寺平 良治

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N-2-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 mu mol/mol creatinine. The present results indicate that the peak found with elution from the C-18 column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.

Two high-molecular-weight forms of GTP cyclohydrolase I in human liver were detected by gel filtration chromatography; the major form had a Mr of about 400 kDa with a specific activity of 1.17 nmol/min/mg, while the other minor form had a Mr of about 600 kDa with a specific activity of 0.25 nmol/min/mg. Each of these two high-molecular-weight forms contained of the same subunit(s) with a Mr of 41 kDa as a component. These two active forms showed homotropic allosteric kinetics at various concentrations of GTP, and their Kd (concentration of ligand needed to produce half-saturation) values were estimated to be about 200 mu M for the lower-molecular-weight form and about 250 mu M for the higher- molecular-weight form. Hill's coefficients were estimated to be 1.95 between 10 and 200 mu M GTP and 0.96 between 200 and 400 mu M GTP for the lower-molecular-weight 400 kDa form and 1.55 between 10 and 200 mu M GTP and 0.75 between 200 and 400 mu M GTP for the higher-molecular-weight (600 kDa) form.

Deoxybiopterin, tentatively named for the new pteridine compound found in urine from patients with malignant lymphoma, was isolated by column chromatography on Dowex 1x2 anion exchange, Dowex 50W cation exchange, and reverse phase columns. The structure of deoxybiopterin was determined as 2-amino-4-hydroxy-6-[(1'S)-1'-hydroxypropyl]pteridine.

An enzyme immunosorbent assay of neopterin and biopterin on a polystyrene microtiter plate has been developed. A conjugate of neopterin or biopterin to bovine serum albumin was used to raise a specific antiserum against neopterin or biopterin in rabbits. An incubation mixture of the antiserum and samples prepared from human serum underwent another antigen-antibody reaction with the hapten fixed on the microtiter plate. The amount of antibody bound to the fixed hapten, which is inverse to the amount of hapten in the sample, was determined by using anti-rabbit IgG-horseradish peroxidase conjugate in a usual manner by measuring absorbance at 490 nm after reaction with o-phenylenediamine and hydrogen peroxide. The minimal detectable amounts of neopterin and biopterin were approximately 0.1 pmol. The specificity of the assay was so high that the assay system for neopterin completely distinguished it from biopterin, as judged from the cross-reaction of 0.002%, and vice versa. The amounts of neopterin and biopterin in human serum determined by the present method agreed well with those determined by high-performance liquid chromatography. We used the present method to determine the concentrations of neopterin in serum from healthy control subjects and patients with cancers and systemic lupus erythematosus; the results were consistent with literature data.

A new pteridine compound, named umanopterin, was isolated from urine of cancer patients. The structure was confirmed to be 2-amino-4-hydroxy-6-[(1'R,2' R)-1',2',3'-trihydroxypropyl]-pteridine, a diastereomer of neopterin.

A new natural pteridine, named oncopterin, was isolated from urine of cancer patients by a combination of DEAE anion exchange, CM cation exchange, and reverse phase column chromatography. Because of its character as a strong base, oncopterin was effectively separated from other unconjugated pteridines by the cation exchange chromatography. The structure of oncopterin was confirmed to be 2-(3-aminopropyl)amino-4-hydroxy-6-[(1'R,2'S)-1',2'-dihydroxypropyl]pteridine, a N2-(3-aminopropyl) derivative of biopterin. Oncopterin could not be isolated from urine of healthy controls, and thus this compound is expected to be a promising biochemical marker of cancers and other