Tetrahydrobiopterin Uptake in Supplemental

Tetrahydrobiopterin Uptake in Supplemental Administration: Elevation of Tissue Tetrahydrobiopterin in Mice Following Uptake of the Exogenously Oxidized Product 7,8-Dihydrobiopterin and Subsequent Reduction by an Anti-folate-Sensitive Process

Keiko Sawabe1,2,*, Kazunori Osuke Wakasugi3, and Hiroyuki Hasegawa1,2 1Department of Biosciences and 2Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Yamanashi 409-0193, Japan 3Internal Medicine Clinical Immunohaematology, Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo 193-0944, Japan

Received April 20, 2004; Accepted August 6, 2004

Abstract. In order to increase the tissue level of tetrahydrobiopterin (BH4), supplementation with 6R-tetrahydrobiopterin (6RBH4) has been widely employed. In this work, the effectiveness of 6RBH4 was compared with 7,8-dihydrobiopterin (7,8BH2) and sepiapterin by administration to mice. Administration of 6RBH4 was the least effective in elevating tissue BH4 levels in mice while sepiapterin was the best. In all three cases, a dihydrobiopterin surge appeared in the blood. The appearance of the dihydrobiopterin surge after BH4 treatment suggested that systemic oxidation of the administered BH4 had occurred before accumulation of BH4 in the tissues. This idea was supported by the following evidences: 1) An increase in tissue BH4 was effectively inhibited by methotrexate, an inhibitor of dihydrofolate reductase which reduces 7,8BH2 to BH4. 2) When the unnatural diastereomer 6SBH4 was administered to mice, a large proportion of the recovered BH4 was in the form of the 6R-diastereomer, suggesting that this BH4 was the product of a dihydrofolate reductase process by which 7,8BH2 converts to 6RBH4. These results indicated that the exogenous BH4 was oxidized and the resultant 7,8BH2 circulated through the tissues, and then it was incorporated by various other tissues and organs through a pathway shared by the exogenous sepiapterin and 7,8BH2 in their uptake. It was demonstrated that maintaining endogenous tetrahydrobiopterin in tissues under ordinary conditions was also largely dependent on an methotrexate-sensitive process, suggesting that cellular tetrahydrobiopterin was maintained both by de novo synthesis and by salvage of extracellular dihydrobiopterin.

Keywords: dihydrofolate reductase, tetrahydrobiopterin, sepiapterin, methotrexate

Introduction are known to function in tissues and organs of neural, circulatory, and immune systems, respectively (7, 8). 6-(R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (6RBH4) Since all NOS isoforms require BH4 as an essential is an essential cofactor of the aromatic amino acid cofactor and regulator, BH4 might be a key substance hydroxylases, phenylalanine- (1), tyrosine- (2), and in ensuring that these enzymes maintain their role in tryptophan-hydroxylase (3, 4). The biosynthesis of various bodily functions. Therefore, an insufficient classic monoamines such as dopamine, noradrenaline, supply of BH4 might result in various disorders related adrenaline, and serotonin is dependent on cofactor BH4. to regulatory systems in the body, as mentioned above. Furthermore, it is also a cofactor of NO-synthase (NOS) Indeed, many studies have appeared demonstrating (5, 6). Three NOS isoforms, nNOS, eNOS, and iNOS, that such disorders have been effectively diminished by BH4 supplementation employed in the treatment of a *Corresponding author. FAX: +81-0554-63-4431 monoamine or NO deficiency. E-mail: [email protected]

