- Email: [email protected]
Tetrahydrobiopterin Loading Test in Xanthine Dehydrogenase and Molybdenum Cofactor Deficiencies
BIOCHEMICAL AND MOLECULAR MEDICINE ARTICLE NO.
58, 199–203 (1996)
0049
Tetrahydrobiopterin Loading Test in Xanthine Dehydrogenase and Molybdenum Cofactor Deficiencies N. BLAU,*,1 J. B. C. DE KLERK,† B. THO¨NY,* C. W. HEIZMANN,* L. KIERAT,* J. A. M. SMEITINK,‡ AND M. DURAN‡ *Division of Clinical Chemistry, Department of Pediatrics, University of Zurich, Zurich, Switzerland; †Sophia Children’s Hospital, Rotterdam, The Netherlands; and ‡Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands Received February 26, 1996, and in revised form March 26, 1996
denum cofactor (MoCo) deficiency (MIM 252150) are characterized by very low uric acid and high xanthine and hypoxanthine in urine caused by deficiency of xanthine oxidase/dehydrogenase (XDH) (1,2). In MoCo deficiency additional enzymes, such as sulfite and aldehyde oxidase, are affected. About one-third of the patients with classic xanthinuria present with xanthine calculi of the urinary tract, and two-thirds of the patients are asymptomatic (3). Patients with the MoCo deficiency present clinically with therapy-resistent seizures, ectopia lentis, and psychomotor retardation (4). The diagnosis is not only based on analysis of purines, but requires measurement of sulfite, thiosulfate, taurine, and S-sulfocysteine in urine and plasma (5,6). Definite diagnosis may be performed by assaying XDH activity in liver (7) or in jejunal biopsy (8), the only tests available so far. MoCo deficiency is also diagnosed by measuring sulfite oxidase activity in cultured fibroblasts. Here we describe an oral BH4 loading test which provides additional diagnostic information.
The objectives of this study were to find additional diagnostic information for the evaluation of xanthine dehydrogenase deficiency and molybdenum cofactor deficiency. Patients were given an oral loading test with 10 mg/kg 5,6,7,8-tetrahydrobiopterin. Urine excretion of pterin and isoxanthopterin was measured by HPLC. Control subjects had a fairly constant ratio of urinary pterin/isoxanthopterin before (0.57–5.32) and after (0.55–4.55) 5,6,7,8-tetrahydrobiopterin loading. These ratios were increased to 33 and 22 in a patient with hereditary xanthinuria and to 570 and 8030 in a patient with molybdenum cofactor deficiency. Obligate heterozygotes had an entirely normal test result. Evidence was obtained for the in vivo involvement of xanthine dehydrogenase in the conversion of pterin to isoxanthopterin. This test could be a sensitive marker for the establishment of residual enzyme activity. q 1996 Academic Press, Inc.
Xanthine dehydrogenase (XDH) is the enzyme responsible for the conversion of xanthine to uric acid. It requires the presence of the molybdenum cofactor for its proper functioning. XDH was reported to have additional functions, i.e., the conversion of pterin to isoxanthopterin, one of the steps in the degradation pathway of 5,6,7,8-tetrahydrobiopterin (BH4) (Fig. 1). Hereditary xanthinuria (MIM 278300) and molyb-
SUBJECTS Control urine samples were obtained from 60 infants screened for hyperphenylalaninemia (mean age 3.3 months; range, 0.5–48) and two healthy adults (ages 38 and 48 years). The hyperphenylalaninemic patients presented with plasma phenylalanine levels between 200 and 1400 mmol/liter. The family with MoCo deficiency had two siblings, a girl and a boy 2 years older. Both pregnancies and deliveries were entirely uncomplicated. Birth
1 To whom correspondence should be addressed at Division of Clinical Chemistry, Department of Pediatrics, University of Zu¨rich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Fax: /411 266 7169; e-mail: [email protected].
199 1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
BMM 2514
/
af02$$$321
07-10-96 11:16:23
bmma
AP: BMM
200
BLAU ET AL.
