J. Inher. Metab. Dis. 15 (1992) 707-712 © SSIEM and KluwerAcademicPublishers. Printed in the Netherlands

Allopurinol Challenge Test in Children A. B. BURLINA1, V. FERRARI1, C. DIONISI-VIcI 2, A. BORDUGO1, F. ZACCHELLO1 and M. TUCHMAN3. 1 Department of Pediatrics, University of Padua, Padua, Italy; 2 Department of Metabolism, Bambino Gesu Children's Hospital, Rome, Italy; 3 Division of Genetics and Metabolism, Departments of Pediatrics and Laboratory Medicine and Pathology, Box 400 Mayo Memorial Building, 420 Delaware Street, SE, University of Minnesota, Minneapolis, M N 55455, USA

Summary: The allopurinol challenge test was performed on 44 healthy subjects (28 children and 16 adolescents) in order to establish normal values of urinary orotic acid excretion following allopurinol ingestion in the paediatric population. The subjects were divided into three groups according to their age: 6 months to 6 years; 6 years to 10 years; and 10 years to 17 years. They were given 100 rag, 200 mg, or 300 mg of atlopurinol, respectively (based on age) in a single oral dose. Maximum peak urinary orotic acid levels following ingestion of allopurinol were 13.0 (n = 14), 9.3 (n = 14), and 10.2 (n = 16) /~mol/mmol creatinine in the three groups, respectively. In all children tested the peak orotic acid level was 3.1 __ 2.7/tmol/mmol creatinine (mean _+ SD, n = 44). This allopurinol challenge test was also performed in six children with ureacycle disorders, including five females with ornithine transcarbamylase (OTC) deficiency, all of whom demonstrated abnormally elevated levels of urinary orotic acid (peak levels of 26-134#mol/mmol creatinine) following allopurinol ingestion. Patients with hyperammonaemia, due to various enzyme deficiencies of the urea cycle often show increased excretion of urinary orotic acid. The urea-cycle disorder most known to be associated with orotic aciduria is ornithine transcarbamylase (OTC) deficiency (EC 2.1.3.3) (McKusick 31125); however, patients with several other disorders related to the urea cycle also exhibit orotic aciduria (Bachmann et al 1980; Rajantie et al 1981; Simell et al 1985; Tuchman et al 1990). These disorders, which are also associated with specific abnormalities in the amino acid profile, are argininaemia (McKusick 20780), citrullinaemia (McKusick 21570), arginosuccinic aciduria (McKusick 20790), lysinuric protein intolerance (McKusick 22270) and hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) (McKusick 23897). The mechanism for orotic aciduria in these disorders is probably the shunting of unmetabolized mitochondrial carbamoyl phosphate into the pyrimidine synthesis pathway augmented by excess aspartate required for de novo synthesis of pyrimidines. *Correspondence MS received 7.10.91 Accepted 27.1.92 707

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A few other physiological and pathological conditions have been associated with orotic aciduria. These include pregnancy (Wood and O'Sullivan 1973), hereditary orotic aciduria and defects in folate metabolism (Suttle et al, 1989) and purine nucteoside phosphorylase deficiency (Cohen et at 1977). Protein loading tests have been used to induce orotic aciduria for diagnostic purposes in OTC-deficient females and in patients with intermittent clinical and biochemical abnormalities suggesting urea-cycle disorders (Hokanson et al, 1978). However, this procedure may give false-negative results and may be harmful because it can induce hyperammonaemia in individuals with urea-cycle defects (Becroft et al, 1984). Orotic aciduria and orotidinuria were observed in individuals treated with allopurinol (Fox et al 1970). Determination of urinary orotidine and orotic acid levels following ingestion of allopurinol has recently been shown to reliably predict female carrier status for OTC deficiency (Hauser et al 1990). However, the data provided in that publication were obtained exclusively from adult individuals, thus limiting the use of this test in children. While evaluating children with suspected urea-cycle disorders, we have encountered several instances in which performance of an allopurinol test would have been useful for establishing a diagnosis, especially for girls who are heterozygous for OTC deficiency. The interpretation of this test in children was difficult owing to the lack of normal values. This work was performed to establish normal values of urinary orotic acid following ingestion of allopurinol in children. We also demonstrate the usefulness of this test in several children with urea-cycle disorders.