124 Tetrahydrobiopterin Uptake in Mice 125

Many patients with a severe deficiency of BH4 have body weight irrespective of the pterin species, sepia- received BH4 therapy and have had their quality of life pterin, 7,8BH2, or 6RBH4. The pterin compounds were greatly improved. In these cases, however, the efficiency dissolved in 0.05 N HCl or 0.9% NaCl just prior to oral of BH4 uptake has not been appreciable (9 – 20). What administration (p.o.) or intraperitoneal injection (i.p.), is known concerning the pharmacodynamics of admin- respectively. When MTX was given, the reagent was istered BH4 cannot sufficiently explain the poor uptake suspended in aqueous 2% CM-cellulose and was admin- of the compound by these recipients. There has been istered 90 min before the pterins. Whole blood was little basic research on the permeability and the meta- collected by heart puncture from mice under anesthesia, bolic fate of exogenously given BH4 as related to various and then the brain and other organs were dissected. The organs of the body. Biosynthesis of tetrahydrobiopterin proximal third of the small intestine was taken and from guanosine 5'-triphosphate (GTP) requires at least opened longitudinally, washed with cold saline, and then three enzymes: GTP-cyclohydrolase I, 6-pyruvoyl tetra- blotted three times with filter paper to remove the mucus hydropterin synthase, and sepiapterin reductase (21, 22). coat. The collected blood was oxidized immediately for Sepiapterin, an oxidation product of the biosynthetic pterin quantitation as described below. The other organs intermediate 6-lactoil-tetrahydropterin, and 7,8-dihydro- were weighed, frozen immediately in liquid N2, and then biopterin (7,8BH2) has long been known to be salvaged stored at 80LC until determination. by sepiapterin reductase and dihydrofolate reductase, Frozen tissues were partially thawed and homog- respectively (23). In our in vitro work using cultured enized immediately in 5 vol of 0.1 N HCl. An aliquot RBL2H3 and PC12 cells, uptake of BH4 was shown to (100 L) of the homogenate was mixed with a 0.5 vol of be hampered by a cellular process resulting in a very an acid-iodine solution (2% I2, 3% KI in 0.1 N HCl) or low level of incorporation, while sepiapterin was with alkaline-iodine (2% I2, 3% KI in 0.2 N NaOH) for incorporated and converted to BH4 in a very efficient oxidation under acid or alkaline conditions, respectively manner (24, 25). In this research, we observed an (26). Aliquots of freshly drawn blood (50 L each) were increase in the BH4 content of tissues when we gave added to a 10:4 mixture of PBS() and the above acid- mice BH4, 7,8BH2, and sepiapterin. In the present study, iodine or alkaline-iodine solutions for the differential we compared 6RBH4, 7,8BH2, and sepiapterin as to how oxidation as above. The oxidation reaction was allowed they are incorporated and delivered to animal organs, to proceed for 1 h at room temperature. After addition of and we found that sepiapterin and 7,8BH2 can efficiently a 0.5 vol of ascorbic acid (2.5%) dissolved in perchloric elevate tissue BH4, to a greater extent than 6RBH4. A acid (0.4 M), a clear solution was obtained by centrifu- new pathway was proposed by which administered gation at 10,000 P g for 10 min. Biopterin and pterin 6RBH4 was somehow oxidized to 7,8BH2, and probably were determined by HPLC equipped with a fluorescence after circulation, it was converted to BH4, resulting in detector (Ex 350 and Em 450 nm). The solid phase appreciable elevation of tissue BH4. was Fine-SIL C18T-5 and the mobile phase was 7% methanol. Since biopterins of various redox states, such Materials and Methods as tetrahydro-, quinonoid dihydro-, and 7,8-dihydrobiopterin, convert to fully oxidized biopterin stoichio- 6RBH4 was donated by Daiichi Suntory Pharma metrically by I2-oxidation under acidic conditions, bio- (Tokyo), and 6-(S)-L-erythro-5,6,7,8-tetrahydrobiopterin measured after acid-I2 oxidation was taken as the pterin (6SBH4), sepiapterin, and 7,8BH2 were purchased “total biopterin”. Tetrahydrobiopterin, and quinonoid from Schircks Laboratories (Jona, Switzerland). Metho- dihydrobiopterin as well if present, convert to pterin in a trexate (MTX) was purchased from Wako (Osaka). selective and stoichiometric manner (26); hence, the CM-cellulose (medium viscosity, sodium salt) was difference between the biopterin quantity after acid- obtained from Fluka (Buchs, Switzerland). C57BL/6J oxidation and that after alkaline-oxidation was termed mice were from Japan SLC (Hamamatsu) and adult “BH4” throughout this paper. Since the amount of fully males 8 – 10 weeks of age were used in the present oxidized biopterin detected in fresh tissue extracts was experiments. The animals were maintained on an ordi- insignificant, the biopterin calculated from the “total nary laboratory chow and tap water ad libitum, under a biopterin” minus “BH4” was termed “BH2” or referred constant 12-h light-dark cycle. The experiments were to as “oxidized biopterin”. When specified in the form conducted in accordance with the ethical guidelines of of 7,8-dihydrobiopterin, “7,8BH2” was used instead of the Teikyo University of Science and Technology “BH2”. For example, the substrate of dihydrofolate Animal Experimentation Committee and the guidelines reductase was “7,8BH2” rather than “BH2”, and a work- of The Japanese Pharmacological Society. ing solution of the reagent BH2 for administration to Pterins were given to animals at a dose of 10 mg/kg mice was termed “7,8BH2”. In order to distinguish 126 K Sawabe et al