FIG. 1. Catabolism of BH4 . Dotted box represents compounds used in the loading test with BH4 .
weight, length, and head circumference were normal. The girl developed convulsions on the second day of life, which were therapy-resistant. No inborn error of metabolism could be established at that time and she died on the 9th day of life. Neuropathological examination of the brain revealed ischemic spongionecrosis of the subcortical areas, somewhat reminiscent of the changes observed in Canavan/van Bogaert-Bertrand disease. The boy started to have convulsions within hours after birth. His EEG showed irritative changes. An MRI of the brain, which was made some weeks later, showed cerebral atrophy, a partial agenesis of the corpus callosum, and hypodensity of the basal ganglia. He developed spastic tetraplegia and had severe psychomotor retardation. Cardiac abnormalities included a sinus tachycardia. There were facial dysmorphia similar to those of many other patients with MoCo deficiency. Death occurred at the age of 4 years. This family belonged to the complementation group A(9), similar to most northwest European patients. The patient with isolated XDH deficiency was born as the second child of healthy parents. Pregnancy (36
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
4/7 weeks) and delivery were uneventful. Jitteriness was observed from the third day of life, which was the trigger to screen for metabolic disease. In retrospect, the clinical symptoms could well be explained by hypocalcemia associated with prematurity. At the age of 20 months the boy showed normal psychomotor development; there was normal renal function with no indications of xanthine calculi. METHODS Tetrahydrobiopterin (10 mg tablets) was purchased from Dr. Schircks Laboratories (Jona, Switzerland). High-performance liquid chromatography (HPLC) of urinary pterins was performed as described previously (10) except that isocratic separation was performed on a C8 Spherisorb, 5-mm (Phase Separation, London, UK) precolumn (4.6 1 40 mm) and ODS-1 Spherisorb, 5-mm (Phase Separation, London, UK) analytical column (4.6 1 250 mm), using 1 mM potassium dihydrogen phosphate buffer, pH 4.6, with 5% (v/v) methanol at a flow rate of 1.2 ml/min. Urine
bmma
AP: BMM
201
TETRAHYDROBIOPTERIN LOADING TEST
TABLE 1 Urinary Pterin Levels before and after (4–8 h) Oral Administration of Tetrahydrobiopterin in Patients with XDH and MoCo Deficiency, Heterozygotes for MoCo Deficiency, and Controls Biopterin (mmol/mol creat.)
Isoxanthopterin (mmol/mol creat.)
Pterin (mmol/mol creat.)
Pterin/isoxanthopterin
Subjects
Before
After
Before
After
Before
After
Before
After
XDH deficiency MoCo deficiency MoCo patient mother MoCo patient father Controls, adults nÅ2 Controls, HPA n Å 60
2.35 2.37
19.74 13.51
0.65 0.57
12.24 8.03
0.02 0.001
0.56 0.001
32.5 570.0
21.9 8030.0
0.65
1.18
0.26
0.16
0.07
0.23
3.7
0.7
0.43
1.85
0.13
0.30
0.06
0.31
2.2
1.0
1.99/1.09 2.34 (0.71–5.47)
22.36/30.77 21.3 (4.00–81.02)
0.28/0.40 1.17 (0.24–4.22)
4.78/5.00 5.32 (0.93–33.71)
0.57/0.24 0.70 (0.10–2.81)
5.69/7.80 4.68 (0.59–17.60)
samples were oxidized with manganese dioxide at pH 1.0–1.5 prior to HPLC. Purines and pyrimidines were analyzed by HPLC with diode-array UV-detection (200–320 nm) according to Van Acker et al. (11) with some modifications. The column was a 250 1 4.6-mm S5 ODS1 (PhaseSep, Deeside, UK). The compounds of interest were eluted with a gradient of (A) 30 mM ammonium acetate (pH 4.3) with 2% (v/v) methanol and (B) methanol/acetonitrile (80:20). The elution started at 100% A and ended at 55% B after 35 min. Pretreatment of the urine samples involved only a 30-fold dilution with buffer A. Oral loading test (10 mg BH4/kg body weight). BH4 tablets were dissolved in 10–20 ml water or orange juice and administered orally about 30 min before a meal. Urine samples were collected before and 4–8 h after the loading and stored in the dark at 0207C. RESULTS Table 1 summarizes the effects of BH4 administration on urinary excretion of biopterin, pterin, and isoxanthopterin in one patient with the XDH deficiency, one patient with the MoCo deficiency and his parents, and in 62 control persons (60 patients with hyperphenylalaninemia and 2 healthy controls). Intestinal absorption of administered BH4 in controls was monitored by increased biopterin excretion (Ç10-fold), 4–8 h after the oral administration. In the parents of a child with MoCo deficiency, loaded with the lower dose of BH4 (5 mg/kg body weight),
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
0.5/1.7 1.31 (0.57–5.32)
0.8/0.6 1.17 (0.55–4.55)
the biopterin increase was only 2- to 4-fold. Increase of pterin (Ç2-fold) and isoxanthopterin (Ç4-fold) concentration in urine was significant and paralleled that of biopterin. Although the formation of pterin and isoxanthopterin from exogenous BH4 depends on different factors, i.e., stability of BH4 and its rate of side-chain cleavage, the ratio of pterin to isoxanthopterin in urine of controls was constant before (0.57–5.32) and after (0.55–4.55) the challenge. These parameters were used to calculate the relative activity of XDH. In the patient with XDH deficiency, urinary biopterin increased about 8-fold, pterin about 19-fold, and the pterin/isoxanthopterin ratio was 21.9, 4–8 h after BH4 administration. Before the challenge, the pterin/isoxanthopterin ratio was already very high with a quotient of 32.5 and only traces of isoxanthopterin were detected. The patient with MoCo deficiency presented a comparable increase in urinary biopterin (Ç5-fold) and pterin (Ç14-fold) after BH4 administration, and the pterin/isoxanthopterin ratio was extremely high, with a quotient of 8030. Isoxanthopterin was present only in traces. These data demonstrate that in both patients XDH activity is markedly reduced and even absent in the patient with the MoCo deficiency. The parents of the patient with MoCo deficiency, loaded with the lower dose of BH4 , showed conversion of pterin to isoxanthopterin similar to that in control persons, suggesting that the theoretical 50% of enzyme activity in the obligate heterozygotes is probably sufficient for the normal BH4 catabolism. Patients with XDH and MoCo deficiencies can be
bmma
AP: BMM
202
BLAU ET AL.
