NA. liver, muscle and spleen) obtained in lactating goats

During fasting evidence does not exist that duode- (Ozdemir Baber, unpublished results) seem to indi-nal flow of PB is stopped, although this would be the cate a similar behaviour in the response modelmethod of choice to reduce exogenous rumen output between goats and sheep in relation to PD metabo-of PB when there is no other methodology available, lism. In sheep, this relationship has been shown to beand in such a way it has been recently applied to curvilinear (Chen et al., 1990a; Balcells et al., 1991)buffaloes (Chen et al., 1996), Zebu cattle (Liang et and it would reflect the degree of biochemical feed-al., 1999) and Zebu crossbreed animals (Ojeda and back on de novo synthesis process by the salvage ofParra, 2000). In this sense, in sheep, endogenous absorbed exogenous purines by tissues (Nolan,

0.75values obtained in fasting animals (7.0 mg-N/W ) 1999).were not too dissimilar from those values obtained in However, these factors are likely to be affected

0.75intragastric fed animals [9.8 mg-N/W ; (Fujihara when the absorption of purine compounds is low, theet al., 1991)]. Results obtained in fasting Kedah– endogenous component being negligible (taken as

0.75Kelantan cattle (274mmol /W ; Liang et al., 1999) zero) when the feeding level of the animals increasedwere also similar to those values reported by Verbic from sub-maintenance to maintenance level (Chen etet al. (1990) and Orellana Boero et al. (2001) in al., 1990a). Similar conclusions were reported inintragastric fed steers (348) or by labelling exogen- sheep also by Balcells et al. (1991).ous PB in cows (235), respectively. Thus, with the If that is accepted, the amount of absorbed purinescaution previously cited, fasting excretion could be (X mmol /day) can simply be estimated as PDtaken as a proximate estimation of endogenous losses excretion (Y) / Incremental recovery (0.76), then:when no other method is available.

X 5 Y /0.76A close relationship was found between urinaryexcretion of purine derivatives and duodenal supplyof purines, as previously reported in other species as Assuming that absorbed purines (X) are equal tosheep (Chen et al., 1990a; Balcells et al., 1991) or PD excreted and that the relation purine bases: total


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urinary excretion of purine derivatives and prediction of rumen microbial outflow in goats

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