6RBH4 from 6SBH4 in tissue extracts, cation exchange pterins as shown in Fig. 1. Sepiapterin administration chromatography was employed according to Bailey and was most effective at increasing the biopterin level in Ayling (27). Freshly dissected tissue was homogenized respective organs. In the case of the oral route (upper in 0.1 N HCl. Proteins were removed by centrifugation panels), the biopterin level continued to rise for 2 h in after addition of perchloric acid (final concentration of the liver (up to 10-fold) and for 1 h in both the kidney 0.1 M). The supernatant was subjected to HPLC with (10-fold) and blood (3-fold). After tissue biopterin UV detection (254 nm). The solid phase was Whatman, reached the peak values, it decreased quasi-exponen- Partisil-10 SCX (HICHROM, England), and the mobile tially. Administration of 6RBH4 resulted in a similar phase was 30 mM NaH2PO4 (pH 3.3). Terms for BH4 of rise in the tissue biopterin level, but the highest level was known C6-streo configuration, that is, BH4 in solutions only about half that of sepiapterin in the liver (53%) and of the purchased 6RBH4 and 6SBH4 as well as those blood (64%), while it was comparable to sepiapterin determined by SCX-HPLC, carried the prefixes 6RBH4 with respect to the kidney. In addition, the peak rise in and 6SBH4, respectively. tissue biopterin in the liver after 7,8BH2 administration was not as high as after sepiapterin or BH4 and the level Results of biopterin reached merely 1.6-fold that of the initial level. In contrast, increases in tissue biopterin in kidney BH4 administration was less effective in elevating the and blood after the oral treatment with 7,8BH2 were tissue BH4 level than sepiapterin or 7,8BH2 comparable to those with 6RBH4. However, the levels We compared sepiapterin to 6RBH4 with respect to of biopterin that were significantly higher than at the the effectiveness of how they elevate the tissue BH4 starting point lasted for 6 h after the oral administration, level when administered to mice. C57BL/6J male mice and in the liver and kidney, they were 122% and 140% received a 10 mg/kg dose of sepiapterin, 7,8BH2, or (P0.05, n 6), respectively. At this time point, levels 6RBH4 and the tissue level of biopterin in liver, kidney, had already returned to the initial values in the case of and blood was monitored for 6 h after the administration. sepiapterin or 6RBH4 treatment. We also compared A remarkable rise was observed after both oral admin- intraperitoneal injection (i.p., lower panels in Fig. 1) to istration (p.o.) and intraperitoneal injection (i.p.) of the the above p.o. experiment. The overall profile of data

