Purine salvage as metabolite and energy saving mechanism in Camelus dromedarius: The recovery of guanine

Comp. Biochem. Physiol. Vol. 87B, No. 1, pp. 157-160, 1987 Printed in Great Britain

0305-0491/87 $3.00+0.00 © 1987 PergamonJournals Ltd

PURINE SALVAGE AS METABOLITE A N D ENERGY SAVING MECHANISM IN CAMELUS DROMEDARIUS: THE RECOVERY OF GUANINE* UMBERTO MURA, AHMED MOHAMUDOSMAN,i" ABDULLAHI SHECK MOHAMED,~" DARIO DI MARTINO and PIeR LUIGI IPATA Department of Physiology and Biochemistry, University of Pisa, via S. Maria, 55-56100 Pisa, Italy and i'Department of Physiology, Faculty of Veterinary Medicine, National University of Somalia, P.O. Box 1738, Mogadishu, Somalia (Received 27 June 1986) Abstract--1. The preservation of purine ring as purine bases appears to be a common feature of camel liver. 2. Hepatic guanine appears to be actively converted into GMP in the camel rather than further degraded. The limiting step of guanine degradation appears to be the lack of hepatic guanase activity. 3. Higher purine bases over uric acid ratios were found in camel urine with respect to those of zebu.

salvaged for anabolic purposes. These observations are in line with the surprisingly high hypoxanthine over uric acid ratios found by us in plasma as well as in urine samples of the camel (Mura et al., 1986). We show here that the features underlying hypoxanthine production and utilization in the camel may be extended to the overall purine turnover, since they hold true also for guanine.

INTRODUCTION It has been clear for centuries that the camel has a degree of independence of water greater than other domestic animals. The first report is that of Aristotle (384-322 BC), who stated more than two thousand years ago that the camel could survive a 4 days period of water deprivation, an underestimate repeated later by Pliny (77 AD). In more recent times, several aspects of the physiological architecture of the camel, such as water turnover (Maefarlane et aL, 1963; Schmidt-Nielsen et al., 1956, 1981a, 1981b), heat storage (SchmidtNielsen et al., 1957a, 1967), polysaccharide metabolism (Chandrasena et al., 1979; Abdalla and Mutasim, 1981; Mutasim and Abdalla, 1982; Kumar et al., 1962; Mura et al., 1985) and blood properties (Banerjee et al., 1962; Banerjee and Bhown, 1964; Eitan et al., 1976; John, 1982; Lewis, 1976; L i n e t al., 1976; Livne and Kuiper, 1973; Ralston, 1975; Yagil et al., 1974) have been investigated. In addition, the capability to store metabolic energy (Gunstone and Patton, 1954) and to preserve nitrogen (SchmidtNielsen et al., 1957b) might contribute to the well documented resistance of the camel to hard environmental conditions (see also Gauthier-Pilters and Dagg, 1981; Yagil, 1985). The retention of purine compounds to minimize cellular metabolism during a fast is a feature already assessed in ruminants (Morris and Ray, 1939). By comparing the levels of liver enzymes of purine degradation in the dromedary and in the zebu bred in Somalia, we have recently shown for the camel a rather low rate of purine catabolism. Moreover the very low hepatic xanthine oxidase activity limits the adenylate degradation, allowing the preservation of hypoxanthine, a molecular species which can be

MATERIALS AND METHODS

Chemicals Purine nucleotides, nucleosides, bases, P R P P and R-I-P were obtained from Sigma Chemical Company. All other chemicals were of reagent grade and were used without further purification.

Extracts preparation Liver samples of both camel and zebu collected from freshly slaughtered animals, in the Public Slaughterhouse of Mogadishu, washed by cold 0.15 M NaC1 and frozen in liquid nitrogen, were thawed and homogenized (2.4 g/10 ml) in 0.2M phosphate buffer pH7 containing 20mM 2-mercaptoethanol. The supematant at 25,000g for 30 min at 4°C was fractionated by ammonium sulphate. The precipitate obtained between 35 and 70% of salt saturation was resuspended in the minimal volume of 50 mM phosphate buffer pH 7.5 containing 20mM 2-mercaptoethanol and dialyzed against 0.15M NaC1 plus 10mM 2-mercaptoethanol. The supematant at 20,000g for 20 min at 4°C, was referred to as "crude extract". Enzyme activity Guanase activity was measured at 37°C in 0.1 M Tris-HC1 buffer pH 7.5, following the decrease in absorbance at 246 nm associated with the conversion of guanine into xanthine. No appreciable interference of xanthine oxidase activity was observed since the wavelength used in the assay is an isosbestic point of xanthine and uric acid.