FIG. 2. Two-dimensional logarithmic plotting of pterin/isoxanthopterin (Pte/Ixa) ratios before and 4–8 h after oral loading test with BH4 (10 mg/kg bw). Two heterozygotes for MoCo were loaded with only 5 mg BH4/kg bw.
separated completely from controls by the logarithmic two-dimensional plotting of the pterin/isoxanthopterin ratios before and 4–8 h after the challenge (Fig. 2). DISCUSSION MoCo deficiency is a serious disorder, associated with intractable neonatal convulsions, feeding difficulties, axial hypotonia, and virtual absence of psychomotor development (12). Cerebral atrophy is a common finding on CT/MRI scanning; it is the cause of progressive microcephaly. The clinical profile is almost indistinguishable from that of severe perinatal asphyxia (13). Accumulating inorganic sulfite, which is neurotoxic, or the abnormal amino acid sulfocysteine, which interferes with brain glutamate receptors, may cause the brain damage. The biochemical abnormalities of the purine catabolic pathway, i.e., hypouricemia and xanthine overflow, do not contribute to the clinical symptoms, as demonstrated by the patients with isolated XDH deficiency (14). The latter patients generally are free of neurological symptoms and the diagnosis of XDH deficiency is often made by chance. The degree of hypouricemia in MoCo deficiency and in XDH deficiency is generally comparable and thus is not a good parameter of residual enzyme activity. Up to now
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
only one MoCo deficient patient had normal uric acid levels (15), which originally resulted in the incorrect diagnosis of sulfite oxidase deficiency (16). This patient had a milder clinical presentation than the vast majority of MoCo-deficient patients. It would be, however, most interesting to examine this patient under BH4 loading. The BH4 challenge is an indicator for the hepatic XDH activity. A portion of the orally administered BH4 is converted by nonenzymatic side chain cleavage to 7,8-dihydropterin, which oxidizes spontaneously to pterin (Fig. 1). The remaining 7,8-dihydropterin and pterin can be oxidized by XDH to 7,8dihydroxanthopterin and isoxanthopterin, respectively. These pterins are the end products of pterin cofactor metabolism and represent the major portion of the urinary pool in mammals (17). Isoxanthopterin is a green-blue fluorescing substance and can be measured by HPLC simultaneously with other pterins. The conversion of pterin to isoxanthopterin is an indicator of XDH activity. In control persons, this conversion is constant and the pterin/isoxanthopterin ratio is close to 1. Challenge with BH4 increases the pterin substrate concentrations for XDH and the ratio increases only slightly although urinary isoxanthopterin increases significantly. In a patient with XDH deficiency the pterin accu-
bmma
AP: BMM
TETRAHYDROBIOPTERIN LOADING TEST
mulates and low isoxanthopterin concentrations in urine are measurable. In this patient residual activity of XDH is probably very low. In a patient with MoCo deficiency XDH is completely absent, and only traces of isoxanthopterin if any are detectable. Obviously, a defect in the cofactor of XDH seems to have more severe consequences for enzyme activity as previously suggested. Although the BH4 challenge mimics the actual XDH activity, obligate heterozygotes for MoCo deficiency can unfortunately not be distinguished from unaffected controls. The theoretical residual activity (Ç50%) seems to be sufficient for complete conversion of pterin to isoxanthopterin. The only limitation for this test is the treatment of gouty patients with allopurinol. Allopurinol may induce xanthinuria due to inhibition of XDH (18). However, the measurement of the pterin/isoxanthopterin ratio is of value in the follow-up of those patients. ACKNOWLEDGMENTS We thank Mr. S. Holms for the excellent technical help and Ms. M. Killen for editorial work. Supported by the Swiss National Science Foundation, Grant 3100-043380.95, and the HartmannMu¨ller Research Foundation (both to N.B.).
