Loading test with BH4

Two types of loading tests have been used in the differential diagnosis of BH4 deficiencies:

• A simple oral BH4 load.
• A combined phenylalanine and BH4 load.

Historically, the first method tried1 and predicted as the most convenient to selectively screen among HPAs, exploited the lowering of plasma phenylalanine in BH4-deficient patients after the administration of exogenous BH4. Intravenous loading with 2 mg/kg BH4 was originally proposed by Danks et al.2. With the increased purity and availability of this synthesized cofactor (BH4), an oral loading test (2.5 mg/kg bw) was introduced by Niederwieser et al.3 and standardized at a dose of 7.5 mg/kg by the same group4. This very simple test discriminated between patients with PAH and BH4 deficiencies.

However, it soon became obvious that some DHPR-deficient patients could be misdiagnosed, in as much as their serum phenylalanine levels was not lowered by BH45, thus introducing an unacceptable limitation if this was the sole test performed. The observation that BH4 nonresponsiveness in DHPR deficiency correlates with the presence of a mutant enzyme5 and that it can be overcome by increasing the dose (20 mg/kg bw) of the administered synthetic cofactor, led to a new standard protocol for the BH4 loading. This tests detects also BH4-responsive variant of PAH deficiency6,7.

Phenylalanine (100 mg/kg bw) plus BH4 (20 mg/kg bw) given orally enables selective screening of all BH4 deficiencies when pterin analysis is not available or when a clear diagnosis has not previously been made8. In its simplest form, this test can be interpreted using only four blood spots from a Guthrie card.

Loading test with BH4 (20 mg/kg bw.)

A simple oral BH4 loading test is performed at least 30 min before the meal. BH4 tablets (Dr. Schircks Lab., Jona, Switzerland, or granulate Suntory Pharmaceuticals, Tokyo, Japan) are dissolved in orange juice or water (ca. 10 ml per 10 mg BH4) and administered immediately at a dose of 20 mg /kg body weight. Blood for the analysis of phenylalanine and tyrosine is collected before, and 4, 8, and 24 hours after administration.

Combined loading test with Phe (100 mg/kg bw.) and BH4 (20 mg/kg bw.)

Tablets of synthetic cofactor (20 mg/kg body weight) are dissolved as described above and administered 3 hours after the Phe load (100 mg/kg body weight). Blood is collected before, and 3, 7, and 11 hours after Phe administration. Store at -20°C.

References

1. Danks DM, Cotton RG, Schlesinger P. Tetrahydrobiopterin treatment of variant form of phenylketonuria. Lancet 1975; 2:1043.
2. Danks DM, Cotton RG, Schlesinger P. Variant forms of phenylketonuria. Lancet 1976; 1:1236-1237.
3. Niederwieser A, Curtius HC, Viscontini M, Schaub J, Schmidt H. Phenylketonuria variants. Lancet 1979; 1:550.
4. Niederwieser A, Curtius HC, Wang M, Leupold D. Atypical phenylketonuria with defective biopterin metabolism. Monotherapy with tetrahydrobiopterin or sepiapterin, screening and study of biosynthesis in man. Eur. J. Pediatr. 1982; 138:110-112.
5. Lipson A, Yu J, O Halloran M, Potter M, Wilken B. Dihydropteridine reductase deficiency: non-response to oral tetrahydrobiopterin load test. J. Inherit. Metab. Dis. 1984; 7:69-71.
6. Kure S, Hou DC, Ohura T, Iwamoto H, S. S, Sugiyama N, Sakamoto O, Fujii K, Matsubara Y, Narisawa K. Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. J Pediatr 1999;135(3):375-378.
7. Blau N, Trefz FK. Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency: Possible regulation of gene expression in a patient with the homozygous L48S mutation. Mol Genet Metabol 2002;75:186-187.
8. Ponzone A, Guardamagna O, Spada M, Ferraris S, Ponzone R, Kierat L, Blau N. Differential diagnosis of hyperphenylalaninemia by a combined phenylalanine-tetrahydrobiopterin loading test. Eur. J. Pediatr. 1993; 152:655-661