6 Sawabe Κ. et ai.·. Intestinal Uptake of Tetrahydrobiopterin Pteridines Vol. 16, 2005, pp. 6-10 Intestinal Uptake of 6(R)-L-ery¿/?ro-Tetrahydrobiopterin is Distinct from the Liver-Type MTX-Sensitive Accumulation Process Keiko Sawabe2, Yasuko Suetake1, Kazunori Osuke Wakasugi\ Hiroyuki Hasegawa1 2 'Department of Biosciences and JBiotechnology Research Center, Teikyo University of Science and Technology. Ucnohara, Yamanashi 409-0193, 'Internal Medicine Clinical Immunohaematology, Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo 193-0944 Abstract In order to increase the tissue level of tetrahydrobiopterin (BH4), supplementation with 6R-tetrahydrobiopterin (6RBH4) has been widely employed. In this work, the profile of BH4 accumulation was compared between the liver and the small intestine after feeding with 10mg/kg of 6RBH4 or 7,8-dihydrobiopterin (7,8BH2 ). Endogenous BH4 was 7.20 ± 0.28 and 1.59 ± 0.13 nmol/g in the liver and the intestine, respectively. BH4 levels rose to 12.5 ± 1.35 (1.7-fold) and 53.0 + 19.6 nmol/g (40-fold) in the liver and the intestine within 30 min, respectively. Administration of 7,8BH2 caused an even higher rise in BH4 (14.4 ± 2.79 nmol/g) in the liver, while it led to a considerable increase in tissue BH2 in the intestine. When dihydrofolate reductase inhibitor MTX was adminis- tered before the pterin feeding, BH4 did not increase in the liver, while it increased 20-fold in the intestine, although the rise was 45% less than that in the absence of MTX. These results suggest that 1) the liver was scarcely able to uptake BH4 in its reduced form; 2) accumulation of BH4 in the liver was achieved through the MTX-sensitive pathway presumably driven by dihydrofolate reductase; 3) a large concentration of BH4 accumu- lated in the intestine, bypassing the MTX-sensitive pathway, that is, it was taken up directly as the fully reduced form, BH4; 4) the other BH4 accumulated in the intestine seemed to have been converted from BH2 through the MTX-sensitive pathway, as in the liver. Key words: tetrahydrobiopterin. dihydrobiopterin, methotrexate Introduction cultured cells and in vivo in mouse. In our in vitro w ork using cultured RBL2H3 and PC 12 cells, uptake 6-(R)-L-erv7/7/O-5,6,7,8-Tetrahydrobiopterin of BH4 was shown to be hampered by some cellular (6RBH4) is an essential cofactor of aromatic amino processes resulting in poor incorporation, while sepia- acid hydroxylases, phenylalanine- (1), tyrosine- (2) pterin and 7.8BH2 were incorporated and converted and tryptophan-hydroxylase (3, 4). The biosynthesis of into BH4 in an efficient manner ( 12-16). We observed classic monoamines such as dopamine, noradrenaline, in mice that orally administered BH4 resulted in an adrenaline as well as serotonin is dependent on the appreciable elevation of tissue BII4 levels in organs cofactor BH4. Furthermore, BH4 is also the cofactor of such as the liver and kidney (17). Remarkably, the NO-synlhase (NOS) (5, 6). An insufficient supply of increased portion of BH4 was revealed to have been BH4 results in a lowered production of monoamines converted from BH by a MTX-sensitive manner in and an uncontrolled release of active oxygen concomi- 2 tant with inadequate NO production, which might lead most organs examined, presumably by the enzymatic to various disorders regarding neural function and action of dihydrofolate reductase (18). In the same work, we noticed a very steep rise in BH2 levels in the blood circulation, respectively (7, 8). Regarding BH4 blood circulation following oral administration of therapy for patients with BH4 insufficiency, however, the efficiency of BH4 uptake has not been appreciated 6RBH4. In this study, we examined biopterin accumu- (9-11 ). We have been working on permeability and the lation (BH4 and BH2) in intestinal mucosa and the liver metabolic fate of exogenously administered BH4 with after feeding 6RBH4 to mice. We analyzed whether the Correspondence to: Dr. Keiko Sawabe, Department of Biosciences, Teikyo University of Science and Technology. Uenohara, Yamanashi 409-0193, Japan, Phone: +(81 (-554-63-441 1, Fax: +(81 (-554-63-443 1, e-mail: k-sawabe(í¿st.ntu.ae.jp Pteridines/Vol. 16/No. 1 Sawabe Κ. et aì.\ Intestinal Uptake of Tetrahydrobiopterin 7 BH4 was directly incorporated in its fully reduced form acid-l2 oxidation was taken as the "total biopterin". or produced in situ from acquired BH2, the oxidation The diffcrcncc between the biopterin quantity after product of exogenous BH4. acid-oxidation and that after alkaline-oxidation was In this work, we report the results that intestinal tis- termed "BH4" throughout this paper. Since the amount sue can uptake BH4 in its reduced form, by passing the of fully oxidized biopterin detected in fresh tissue MTX-sensitive route, while the liver uptakes BH4 extracts was insignificant, the biopterin calculated through the MTX-sensitive pathway. from the "total biopterin" minus "BH4" was termed "BH:". When 6-(R)-L-erv?