HOME

CONTACT

SITE MAP

GTPCH		GFRP		PTPS		SR		PCD		DHPR

Enzymatic Assay - 6-Pyruvoyl-tetrahydropterin synthase (PTPS)

PTPS is expressed in almost all tissues and cells used for diagnostic purpose. The most common method for enzymatic diagnosis is to measure PTPS in red blood cells (RBC). PTPS activity is higher in young erythrocytes, including reticulocytes, in cord blood during the first few days of life. Relating the expected values of PTPS activity to the relative reticulocyte count (o/oo), PTPS values increase by a factor of two from the first to the ninth day of life. In order to be able to discriminate between affected homozygotes, heterozygotes, and controls, neopterin and biopterin in urine should be measured simultaneously.

Assays are also available for liver, skin fibroblasts and amniocytes.

Method

The method is based on the measurement of tetrahydrobiopterin (BH4) derived from the substrate, dihydroneopterin triphosphate (110 uM), in the presence of NADPH (1 mM), NADH (1 mM), dihydropteridine reductase (220 mU), magnesium (10 mM), sepiapterin reductase (5 mU), and Tris-HCl buffer, pH 7.4 (0.1 M). Biopterin is measured fluorometrically by HPLC.

Material

• RBC: ~0.5 ml RBC separated from heparinized blood (do not use EDTA). Keep frozen at -20°C.
• Liver biopsy: ~10 mg. Keep frozen at -20°C.
• Fibroblasts / amniocytes: 25 ml flask with cultured cells. Send at RT.

• In addition to the RBC (see above) a portion (5 ml) of urine is required.

• RBC (mU/g Hb)
• Adults: 11-29
• Newborns: 34-64
• Fetus (16-20 WG): 35-77
• Liver biopsy (mU/mg prot.) : 0.5-1.0
• Fibroblasts (mU/mg prot.) : 1.9-2.6

• Shintaku H, Niederwieser A, Leimbacher W, Curtius HC. Tetrahydrobiopterin deficiency: Assay for 6-pyruvoyl-tetrahydropterin synthase activity in erythrocytes, and detection of patients and heterozygous carriers. Eur. J. Pediatr. 1988;147:15-19.
• Blau N, Kierat L, Matasovic A, Leimbacher W, Heizmann CW, Guardamagna O, Ponzone A. Antenatal diagnosis of tetrahydrobiopterin deficiency by quantification of pterins in amniotic fluid and enzyme activity in fetal and extrafetal tissue. Clin. Chim. Acta. 1994;226:159-169.
• Blau N, Blaskovics M. Hyperphenylalaninemia. In: Blau N, Duran M, Blaskovics M, eds. Physician's Guide to the Laboratory Diagnosis of Metabolic Diseases. London: Chapman & Hall, 1996: 65-78.
• Werner ER, Werner-Felmayer G, Wachter H. High-performance liquid chromatographic methods for the quantification of tetrahydrobiopterin biosynthetic enzymes. J Chromatogr Biomed Appl 1996;684(1-2):51-58.

Confidentiality of data relating to individual patients and visitors to the BH4 Web site, including their identity, is respected by this Web site. The Web site owners undertake to honour or exceed the legal requirements of medical/health information privacy that apply in the country and state where the Web site and mirror sites are located.

Web site and all contents © BH4.org 2005