Sadao Matsuura

Biopterin in human brain and urine from controls and parkinsonian patients: Application of a new radioimmunoassay

Total biopterin concentrations in the post-mortem human brain (caudate nucleus) and in the urine of controls and parkinsonian patients were measured by a newly developed radioimmunoassay. There was good correlation between the total biopterin level and tyrosine hydroxylase activity in the human brain. Biopterin concentrations in the caudate nucleus were greatly reduced in parkinsonian patients. In contrast, the reduction of urinary biopterin in parkinsonian patients was slight and not statistically significant, as compared with normal controls.

A N-methyltransferase in human brain catalyses N-methylation of 1,2,3,4-tetrahydroisoquinoline into N-methyl-1,2,3,4-tetrahydroisoquinoline, a precursor of a dopaminergic neurotoxin, N-methylisoquinolinium ion.

N-methylation of 1,2,3,4-tetrahydroisoquinoline (TIQ) present in human brain was found by a N-methyltransferase in human brain homogenate. Formation of N-methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) from TIQ was quantitatively assayed by high-performance liquid chromatography with electrochemical detection. The reaction required S-adenosyl-L-methionine (SAM) as a methyl donor and in terms of SAM the value of the Michaelis constant, Km, and of the maximal velocity, Vmax, were 5.11 +/- 1.69 microM and 7.31 +/- 0.21 pmol/min/mg protein, respectively. The value of Km and Vmax in terms of TIQ were 20.9 +/- 5.5 microM and 7.98 +/- 1.21 pmol/min/mg protein, respectively. The optimal pH of the reaction was 8.25. A major part of the N-methyltransferase activity was found in the cytosolic fraction of human cortex. Enzymatic formation of NMTIQ indicates that in human brain this compound may be an intermediate of biosynthesis of a potent neurotoxin of dopamine metabolism, N-methylisoquinolinium ion, from naturally-occurring TIQ.

Oxidation of N-Methyl-1,2,3,4-Tetrahydroisoquinoline into the N-Methyl-Isoquinolinium Ion by Monoamine Oxidase

Abstract: N‐Methyl‐1,2,3,4‐tetrahydroisoquinoline (NMTIQ) was found to be oxidized by monoamine oxidase (MAO) into N‐methylisoquinolinium ion, which was proved to inhibit enzymes related to the metabolism of catecholamines, such as tyrosine hydroxylase, aromatic‐L‐amino acid decarboxylase, and MAO. NMTIQ was oxidized by both types A and B MAO in human brain synaptosomal mitochondria. Oxidation was dependent on the amount of MAO sample and the reaction time. Enzyme activity with respect to NMTIQ reached optimum at a pH of ∼7.25, as was the case with other substrates. Type A MAO had higher activity for this substrate than type B. The Km and Vmax values of the oxidation by types A and B MAO were 571 ± 25 μM and 0.29 ± 0.06 pmol/min/mg protein, and 463 ± 43 μM and 0.16 ± 0.03 pmol/min/mg protein, respectively. The Vmax values of types A and B MAO for NMTIQ were much smaller than those for other substrates such as kynuramine. NMTIQ was the first tetrahydroisoquinoline shown to be oxidized into the isoquinolinium ion by MAO in the brain.

Presence of tetrahydroisoquinoline, a parkinsonism-related compound, in foods

We detected 1,2,3,4-tetrahydroisoquinoline (TIQ) in the various foods studied. Makino et al. reported that TIQ was detected in cheese, wine and cocoa. We confirmed their findings, except for the presence of TIQ in cocoa, which could not be detected because of impurities in the extract

Radioimmunoassay for biopterin in body fluids and tissues

Specific antibodies against l-erythro-biopterin have been prepared in rabbits using the conjugates to bovine serum albumin. The antiserum against l-erythro-biopterin distinguished among l-erythro-tetrahydro- or 7,8-dihydro-biopterin, the other three stereoisomers of biopterin, d-erythro-neopterin, folic acid, and other synthetic pteridines. Using the specific antiserum against l-erythro-biopterin, a radioimmunoassay has been developed to measure the biopterin concentrations in urine, serum, cerebrospinal fluid, and tissues. The conjugate of l-erythro-biopterin with tyramine, 4-hydroxy-2-[2-(4-hydroxyphenyl)ethylamino]-6-(l-erythro-1,2-dihydroxypropyl)pteridine (BP-TYRA), was synthesized and labeled with 125I as the labeled ligand for the radioimmunoassay. BP-125I-TYRA had similar binding affinity as the natural l-erythro-biopterin and was thus permitted to establish a highly sensitive radioimmunoassay for biopterin. The limit of sensitivity of the radioimmunoassay with BP-125I-TYRA as labeled ligand was 0.5 pmol. The total concentration of biopterins, i.e., biopterin, 7,8-dihydro-, quinonoid dihydro and tetrahydrobiopterins, in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, whereas iodine oxidation under alkaline conditions gave the concentration only of the former two. Biopterin in urine could be measured directly using 1 μl of urine, but a pretreatment with a small Dowex 50-H+ column was required for serum, cerebrospinal fluid, and brain tissues.