/7ro-tetrahydrobioptcrin was specified as a reagent, "6RBH4" was used instead Materials and Methods of "BH4". For example, a working solution of the reagent BH, for administration to mice was termed 6-( R )-L-erythro-5,6,7,8-Tetrahydrobiopterin "6RBH4". (6RBH4) was donated by Daiich Suntory (Tokyo) and 7,8-dihydrobiopterin (7.8BH2) was purchased from Schirks Laboratories (Jona, Switzerland). Results and Discussion Methotrexate (MTX) was purchased from Wako (Osaka, Japan). CM-cellulose (medium viscosity, In order to measure intestinal absorption of orally sodium salt) was obtained from Fluka (Buchs, administered BH4, we gave 6RB114 at a dose of 10 Switzerland). C57BL/6J mice were from Japan SLC mg/kg to mice (C57BL/6J 10 week, male), then (Hamamatsu, Japan) and adult males 10 weeks of age biopterin uptake (BH4 and BH2) was measured in the were used in the present experiments. The animals liver and intestine after administration. were maintained on an ordinary laboratory chow and Increase in biopterin (BH4 and BH2) in the liver was tap water ad libidunu under a constant 1 2-h light-dark first examined after administration. The liver con- cycle. The experiments were conducted in accordance tained endogenous biopterin at a level of 7.64 ± 0.32, with the ethical guidelines of the Teikyo University of of which 94.2% was the fully reduced form, BH4. Science and Technology Animal Experimentation When 6RBH4 was given orally, biopterin content Committee. increased to about 2-fold within 30 min. continued to Pterins were given to animals at a dose of 10mg/kg increase, and reached more than 5-fold at 120 min, body weight. The pterin compounds were dissolved in then decreased gradually. As mentioned previously 0.05 Ν HCl just prior to oral administration (p.o.). (18), a steep BH2 surge appeared in the blood 30-60 When MTX was given, the reagent was suspended in min after 6RBH4 administration. Since liver cells con- aqueous 2% CM-cellulose and was administered 90 tain dihydrofolate reductase DHFR which converts min before the pterins. The liver and small intestine 7.8BH2 to BH4. some portion of the BH4 might be were dissected under anaesthesia. The proximal third derived from 7,8BH2. In order to analyze the contribu- of the small intestine was taken and opened longitudi- tion of the DHFR-pathway, an inhibitor, MTX, was nally, washed with cold saline, blotted three times with administered 90 min prior to 6RBH4 administration, filter paper to remove the mucus coat, weighed, frozen then incorporation of biopterin in the liver was meas- ured, as shown in Fig. I b. MTX administration did not immediately in liquid N2. and then stored at -80°C until determination. alter the endogenous content of total biopterin but sig- Frozen tissues were partially thawed and homoge- nificantly lowered the percentage of BH4 (designated as "%BH ") to 76.6%, suggesting that the MTX-sensi- nized immediately in a 5 vol of 0.1 Ν HCl. An aliquot 4 tive pathway was involved to a considerable extent in ( 100 μι.) of the homogenate was mixed with a 0.5 vol maintaining the endogenous level. When 6RBH4 was of an acid-iodine solution (2% I2. 3% KI in 0.1 Ν HCl), given to the animals in the absence of MTX, liver BH or with alkaline-iodine (2% l , 3% KI in 0.2 Ν NaOH) 4 2 rose up to 1.7-fold with a constant %BH (95.4%). In for oxidation under acid or alkaline conditions, respec- 4 the presence of MTX, total biopterin also increased up tively ( 19). The oxidation reaction was allowed to pro- to 1.58-fold, however. BH scarcely rose and conse- ceed for 1 h at room temperature. After addition of a 4 quent %BH decreased to 57.7%, therefore, a large 0.5 vol of ascorbic acid (2.5%) dissolved in perchloric 4 quantity of 7,8BH2 appeared. The large quantity of acid (0.4 M), a clear solution was obtained by cen- BH2 might otherwise be converted to BH4 by an trifugation at 1 (),()()() χ g for 10 min. Biopterin and MTX-sensitive pathway. 7,8BH2 administration at the pterin were determined by HPLC equipped with a flu- same dose raised the liver BH4 level 1.97-fold (%BH4 orescence detector (Ex=350 and Em=450 nm). The - 95.4) within 30 min in the absence of MTX. The solid phase was Fine-SIL C18T-5 and the mobile increase in BH4 caused by BH2 administration was phase was 7% methanol. Biopterin measured after Pteridincs/Vol. 16/No. 1 8 Sawabe Κ. et αι.: Intestinal Uptake of Tetrahydrobiopterin 10 (a) 8 Pterin Non BH4 7,8BH2 Pterin Non BH4 7,8BH2 MTX - + - + - + MTX -+-+-+ Figure 1.