REFERENCES 1. Simmonds HA, Reiter S, Nishino T. Hereditary xanthinuria. In The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS, Valle D, Eds). New York: McGraw-Hill, 1995, Vol 2, pp 1781–1797. 2. Johnson JL, Wadman SK. Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency. In The Metabolic and Molecular Bases of Inherited Disease. (Scriver CR, Beaudet AL, Sly WS, Valle D, Eds). New York: McGraw–Hill, 1995, Vol 2, pp 2271–2283. 3. Higashino K, Yamamoto T, Hada T, Kono N, Kawachi M, Nanahoshi M, Takahashi S, Suda M, Naka M. Hereditary xanthine oxidase deficiency consisting of at least two subgroups. Adv Exp Med Biol 253A:87–91, 1989. 4. Beemer FA, Duran M, Wadman SK, Cats BP. Absence of hepatic molybdenum cofactor. An inborn error of metabolism associated with lens dislocation. Ophthalm Paediatr Genet 5:191–195, 1985.
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
203
5. Skovby F. Disorders of sulfur amino acids. In Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases (Blau N, Duran M, Blaskovics M, Eds.). London: Chapman and Hall, 1996, pp 187–200. 6. Simmonds A. Purine and pyrimidine disorders. In Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases (Blau N, Duran M, Blaskovics M, Eds.). London: Chapman and Hall, 1996, pp 341–357. 7. Mousson B, Desjacques P, Baltassat P. Measurement of xanthine oxidase activity in some human tissues. An optimized method. Enzyme 29:32–43, 1983. 8. Kawachi M, Kono N, Mineo I, Yamada Y, Tarui S. Decreased xanthine oxidase activities and increased urinary oxypurines in heterozygotes for hereditary xanthinuria. Clin Chim Acta 188:137–146, 1990. 9. Johnson JL, Wuebbens MM, Mandell R, Shih VE. Molybdenum cofactor biosynthesis in humans. Identification of two complementation groups of cofactor-deficient patients and preliminary characterization of a diffusible molybdopterin precursor. J Clin Invest 83:897–903, 1989. 10. Curtius HC, Blau N, Kuster T. Pterins. In Techniques in Diagnostic Human Biochemical Genetics (Hommes FA, Ed.). New York: Wiley–Liss, 1991, pp 377–396. 11. Van Acker K, Eyskens FJ, Verkerk RM, Scharpe SS. Urinary excretion of purine and pyrimidine metabolites in neonate. Pediatr Res 34:762–766, 1993. 12. Duran M, de Bree PK, de Klerk JBC, Dorland L, Berger R. Molybdenum-cofactor deficiency: Clinical presentation and laboratory diagnosis. Int Pediatr, in press. 13. Schuierer G, Kurlemann G, Bick U, Stephani U. Molybdenum cofactor deficiency: CT and MR findings. Neuropediatrics 26:51–54, 1995. 14. Frayha RA, Salti IS, Araout RA, Katchadurian A, Uhtman SM. Hereditary xanthinuria, report on three patients and short review of the literature. Nephron 19:328–332, 1977. 15. Johnson JL, Wuebbens MM, Mandell R, Shih VE. Molybdenum cofactor deficiency in a patient previously characterized as deficient in sulfite oxidase. Biochem Med Metab Biol 40:86–93, 1988. 16. Shih VE, Abroms IF, Johnson JL, Carney M, Mandell R, Robb RM, Cloherty JP, Rajagopalan KV. Sulfite oxidase deficiency. Biochemical and clinical investigations of a hereditary metabolic disorder in sulfur metabolism. N Engl J Med 197:1022–1028, 1977. 17. Rembold H. Pteridine catabolism. In Biochemical and Clinical Aspects of Pteridines (Curtius HC, Pfleiderer W, Wachter H, Eds). Berlin: Walter de Gruyter, 1983, Vol 2, pp 107– 122. 18. Cameron JS, Moro F, Simmonds HA. Gout, uric acid and purine metabolism in paediatric nephrology. Pediatr Nephrol 7:105–18, 1993.