Kinetic properties of tyrosine hydroxylase purified from bovine adrenal medulla and bovine caudate nucleus

Abstract Tyrosine hydroxylase ( l -tyrosine,tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) was highly purified from the soluble fraction of either bovine adrenal medulla or bovine caudate nucleus and the kinetic properties were compared using 6-methyltetrahydropterin or natural (6R)- l -erythro- tetrahydrobiopterin . Two enzyme fractions with different molecular weights from bovine adrenal medulla were separated on a DEAE-Sephacel column; the apparent molecular weights of the first and second enzyme fractions (Fraction I and Fraction II) were estimated to be 280 000 and 390 000, respectively, by Bio-Gel A-1.5 m chromatography. Fraction II was judged to be composed of several enzyme forms with different molecular weights. The molecular weight of the subunit of Fraction I was estimated to be 60 000 by SDS-polacrylamide slab gel electrophoresis. Therefore, the enzyme may be a tetramer. The kinetic properties of the low molecular weight form (Fraction I) and the high molecular weight form (Fraction II) were compared. When 6-methyltetradropterin was used as an artificial cofactor, Fraction II showed two distinct apparent Km values for the pterin cofactor, whereas Fraction I showed a single apparent Km value. Apparent Km values for tyrosine and oxygen were similar in the two enzyme preparations, and neither tyrosine nor oxygen was inhibitory. When (6R)- l -erythro- tetrahydrobiopterin was used as the natural cofactor, Fraction I showed two distinct apparent Km values for tetrahydrohiopterin in the presence of 217 μM oxygen and 50 μM tyrosine, but a single apparent Km value in the presence of 49 μM oxygen and 50 μM tyrosine. Tyrosine at 100 μM and oxygen at 94 μM were inhibitory on Fraction I with tetrahydrobiopterin as cofactor. These results show that two apparent Km values for natural (6R)- l -erythro- tetrahydrobiopterin cofactor can be observed only in air (217 μM oxygen), and that the different enzyme forms have different apparent Km values for the pterin cofactor. Tyrosine hydroxylase activity in bovine caudate nucleus was also separated into two fractions on a DEAE-Sepahcel column; the major peak was purified further and the apparent molecular weight was estimated to be 310 000 on a Bio-Gel A-1.5 m column. When natural (6R)- l -erythro- tetrahydrobiopterin was used as a cofactor, the enzyme showed a single apparent Km value for tetrahydropterin cofactor in the presence of 217 μM oxygen and 20 μM tyrosine. This result shows that tyrosine hydroxylase from caudate nucleus has a similar molecular weight but different kinetic properties compared with the enzyme from the adrenal medulla.

Migration of tetrahydroisoquinoline, a possible parkinsonian neurotoxin, into monkey brain from blood as proved by gas chromatography-mass spectrometry.

1,2,3,4-Tetrahydroisoquinoline (TIQ) was quantitated by use of gas chromatography-mass spectrometry in brains and livers of marmosets which showed parkinsonism after daily subcutaneous injection of TIQ. TIQ showed greatly increased levels in the brains and livers of the TIQ-treated marmosets, with no detectable metabolites of TIQ. TIQ was present as an endogenous amine in the brains and livers of saline-treated marmosets at very low concentrations. It thus seems that TIQ can pass easily through the blood-brain barrier but cannot be metabolized in the brain or the liver. It is possible that TIQ accumulated in the brain may produce parkinsonism.