*Abbreviations: PRPP, 5-phosphoribosyl-l-pyrophosphate; RIP, ~tDribose-l-Phosphate; Hyp, hypoxanthine; Xn, xanthine; Gua, guanine; Guanosine; HGPRT, hypoxanthine, guanine: phosphoribosyl transferase.

HPLC analysis of purine bases in urine Urine samples of zebu and the 1 to 5 water diluted urine samples of dromedary were heated for 2 min at 80°C, cooled to room temperature and then filtered through mixed ester cellulosefilters (Amicon, 0.2 micron pore size). One hundred

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UMBERTO MuP.A et al.

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Fig. 1. Guanase activity in liver extracts. Fifty/~ M guanine was incubated in a cuvette with either camel (O) or zebu ( x ) liver crude extract (0.3 mg/ml final protein concentration) and the absorbance at 246 nm recorded.

#1 of filtered samples were applied on I ml Bond Elut C18 columns and eluted by two times 200 #1 aliquots of 50 mM potassium phosphate pH2.75 containing 3% methanol. Twenty #1 of the eluted samples were applied on a reversed phase Spherisorb $50DS2 column and eluted by 50 mM potassium phosphate pH 2.75 (1.5 ml/min flux and 2500 psi). The elution peaks were monitored at 254 nm and analyzed.

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camel

[ ] zebu

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Fig. 3. Ribose-l-phosphate dependent fate of guanine in liver extracts. Fifty # M guanine was incubated at 37°C with either camel or zebu liver extract in the presence of 2 mM RIP. For details of the incubation conditions and analysis of the products, see legend of Fig. 2.

HPLC analysis of guanine derivatives in incubation mixtures of liver extracts

Incubation mixtures of either zebu or camel liver extracts heated for 2 min at 100°C, were centrifuged and the supernatant filtered through a Centricon-30 micro concentrator (Amicon). One hundred and fifty #1 of the filtered samples were applied on I ml Bond Elut C18 columns (Analytichem International) and eluted by two times 200#1 aliquots of 0.25 M ammonium acetate pH 6 containing 25% methanol. The samples were then evaporated to dryness with a centrifugal evaporator (Speedvac, Savant) and resuspended by 100/zl of water. Twenty #1 of the resuspended sample was applied by a Beckman rood. 332 equipment, on a reversed phase Spherisorb $50DS2 column (Phase Sop) and eluted by 50 mM potassium phosphate pH 2.75 (8 rain) followed by a linear 0-20% methanol gradient (10 rain) (1.3 ml/min flux and 2500 psi). The elution peaks were monitored at 254 nm (Beckman rood. 153) and analyzed by Chromatopac CR3A integrator (Shimadzu).

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Guo

Fig. 2. PRPP dependent fate of guanine in liver extracts. Fifty # M guanine was incubated at 37°C in the presence of 2.5mM PRPP with either camel or zebu liver extract (3 mg/ml final protein concentration) in 40 mM Tris-HCl buffer pH7.8 supplemented with 0.4mM MgCI2 and 1.6 mM of dithiothreitol (final volume 0.5 ml). After 5 min of incubation the mixtures were heated for 2 rain at 100°C and analyzed by HPLC as described in the materials and methods section. Results are reported as the relative contents (%) of guanine derivatives (Gua, Guo, GMP, Xn, uric acid).

