Neopterin and biopterin production and GTP cyclohydrolase I (GTPCH) activity were measured in cytokine-stimulated fibroblasts; 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SR), and dihydropteridine reductase (DHPR) activities were measured in unstimulated fibroblasts.

Fibroblasts from patients with DRD and autosomal recessive GTPCH deficiency showed reduced GTPCH activity (15.4% and 30.7% of normal activity, respectively) as compared with controls (P < 0.001). Neopterin production was very low and biopterin production was reduced in both disorders. PTPS and DHPR deficient cells showed no enzyme activities; in PTPS deficiency the pattern of pterins production was typical (neopterin: 334-734 pmol/mg, controls 18-98; biopterin: 0 pmol/mg, controls 154-303). Reference values of all enzyme activities and pterins production were measured in fibroblasts and also in amniocytes for prenatal diagnosis.

Cell culture and treatment with cytokines.

Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum, 50 units of penicillin/mL and 50 mg of streptomycin/mL. Amniocytes were cultured in AmnioMax C-100 medium. Cells were passaged by trypsinization and studied at low passage numbers (3-12). All cells were Mycoplasma negative, checked by the Hoechst staining method (31). Cultures were kept at 37°C in humidified air containing 5% CO2.

In order to induce the expression of GTPCH, confluent cell monolayers in 78 cm2 plates were stimulated with recombinant human IFN-g and TNF-a at concentrations of 250 U/mL and 100 U/mL, respectively, in fresh medium. After incubation for 24 hours, cells were harvested by trypsinization, washed with phosphate-buffered saline and immediately lysed for neopterin and biopterin measurement and for GTPCH activity assay. PTPS, DHPR, and SR are constitutively expressed in fibroblasts, therefore their activities were tested in unstimulated cells. Confluent cells in 78 cm2 plates were harvested, washed with phosphate-buffered saline, and kept at –80°C until analyzed for PTPS, DHPR and SR.

Measurement of neopterin and biopterin production in cell extracts.

Cytokine-stimulated cells from one confluent 78 cm2 plate were lysed in 150 mL of 50 mmol/L Tris-HCl (pH 7,4) containing 1 mmol/L EDTA and 1 mmol/L DTE, by freezing and thawing 6 times. Lysate was centrifuged at 15000 x g and 100 mL of the supernatant were oxidized with 6 mL of 1 mol/L HCl and 20 ml of 1% (w/v) iodine solution for 15 minutes at room temperature in the dark. Oxidation was stopped by adding 20 mL of 1% ascorbic acid (w/v, freshly prepared). Ten microliters of buffer (1 mol/L Tris-HCl, pH 9.6 containing 80 mmol/LMgCl2) were added to the sample to adjust to pH 8.0-9.0. Dephosphorylation of neopterin triphosphate was achieved by hydrolysis with alkaline phosphatase (16,8 U) for 60 minutes at 37°C. The reaction was stopped by acidification to pH 2.0 with 7 mL of 2 mol/L HCl. The sample (final volume: 175 mL) was subsequently deproteinized on Ultrafree-MC and analyzed by HPLC (32). The intracellular concentrations of neopterin and biopterin after 24 hours stimulation with cytokines are expressed as pmol per mg of protein.

GTPCH assay.

The assay was performed as previously described by Hatakeyama (33), with some modifications. The assay monitors the conversion of the substrate GTP under saturating conditions to neopterin triphosphate, which is detected as neopterin, the oxidized and dephosphorylated product (Figure 1).

Fibroblasts were analyzed immediately after 24 hours incubation with cytokines. Cells from one confluent 78 cm2 plate were lysed in 200 mL of freshly prepared homogenization buffer (50 mmol/L Tris-HCl, pH 7.5 containing 0.1 mol/L KCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L leupeptin, and 1 mmol/L pepstatin) by freezing and thawing 6 times and were subsequently centrifuged at 15000 x g for 5 minutes. One hundred and fifty microliters of the supernatant were desalted on a spin column (Nick spin column, Sephadex G50, Pharmacia Biotech). Fifty microliters of filtrate were added to 148 mL of reaction buffer (50 mmol/L Tris-HCl, pH 7.5 containing 0.1 mol/L KCl and 1 mmol/L EDTA) and 2 mL of 100 mmol/L GTP. One hundred microliters of the mixture were incubated for 60 minutes at 37°C, whereas the remaining 100 mL were immediately oxidized (blank with cell extract). A blank without cell extract (25 mL of homogenization buffer containing 1 mL 100 mmol/L GTP, and 74 mL of reaction mixture) was incubated together with the sample. The reaction was stopped by cooling the sample on ice and adding 10 mL of oxidizing solution (0.5% w/v iodine/1% w/v potassium iodide in 1 mol/L HCl). After oxidation in the dark for 60 minutes, the reaction was stopped by adding 10 mL of 2% ascorbic acid (w/v, freshly prepared). The mixture was adjusted to pH 8.5 by adding 14 mL of 1 mol/L NaOH and the sample was incubated with 20 mL of alkaline phospatase solution (300 U/mL of calf intestine alkaline phospatase in 0.1 mol/L Tris-HCl, pH 8.0 containing 1 mmol/L MgCl2, and 0.1 mmol/L ZnCl2) for 60 minutes at 37°C. Neopterin was measured, after deproteinization through an Ultrafree-MC filter, by HPLC (32). One unit of GTPCH produces 1 mmol neopterin per minute at 37°C.

Figure 1 - Pterins production in Cytokine-Stimulated Fibroblasts

Figure 2 - GTPCH activity

Reference

• Bonafé L, Thöny B, Leimbacher W, Kierat L, Blau N. Diagnosis of Dopa-responsive dystonia and other tetrahydrobiopterin disorders by the study of biopterin metabolism in fibroblasts. Clin Chem 2001;47:477-485.