bmma
AP: BMM
58, 199–203 (1996)
0049
Tetrahydrobiopterin Loading Test in Xanthine Dehydrogenase and Molybdenum Cofactor Deficiencies N. BLAU,*,1 J. B. C. DE KLERK,† B. THO¨NY,* C. W. HEIZMANN,* L. KIERAT,* J. A. M. SMEITINK,‡ AND M. DURAN‡ *Division of Clinical Chemistry, Department of Pediatrics, University of Zurich, Zurich, Switzerland; †Sophia Children’s Hospital, Rotterdam, The Netherlands; and ‡Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands Received February 26, 1996, and in revised form March 26, 1996
denum cofactor (MoCo) deficiency (MIM 252150) are characterized by very low uric acid and high xanthine and hypoxanthine in urine caused by deficiency of xanthine oxidase/dehydrogenase (XDH) (1,2). In MoCo deficiency additional enzymes, such as sulfite and aldehyde oxidase, are affected. About one-third of the patients with classic xanthinuria present with xanthine calculi of the urinary tract, and two-thirds of the patients are asymptomatic (3). Patients with the MoCo deficiency present clinically with therapy-resistent seizures, ectopia lentis, and psychomotor retardation (4). The diagnosis is not only based on analysis of purines, but requires measurement of sulfite, thiosulfate, taurine, and S-sulfocysteine in urine and plasma (5,6). Definite diagnosis may be performed by assaying XDH activity in liver (7) or in jejunal biopsy (8), the only tests available so far. MoCo deficiency is also diagnosed by measuring sulfite oxidase activity in cultured fibroblasts. Here we describe an oral BH4 loading test which provides additional diagnostic information.
The objectives of this study were to find additional diagnostic information for the evaluation of xanthine dehydrogenase deficiency and molybdenum cofactor deficiency. Patients were given an oral loading test with 10 mg/kg 5,6,7,8-tetrahydrobiopterin. Urine excretion of pterin and isoxanthopterin was measured by HPLC. Control subjects had a fairly constant ratio of urinary pterin/isoxanthopterin before (0.57–5.32) and after (0.55–4.55) 5,6,7,8-tetrahydrobiopterin loading. These ratios were increased to 33 and 22 in a patient with hereditary xanthinuria and to 570 and 8030 in a patient with molybdenum cofactor deficiency. Obligate heterozygotes had an entirely normal test result. Evidence was obtained for the in vivo involvement of xanthine dehydrogenase in the conversion of pterin to isoxanthopterin. This test could be a sensitive marker for the establishment of residual enzyme activity. q 1996 Academic Press, Inc.
Xanthine dehydrogenase (XDH) is the enzyme responsible for the conversion of xanthine to uric acid. It requires the presence of the molybdenum cofactor for its proper functioning. XDH was reported to have additional functions, i.e., the conversion of pterin to isoxanthopterin, one of the steps in the degradation pathway of 5,6,7,8-tetrahydrobiopterin (BH4) (Fig. 1). Hereditary xanthinuria (MIM 278300) and molyb-
SUBJECTS Control urine samples were obtained from 60 infants screened for hyperphenylalaninemia (mean age 3.3 months; range, 0.5–48) and two healthy adults (ages 38 and 48 years). The hyperphenylalaninemic patients presented with plasma phenylalanine levels between 200 and 1400 mmol/liter. The family with MoCo deficiency had two siblings, a girl and a boy 2 years older. Both pregnancies and deliveries were entirely uncomplicated. Birth
1 To whom correspondence should be addressed at Division of Clinical Chemistry, Department of Pediatrics, University of Zu¨rich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. Fax: /411 266 7169; e-mail: [email protected].
199 1077-3150/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
BMM 2514
/
af02$$$321
07-10-96 11:16:23
bmma
AP: BMM
200
BLAU ET AL.
FIG. 1. Catabolism of BH4 . Dotted box represents compounds used in the loading test with BH4 .