METHODS

The normal values were obtained from children who were tested during a visit to a regional health screening programme in Padua and Rome, Italy. The study was approved by the University of Padua and by the Hospital Bambino Gesu in Rome. Consent of the parents to perform the test was obtained. All children studied were healthy at the time of testing. We studied 44 children aged 6 months to 17 years. They were divided into three age groups: (1) 14 children (7 males and 7 females) who were between 6 months and 6 years of age, (2) 14 children (7 males and 7 females) who were between the ages of 6 and 10 years; and (3) 16 children (3 males and 13 females) who were between the ages of 10 and 17 years. Dietary intake was kept constant throughout the test and they did not consume ethanol-, caffeine- or benzoatecontaining foods during the test period. No side-effects were observed following ingestion of a single dose of allopurinol. Five females with OTC deficiency and one male with HHH syndrome between the ages of 1 and 14 years were also tested. All of them were on therapy for hyperammonaemia and had normal urinary orotic acid levels prior to testing. A spot urine sample was collected immediately prior to the ingestion of a single oral dose of allopurinol containing 100, 200, or 300 mg for children in each of the three groups, respectively. Following allopurinol ingestion, four separate 6-h urine collections were obtained: from 0 to 6h, from 6 to 12h, from 12 to 18h, and from 18 to 24h after allopurinol was given. An aliquot was taken from each collection J. Inher. Metab. Dis. 15 (1992)

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and analysed. Orotic acid concentration was determined by a reversed-phase highperformance liquid chromatography (HPLC) method employing direct injection of urine into the column as described before (Ferrari et al 1989). Briefly, the method involves a procedure of cleaning the urine using a C-18 cartridge, acidification to pH 1.5-2.0 with hydrochloric acid, and injection of 25 #1 of urine into the HPLC column. The acids are separated on two coupled C-18 columns (5 #m particles) using a mobile phase of 3.2 mmol/L hydrochloric acid. Peaks are detected by spectrophotometric absorption at 280 nm. Quantitation is achieved by calibration with known amounts of orotic acid. This method can also be used to measure orotidine, which elutes as an earlier peak. Orotidine was not measured in this study because we previously found orotic acid to be as good a marker for the allopurinol test (Figure 1, showing comparison of orotic acid vs. orotidine following allopurinol ingestion in five patients with urea-cycle disorders). Urinary creatinine concentration was determined by the Folin method (Folin and Wu, cited in Hawk et al 1947). Results of orotic acid concentrations were normalized to the urinary creatinine and expressed as #mol/mmol creatinine.

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Figure 1 Urinary orotic acid and orotidine in the allopurinol test performed in four female patients with OTC deficiency(I-IV) and one male patient with HHH syndrome (V). RESULTS Table 1 summarizes the allopurinol challenge test results obtained from 44 healthy children divided into three groups according to age. In most chitdren (23 out of 44), peak orotic acid levels were found in urine collected 6-12 h following ingestion of allopurinol; in 7 children the peak was 0-6 h post ingestion; in 10 children it was at

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Table 1 Age-related urinary orotic acid levels (lxmol/mmol urinary creatinine) in normal children following aliopurinnl ingestion Period 1

Period 2

Period 3

Period 4

Baseline

(0-6h)

(6-12h)

(12-18h)

(18-24h)

Median Minimum Maximum (n)

0.3 0.1 2.3 (14)

1.3 0.2 6.5 (14)

Median Minimum Maximum (n)

0.2 < 0.01 1.8 (14)

t.3 0.1 7.4 (13)

Median Minimum Maximum (n)

0.2 < 0.01 0.8 (16)

0.8 0.2 4.0 (16)