3/ "~"Hyp I I I

~ G u a ~ I I

xn~ UriSc~ a c i d Fig. 4. Scheme of purine nucleotide monophosphate catabolism and salvage in camel and zebu liver extracts. (1) 5'-nucleotidase; (2) Ado deaminase; (3) purine nucleoside phosphorylasc; (4) HGPRT; (5) xanthine oxidase; (6) guanase. The bar indicates the block of the catabolic process of purine bases due to the virtual lack of xanthine oxidase and guanase in the camel liver extract.

Purinc salvage in the camel RESULTS AND DISCUSSION The results shown in Fig. 1 show that camel liver is virtually devoid of any detectable guanase activity, even though guanine may be formed from G M P through the sequential action of 5'-nucleotidase and purine nucleoside pbosphorylase (Mura et al., 1986). In contrast, guanine is actively deaminated in zebu liver where xanthine, the reaction product of guanase action, is readily oxidized to uric acid by a potent xanthine oxidase activity. The lack of guanase activity in camel liver would determine a preservation of the purine base for further catabolism. This situation is quite similar to

159

that observed in this animal for hypoxanthine in the hepatic adenylat¢ degradative pathway. In that case, even though slightly detectable in the serum (AlKhalidi and Changlassian, 1965), xanthine oxidase could not be detected in camel liver extracts as well as in other organs tested by us in conditions revealing a marked oxidase activity in zebu liver. As a consequence of such limiting catabolic step, hypoxanthine in camel liver becomes much easily available to be salvaged. The purine base is readily reconverted to IMP in the presence of PRPP through the action of H G P R T rather than being oxidized to uric acid (Mura et al., 1986).

Hyp i

A

.Uric acid

Xn_. ®

rain

Gua

B

Hyp "L Xn__.~, ...............

Uric acid ~-. - . . . .

I

G~a i

0

4

unit

8

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Fig. 5. Typical chromatographic separation by reversed phase HPLC of purine bases and uric acid in camel (A) and zebu (B) urine: (I) Gua; (2) uric acid; (3) Hyp; (4) Xn. On the right the relative content of Hyp, Gua and Xn with respect to uric acid (unit) is depicted on three independent axes. The shaded areas represent the standard deviation of the mean evaluated on 20 camel samples and 10 zebu samples, where uric acid level ranged between 0.12-0.78 and 0.05-2.8mM respectively. The chromatographic analysis was performed as described in the Materials and Methods section. The elution peaks were monitored at 254nm with 0.16 and 0.64AUFS for A and B respectively.

UMBERTO MURAet al.

160

A higher ratio of uric acid over various guanine derivatives was observed in zebu with respect to camel when liver extracts were incubated with guanine in the presence of either R I P or PRPP. A remarkable difference in the relative content of uric acid and G M P is observed between the two species, in conditions favoring the PRPP-dependent guanine salvage (Fig. 2). G M P mainly accumulates in incubation with camel extracts, while uric acid is the main product formed with zebu extracts. Guanine was less utilized by camel extracts, when PRPP was substituted by R I P (Fig. 3) As expected, in this condition there was an increase of guanosine and a decrease of GMP formation in both animal species. However, uric acid still accumulated in incubations with zebu extracts. It is evident that in zebu the metabolic step of guanine deamination actively competes with the salvage of the purine base which may be mediated either by H G P R T or by nucleoside formation through purine nucleoside phosphorylase (see Fig. 4). In line with these observations, the HPLC analyses of camel and zebu urine samples evidence that the pattern of purine base excretion of the two animal species is markedly different. The results reported in Fig. 5, as purine bases over uric acid ratios, evidence for the camel a higher value of both hypoxanthine and guanine over uric acid, reflecting the observed metabolic block at the level of xanthine oxidase and guanase. Overall it appears that camel tends to minimize the metabolite and energy expenditure needed to synthesize purine nucleotides "ex novo" (5mol of ATP/mol of purine ring) both by reducing the purine catabolism rate and by recycling preformed purine bases. Circulating purine bases could represent a reservoir of metabolites the availability of which in conditions of nutrient limitation, may contribute to the special feature of the camel as an animal compatible with desert life. Acknowledgements--We thank Prof. A. Mura-Falcone and Mr. G. Falcone for historical bibliographic research. This work was supported by a grant from the Italian C.N.R. and Board of Education.

REFERENCES

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