weight, length, and head circumference were normal. The girl developed convulsions on the second day of life, which were therapy-resistant. No inborn error of metabolism could be established at that time and she died on the 9th day of life. Neuropathological examination of the brain revealed ischemic spongionecrosis of the subcortical areas, somewhat reminiscent of the changes observed in Canavan/van Bogaert-Bertrand disease. The boy started to have convulsions within hours after birth. His EEG showed irritative changes. An MRI of the brain, which was made some weeks later, showed cerebral atrophy, a partial agenesis of the corpus callosum, and hypodensity of the basal ganglia. He developed spastic tetraplegia and had severe psychomotor retardation. Cardiac abnormalities included a sinus tachycardia. There were facial dysmorphia similar to those of many other patients with MoCo deficiency. Death occurred at the age of 4 years. This family belonged to the complementation group A(9), similar to most northwest European patients. The patient with isolated XDH deficiency was born as the second child of healthy parents. Pregnancy (36
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
4/7 weeks) and delivery were uneventful. Jitteriness was observed from the third day of life, which was the trigger to screen for metabolic disease. In retrospect, the clinical symptoms could well be explained by hypocalcemia associated with prematurity. At the age of 20 months the boy showed normal psychomotor development; there was normal renal function with no indications of xanthine calculi. METHODS Tetrahydrobiopterin (10 mg tablets) was purchased from Dr. Schircks Laboratories (Jona, Switzerland). High-performance liquid chromatography (HPLC) of urinary pterins was performed as described previously (10) except that isocratic separation was performed on a C8 Spherisorb, 5-mm (Phase Separation, London, UK) precolumn (4.6 1 40 mm) and ODS-1 Spherisorb, 5-mm (Phase Separation, London, UK) analytical column (4.6 1 250 mm), using 1 mM potassium dihydrogen phosphate buffer, pH 4.6, with 5% (v/v) methanol at a flow rate of 1.2 ml/min. Urine
bmma
AP: BMM
201
TETRAHYDROBIOPTERIN LOADING TEST
TABLE 1 Urinary Pterin Levels before and after (4–8 h) Oral Administration of Tetrahydrobiopterin in Patients with XDH and MoCo Deficiency, Heterozygotes for MoCo Deficiency, and Controls Biopterin (mmol/mol creat.)
Isoxanthopterin (mmol/mol creat.)
Pterin (mmol/mol creat.)
Pterin/isoxanthopterin
Subjects
Before
After
Before
After
Before
After
Before
After
XDH deficiency MoCo deficiency MoCo patient mother MoCo patient father Controls, adults nÅ2 Controls, HPA n Å 60
2.35 2.37
19.74 13.51
0.65 0.57
12.24 8.03
0.02 0.001
0.56 0.001
32.5 570.0
21.9 8030.0
0.65
1.18
0.26
0.16
0.07
0.23
3.7
0.7
0.43
1.85
0.13
0.30
0.06
0.31
2.2
1.0
1.99/1.09 2.34 (0.71–5.47)
22.36/30.77 21.3 (4.00–81.02)
0.28/0.40 1.17 (0.24–4.22)
4.78/5.00 5.32 (0.93–33.71)
0.57/0.24 0.70 (0.10–2.81)
5.69/7.80 4.68 (0.59–17.60)
samples were oxidized with manganese dioxide at pH 1.0–1.5 prior to HPLC. Purines and pyrimidines were analyzed by HPLC with diode-array UV-detection (200–320 nm) according to Van Acker et al. (11) with some modifications. The column was a 250 1 4.6-mm S5 ODS1 (PhaseSep, Deeside, UK). The compounds of interest were eluted with a gradient of (A) 30 mM ammonium acetate (pH 4.3) with 2% (v/v) methanol and (B) methanol/acetonitrile (80:20). The elution started at 100% A and ended at 55% B after 35 min. Pretreatment of the urine samples involved only a 30-fold dilution with buffer A. Oral loading test (10 mg BH4/kg body weight). BH4 tablets were dissolved in 10–20 ml water or orange juice and administered orally about 30 min before a meal. Urine samples were collected before and 4–8 h after the loading and stored in the dark at 0207C. RESULTS Table 1 summarizes the effects of BH4 administration on urinary excretion of biopterin, pterin, and isoxanthopterin in one patient with the XDH deficiency, one patient with the MoCo deficiency and his parents, and in 62 control persons (60 patients with hyperphenylalaninemia and 2 healthy controls). Intestinal absorption of administered BH4 in controls was monitored by increased biopterin excretion (Ç10-fold), 4–8 h after the oral administration. In the parents of a child with MoCo deficiency, loaded with the lower dose of BH4 (5 mg/kg body weight),
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
0.5/1.7 1.31 (0.57–5.32)
0.8/0.6 1.17 (0.55–4.55)
the biopterin increase was only 2- to 4-fold. Increase of pterin (Ç2-fold) and isoxanthopterin (Ç4-fold) concentration in urine was significant and paralleled that of biopterin. Although the formation of pterin and isoxanthopterin from exogenous BH4 depends on different factors, i.e., stability of BH4 and its rate of side-chain cleavage, the ratio of pterin to isoxanthopterin in urine of controls was constant before (0.57–5.32) and after (0.55–4.55) the challenge. These parameters were used to calculate the relative activity of XDH. In the patient with XDH deficiency, urinary biopterin increased about 8-fold, pterin about 19-fold, and the pterin/isoxanthopterin ratio was 21.9, 4–8 h after BH4 administration. Before the challenge, the pterin/isoxanthopterin ratio was already very high with a quotient of 32.5 and only traces of isoxanthopterin were detected. The patient with MoCo deficiency presented a comparable increase in urinary biopterin (Ç5-fold) and pterin (Ç14-fold) after BH4 administration, and the pterin/isoxanthopterin ratio was extremely high, with a quotient of 8030. Isoxanthopterin was present only in traces. These data demonstrate that in both patients XDH activity is markedly reduced and even absent in the patient with the MoCo deficiency. The parents of the patient with MoCo deficiency, loaded with the lower dose of BH4 , showed conversion of pterin to isoxanthopterin similar to that in control persons, suggesting that the theoretical 50% of enzyme activity in the obligate heterozygotes is probably sufficient for the normal BH4 catabolism. Patients with XDH and MoCo deficiencies can be
bmma
AP: BMM
202
BLAU ET AL.