Age 6 months to 6 years

2.7 0.7 13.0 (14)

1.1 0.1 5.0 (8)

0,9 0.1 3.0 (14)

Age 6 years to 10 years

1.7 0.3 9.3 (14)

1.9 0.3 6.3 (8)

1.1 0.3 4.6 (13)

Age 10 years to 17 years

2.0 0. I 4.8 (16)

1.4 0.1 10.2 (15)

0.7 0.1 2.8 (16)

12-18h; and in 3 children it was at 18-24h. In one child, urinary orotic acid failed to increase. Peak urinary orotic acid levels observed following ingestion of allopurinol were 13.0 ( n - - 14), 9.3 (n = 14), and 10.2 (n = 16) (#mol/mmol creatinine) in the three age groups, respectively. In all subjects tested peak orotic acid level was 3.1 +__2.7#mol/mmot creatinine (mean _+ SD, n = 44). N o significant differences between males and females were found in the results. Table 2 summarizes the allopurinol challenge test results for six children with proven urea-cycle defects. As expected, abnormal results were clearly obtained in five females with O T C deficiency and in one male with H H H . Peak orotic acid levels in these patients ranged from 26 to 134 #mol/mmol creatinine. DISCUSSION Knowledge of the results of altopurinol challenge tests in healthy children described here is essential if one is to use this diagnostic test in children suspected to have Table 2 Urinary orotic acid levels (pmol/mmoi creatinine) in children with ure~ ..... le related defects following allopurinol ingestion

1 2 3 4 5 6

Disease ~

Sex

OTC OTC OTC OTC OTC HHH

F F F F F M

Age

ly 2y 6y 8y 13y 13y

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Period 1

Period 2

Period 3

Period 4

Baseline

(0-6h)

(6-12 h)

(12-18 h)

(18-24h)

3.8 2.3 0.3 0.3 1.0 0.5

50.4 26.4 17.1 5.9 22.8 20.4

97.8 25.4 133.6 51.0 108.0 44.9

32.4 6.0 92.7 15.4 36.6 11.4

33.7 5.9 2t.7 t.3 99.4 2.2

aOTC = ornithine transcarbamylase deficiency; HHH = hyperornithineamia, hyperammonaemia, and homocitrullinuria J. lnher. Metab. Dis. 15 (1992)

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inherited urea-cycle disorders. When interpreting the results of this test, the age of the child has to be taken into account. This is especially important in the very young infants, who would show higher values when normalized to the urinary creatinine concentrations. The maximal peak level of orotic acid obtained in this study (13#mol/mmol creatinine) was in the youngest child tested (6 months old). Until normal values in very young infants can be obtained, we suggest using the maximum age-related normal levels reported in this study as maximum values for peak orotic acid for children older than 1 year. No reliable normal values are currently available for infants less than 1 year of age. As was shown in all five children with OTC deficiency and in the child with HHH syndrome, peak orotic acid levels were much higher than in any of the normals. The preferred method for determining orotic acid levels uses high-performance liquid chromatography (HPLC), which allows analysis of urine directly as well as offering efficient separation from other urinary compounds, thus avoiding technical problems and inaccuracies of other methods (Harris and Oberholzer 1980). It was suggested that orotidine is a better marker than orotic acid in this test (Hauser et al 1990). We believe, however, that orotic acid is as good a marker for it because, in several of our patients, orotic acid increased earlier and to a higher degree than orotidine after allopurinol ingestion. This is illustrated in Figure 1, which displays our experience with orotic acid vs orotidine levels following.ingestion of allopurinol in patients with OTC deficiency and HHH syndrome. Moreover, when using reversedphase HPLC, it is advantageous to quantitate orotic acid over orotidine because the latter acid has a very short retention time on the reversed-phase column and may be interfered with by other acids co-eluting early. Several inborn errors of metabolism show intermittent clinical and biochemical signs (Saudubray and Ogier 1990). Therefore, urinary orotic acid levels could be normal in children with urea-cycle disorders when measured randomly. Girls with partial OTC deficiency may not show spontaneous orotic aciduria unless stimulated with allopurinol. We encountered several cases of children with suspected urea-cycle defects in whom the diagnosis was not clear even after enzymatic studies of the liver. The interpretation of a partial deficiency of liver OTC enzymatic activity caused by some mutations, low-protein diet or non-optimal handling of the specimen could be difficult. The variable mosaicism in different areas of the liver of females with OTC deficiency may also be a potential problem, especially when the residual activity is substantial. In these cases, the allopurinol test may help in diagnosis. ACKNOWLEDGEMENTS We thank Dr Claude Bachmann for his input and helpful suggestions and to Mr Robert Holzknecht for his help in preparing the manuscript. REFERENCES