(6R)-Tetrahydrobiopterin Increases the Activity of Tryptophan Hydroxylase in Rat Raphe Slices

Abstract The effects of (6R)‐ and (6S)‐tetrahydrobiopterin (BPH4), tetrahydroneopterin, and 6‐methyltetrahydropterin on the activity of tryptophan hydroxylase were investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measurement of 5‐hydroxytryptophan (5‐HTP) formation under inhibition of aromatic L‐amino acid decarboxylase with use of HPLC‐fluorometric detection. (6R)‐BPH4 (the naturally occurring form) at 42 μM, tetrahydroneopterin at 50 μM, and 6‐methyltetrahydropterin at 100 μM increased tryptophan hydroxylase activity to 350, 145, and 146% of control values, respectively. (6S)‐BPH4, however, had no significant effects on tryptophan hydroxylase activity. These results suggest that tryptophan hydroxylase is subsaturating in vivo for the naturally occurring cofactor, (6R)‐BPH4, and that the concentration of (6R)‐BPH4 may play an important role for the regulation of tryptophan hydroxylase activity in vivo.

Effects of stereochemical structures of tetrahydrobiopterin on tyrosine hydroxylase.

1. Four stereochemical isomers of tetrahydrobiopterin, i.e., 6-L-erythro-, 6-D-erythro-, 6-L-threo-, or 6-D-threo-1,2-dihydroxypropyltetrahydropterin, have been synthesized and used as cofactors for tyrosine hydroxylase (EC 1.14.18.-) purified from the soluble fraction of bovine adrenal medulla. The L-erythro- (the putative natural cofactor) and D-threo isomers showed a striking similarity in their cofactor activities for tyrosine hydroxylase; the remaining two isomeric tetrahydrobiopterins, D-erythro and L-threo isomers, also had very similar cofactor characteristics. 2. The Km values of the L-erythro and D-threo isomers as cofactor were found to be dependent on their concentrations. When their concentrations were below 100 muM, the Km values of the L-erythro and D-threo isomers were fairly low (about 20 muM). However, the Km values were markedly higher (about 150 muM) at concentrations above 100 muM. The same kinetic behavior was also observed with the tetrahydrobiopterin prepared from a natural source (bullfrog). In contrast, the Km value of the L-threo or D-erythro isomer was found to be independent of the concentration and remained constant throughout the concentration examined. 3. The Km values of tyrosine did not show much difference (from 20 muM to 30 muM) with respect to the structure of the four isomeric cofactors. At high concentrations tyrosine inhibited the enzymatic reaction with any one of the four tetrahydrobiopterin cofactors. 4. Oxygen at high concentrations was also inhibitory with any one of the four stereochemical isomers as cofactor. Approximate Km values for oxygen with the tetrahydrobiopterins as cofactor were 1-5%. 5. In contrast to the four isomers of tetrahydrobiopterin, when 6-methyltetrahydropterin or 6,7-dimethyltetrahydropterin was used as cofactor tyrosine or oxygen did no inhibit the enzymatic reaction at high concentrations, and the Km values toward the pterin cofactor, tyrosine, and oxygen were significantly higher than the Km values with the tetrahydrobiopterins as cofactor.

Radioimmunoassay for neopterin in body fluids and tissues

Specific antibodies against D-erythroneopterin have been prepared in rabbits using a conjugate of D-erythroneopterin to bovine serum albumin (D-erythroneopterinylcaproyl-bovine serum albumin). The antiserum distinguished D-erythroneopterin from other pteridines, i.e., three stereoisomers of neopterin, L-erythrobiopterin, folic acid, xanthopterin, and four other synthetic pteridines. Using this specific antiserum, a radioimmunoassay for D-erythroneopterin has been developed to measure the neopterin concentrations in urine and tissues. The conjugate of D-erythroneopterin with tyramine (NP-Tyra) was synthesized and labeled with 125I as the labeled ligand NP-[125I]tyra for the radioimmunoassay. The minimal detectable amount of neopterin was about 0.1 pmol. The concentration of total neopterin (neopterin, 7,8-dihydroneopterin, quinonoid dihydroneopterin, and tetrahydroneopterin) in the biological samples was obtained by iodine oxidation under acidic conditions prior to the radioimmunoassay, and that of neopterin plus 7,8-dihydroneopterin by oxidation under alkaline conditions. Total neopterin values in human urine obtained by this new radioimmunoassay showed a good agreement with those obtained by high-performance liquid chromatography with fluorescence detection. With rat tissue samples which contained very low concentrations of neopterin as compared to biopterin, biopterin was simultaneously determined by our previously reported radioimmunoassay, and neopterin values were corrected for the cross-reactivity (0.1%). The neopterin concentrations obtained by this method agreed with the values obtained by the radioimmunoassays for neopterin and biopterin after their separation by high-performance liquid chromatography. This very small amount of neopterin, as compared with biopterin, in rat tissues could not be determined by high-performance liquid chromatography-fluorometry alone due to the masking of the neopterin peak by a large biopterin peak.(ABSTRACT TRUNCATED AT 250 WORDS)