FIG. 2. Two-dimensional logarithmic plotting of pterin/isoxanthopterin (Pte/Ixa) ratios before and 4–8 h after oral loading test with BH4 (10 mg/kg bw). Two heterozygotes for MoCo were loaded with only 5 mg BH4/kg bw.
separated completely from controls by the logarithmic two-dimensional plotting of the pterin/isoxanthopterin ratios before and 4–8 h after the challenge (Fig. 2). DISCUSSION MoCo deficiency is a serious disorder, associated with intractable neonatal convulsions, feeding difficulties, axial hypotonia, and virtual absence of psychomotor development (12). Cerebral atrophy is a common finding on CT/MRI scanning; it is the cause of progressive microcephaly. The clinical profile is almost indistinguishable from that of severe perinatal asphyxia (13). Accumulating inorganic sulfite, which is neurotoxic, or the abnormal amino acid sulfocysteine, which interferes with brain glutamate receptors, may cause the brain damage. The biochemical abnormalities of the purine catabolic pathway, i.e., hypouricemia and xanthine overflow, do not contribute to the clinical symptoms, as demonstrated by the patients with isolated XDH deficiency (14). The latter patients generally are free of neurological symptoms and the diagnosis of XDH deficiency is often made by chance. The degree of hypouricemia in MoCo deficiency and in XDH deficiency is generally comparable and thus is not a good parameter of residual enzyme activity. Up to now
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
only one MoCo deficient patient had normal uric acid levels (15), which originally resulted in the incorrect diagnosis of sulfite oxidase deficiency (16). This patient had a milder clinical presentation than the vast majority of MoCo-deficient patients. It would be, however, most interesting to examine this patient under BH4 loading. The BH4 challenge is an indicator for the hepatic XDH activity. A portion of the orally administered BH4 is converted by nonenzymatic side chain cleavage to 7,8-dihydropterin, which oxidizes spontaneously to pterin (Fig. 1). The remaining 7,8-dihydropterin and pterin can be oxidized by XDH to 7,8dihydroxanthopterin and isoxanthopterin, respectively. These pterins are the end products of pterin cofactor metabolism and represent the major portion of the urinary pool in mammals (17). Isoxanthopterin is a green-blue fluorescing substance and can be measured by HPLC simultaneously with other pterins. The conversion of pterin to isoxanthopterin is an indicator of XDH activity. In control persons, this conversion is constant and the pterin/isoxanthopterin ratio is close to 1. Challenge with BH4 increases the pterin substrate concentrations for XDH and the ratio increases only slightly although urinary isoxanthopterin increases significantly. In a patient with XDH deficiency the pterin accu-
bmma
AP: BMM
TETRAHYDROBIOPTERIN LOADING TEST
mulates and low isoxanthopterin concentrations in urine are measurable. In this patient residual activity of XDH is probably very low. In a patient with MoCo deficiency XDH is completely absent, and only traces of isoxanthopterin if any are detectable. Obviously, a defect in the cofactor of XDH seems to have more severe consequences for enzyme activity as previously suggested. Although the BH4 challenge mimics the actual XDH activity, obligate heterozygotes for MoCo deficiency can unfortunately not be distinguished from unaffected controls. The theoretical residual activity (Ç50%) seems to be sufficient for complete conversion of pterin to isoxanthopterin. The only limitation for this test is the treatment of gouty patients with allopurinol. Allopurinol may induce xanthinuria due to inhibition of XDH (18). However, the measurement of the pterin/isoxanthopterin ratio is of value in the follow-up of those patients. ACKNOWLEDGMENTS We thank Mr. S. Holms for the excellent technical help and Ms. M. Killen for editorial work. Supported by the Swiss National Science Foundation, Grant 3100-043380.95, and the HartmannMu¨ller Research Foundation (both to N.B.).