Bachmann C, Colombo JP (1980). Diagnostic value of orotic acid excretion in heritable disorders of the urea cycle and in hyperammonemiadue to organic acidurias. Eur J Pediatr 134: 109-113. d. Inher. Metab. Dis. 15 (1992)

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Becroft DMO, Barry, DMJ, Webster DR, Simmonds HA (1984). Failure of protein loading tests to identify heterozygosity for ornithine carbamyltransferase deficiency. J Inher Metab Dis 7: 157-159. Cohen A, Staal GEJ, Ammann AJ, Martin DW Jr (1977). Orotic aciduria in two unrelated patients with inherited deficiencies of purine nucleoside phosphorylase. J Clin Invest 60: 491-494. Ferrari V, Giordano G, Gracco AT, Dussini N, Chiandetti L, Zacchello F (1989). Determination of urinary orotate excretion by high-performance liquid chromatography. J Chromatogr 497: 101-107. Fox RM, Royse-Smith D, O'Sullivan WJ (1970). Orotidinuria induced by allopurinol. Science 168: 861-862. Harris ML, Oberholzer VG (1980). Conditions affecting the colorimetry of orotic acid and orotidine in urine. Clin Chem 26: 473-479. Hauser ER, Finkelstein JE, Valle D, Brusilow SW (1990). Allopurinol-induced orotindinuria. A test for mutations at the ornithine carbamoyltransferase locus in women. N Engl J Med 322: 1641-1645. Hawk PB, Oser BL, Summerson WH (eds) (1947). Practical Physiological Chemistry. Philadelphia: Blakiston, 115-120. Hokanson JT, O'Brien WE, Idemoto J, Schafer IA (1978). Carrier detection in ornithine transcarbamylase deficiency. J Pediatr 93: 75-78. Rajantie 'J (1981). Orotic aciduria in lysinuric protein intolerance: dependence on the urea cycle intermediates. Pediatr Res 15: 115-119. Saudubray JM, Ogier H (1990). Clinical approach to inherited metabolic diseases. In Fernandes J, Saudubray JM, Tada K, eds. Inborn Metabolic Diseases, Diagnosis and Treatment. Berlin: • Springer-Verlag, 3-25. Simell O, Mackenzie S, Clow CL, Scriver CR (1985). Ornithine loading did not prevent induced hyperammonemia in a patient with hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. Pediatr Res 19: 1283-1287. Suttle DP, Becroft DMO, Webster DR (1989). Hereditary orotic aciduria and other disorders of pyrimidine metabolism. In Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Basis of Inherited Disease, 6th edn. New York: McGraw-Hill, 1095-i126. Tuchman M, Knopman DS, Shih VE (1990). Episodic hyperammonemia in adult siblings with hyperornithinemia, hyperammonemia, and homocitrullinuria syndrome. Arch Neurol 47: 1134-1137. Wood MH, O'Sullivan WJ (1973). The orotic aciduria of pregnancy. Am J Obstet Gynecol 116: 57-61.

J. Inher. Metab. Dis. 15 (1992)

Allopurinol challenge test in children.

The allopurinol challenge test was performed on 44 healthy subjects (28 children and 16 adolescents) in order to establish normal values of urinary or...
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