REFERENCES 1. Simmonds HA, Reiter S, Nishino T. Hereditary xanthinuria. In The Metabolic and Molecular Bases of Inherited Disease (Scriver CR, Beaudet AL, Sly WS, Valle D, Eds). New York: McGraw-Hill, 1995, Vol 2, pp 1781–1797. 2. Johnson JL, Wadman SK. Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency. In The Metabolic and Molecular Bases of Inherited Disease. (Scriver CR, Beaudet AL, Sly WS, Valle D, Eds). New York: McGraw–Hill, 1995, Vol 2, pp 2271–2283. 3. Higashino K, Yamamoto T, Hada T, Kono N, Kawachi M, Nanahoshi M, Takahashi S, Suda M, Naka M. Hereditary xanthine oxidase deficiency consisting of at least two subgroups. Adv Exp Med Biol 253A:87–91, 1989. 4. Beemer FA, Duran M, Wadman SK, Cats BP. Absence of hepatic molybdenum cofactor. An inborn error of metabolism associated with lens dislocation. Ophthalm Paediatr Genet 5:191–195, 1985.
AID
BMM 2514
/
af02$$$322
07-10-96 11:16:23
203
5. Skovby F. Disorders of sulfur amino acids. In Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases (Blau N, Duran M, Blaskovics M, Eds.). London: Chapman and Hall, 1996, pp 187–200. 6. Simmonds A. Purine and pyrimidine disorders. In Physician’s Guide to the Laboratory Diagnosis of Metabolic Diseases (Blau N, Duran M, Blaskovics M, Eds.). London: Chapman and Hall, 1996, pp 341–357. 7. Mousson B, Desjacques P, Baltassat P. Measurement of xanthine oxidase activity in some human tissues. An optimized method. Enzyme 29:32–43, 1983. 8. Kawachi M, Kono N, Mineo I, Yamada Y, Tarui S. Decreased xanthine oxidase activities and increased urinary oxypurines in heterozygotes for hereditary xanthinuria. Clin Chim Acta 188:137–146, 1990. 9. Johnson JL, Wuebbens MM, Mandell R, Shih VE. Molybdenum cofactor biosynthesis in humans. Identification of two complementation groups of cofactor-deficient patients and preliminary characterization of a diffusible molybdopterin precursor. J Clin Invest 83:897–903, 1989. 10. Curtius HC, Blau N, Kuster T. Pterins. In Techniques in Diagnostic Human Biochemical Genetics (Hommes FA, Ed.). New York: Wiley–Liss, 1991, pp 377–396. 11. Van Acker K, Eyskens FJ, Verkerk RM, Scharpe SS. Urinary excretion of purine and pyrimidine metabolites in neonate. Pediatr Res 34:762–766, 1993. 12. Duran M, de Bree PK, de Klerk JBC, Dorland L, Berger R. Molybdenum-cofactor deficiency: Clinical presentation and laboratory diagnosis. Int Pediatr, in press. 13. Schuierer G, Kurlemann G, Bick U, Stephani U. Molybdenum cofactor deficiency: CT and MR findings. Neuropediatrics 26:51–54, 1995. 14. Frayha RA, Salti IS, Araout RA, Katchadurian A, Uhtman SM. Hereditary xanthinuria, report on three patients and short review of the literature. Nephron 19:328–332, 1977. 15. Johnson JL, Wuebbens MM, Mandell R, Shih VE. Molybdenum cofactor deficiency in a patient previously characterized as deficient in sulfite oxidase. Biochem Med Metab Biol 40:86–93, 1988. 16. Shih VE, Abroms IF, Johnson JL, Carney M, Mandell R, Robb RM, Cloherty JP, Rajagopalan KV. Sulfite oxidase deficiency. Biochemical and clinical investigations of a hereditary metabolic disorder in sulfur metabolism. N Engl J Med 197:1022–1028, 1977. 17. Rembold H. Pteridine catabolism. In Biochemical and Clinical Aspects of Pteridines (Curtius HC, Pfleiderer W, Wachter H, Eds). Berlin: Walter de Gruyter, 1983, Vol 2, pp 107– 122. 18. Cameron JS, Moro F, Simmonds HA. Gout, uric acid and purine metabolism in paediatric nephrology. Pediatr Nephrol 7:105–18, 1993.
bmma
AP: BMM