Fig. 1. Elevation of biopterin level after administration of BH4 precursors, 6RBH4, sepiapterin, and 7,8BH2. Mice (C57BL/6J, male, 10 weeks of age) received 6RBH4 (open circles), 7,8BH2 (closed diamonds), or sepiapterin (open diamonds) each at a dose of 10 mg/kg. Administration was performed p.o. (upper panels) or i.p. (lower panels). After the indicated time periods, mice were sacrificed and organs were dissected and then frozen in liquid nitrogen and stored at 80LC until analysis. Biopterin was measured after oxidation with I2 in acidic and alkaline conditions. Total biopterin and BH4 were calculated as described in Materials and Methods. Note that the amounts diverged widely among organs, and the scales of the y-axes differ. Data are means M S.E.M. (n 6). Tetrahydrobiopterin Uptake in Mice 127 from i.p. injection of sepiapterin or 6RBH4 was similar converted to BH2 by sepiapterin reductase and then to to that for p.o. administration, but the quantity of BH4 by the action of dihydrofolate reductase (DHFR), biopterin detected in the respective organs was 2- to 3- but it was possible that the latter enzyme was not power- fold greater than with oral administration. It was noted ful enough to prevent accumulation of the substrate that BH4 administration i.p. lead to a much lower eleva- 7,8BH2. Therefore, administration of BH4 was not tion of liver biopterin compared to after sepiapterin expected to be followed by a BH2 surge. However, a injection. The biopterin level in the brain was quite remarkable amount of BH2 (0.22 M 0.03 nmol/g blood, stable after 6RBH4 administration while a slight eleva- 50% of the net increase in biopterin) appeared 30 min tion of biopterin was observed after both sepiapterin after the oral 6RBH4 treatment (Fig. 2c). Intraperitoneal and 7,8BH2 loading; roughly 1.5-fold (i.p.) at the peak injection of 6RBH4 also caused a BH2 surge (0.41 M (1 h) and 1.2-fold (p.o.) at 2 h (P0.05) (data not 0.03 nmol/g blood, 27.9% of the net increase, not shown). As to the redox state of the biopterin measured plotted in the figure). This indicated that 6RBH4, admin- in the liver and kidney, BH4 consistently comprised istered either p.o. or i.p., was oxidized somewhere in the around 95% of the total biopterin regardless of which- animal and was then delivered throughout the body ever of 6RBH4, 7,8BH2, or sepiapterin was administered. through the blood circulation. The appearance of the It was noted that virtually no oxidized biopterin (BH2) BH2 surge in the blood after 6RBH4 administration was detected in the brain throughout the experiment. strongly suggested the systemic oxidation of 6RBH4. In In contrast, the relative amount of BH4 in the blood contrast, oral administration of 7,8BH2 appeared to changed in response to pterin administration as cause the least BH2 surge (Fig. 2b). As a consequence, described below. the drop in the percentage of BH4 after loading with 7,8BH2 was smallest among the three pterins examined The BH2 surge appeared in the blood after BH4 admin- (inset panels of Fig. 2). Following the BH2 surge, a istration as well as after sepiapterin or 7,8BH2 proportion of BH4 was recovered to its original percent- The percentage of BH4 in the blood was 80% to age of 82% within 1 h, and the relative amount then 85% under ordinary conditions. After oral administra- continued to increase at least for the following 5 h, tion of sepiapterin, the net increase in blood biopterin reaching around 94% (see the insets). The concomitant was 0.81 M 0.43 nmol/g over the initial 0.33 M 0.02 decrease in BH2 suggested either that conversion of BH2 nmol/g. As shown in Fig. 2a, a great surge in oxidized to BH4 catalyzed by DHFR occurred throughout the biopterin (BH2, 49% of the biopterin increase) appeared experimental period or that BH2 selectively passed at around 30 min. The administered sepiapterin was beyond our detection by such means as through urinary

Fig. 2. BH2 appeared in the blood after administration of biopterin precursors, 6RBH4, 7,8BH2, and sepiapterin. Blood biopterin was monitored after oral administration of sepiapterin (a), 7,8BH2 (b), and 6RBH4 (c). Pterins were administered the same dose of 10 mg/kg body weight. Total biopterin (closed circles) and BH4 (open circles) were determined with the same samples. The dotted area represents the relative amount of BH2 in the total biopterein. Inset: The percentage of BH4 to the total biopterin in the blood. Data are mean M S.E.M. values (n 5). 128 K Sawabe et al excretion. Pretreatment of animals with the DHFR inhibitor MTX blocked BH4 accumulation, but a very large accumula- Distribution of exogenous BH4 involved an MTX-sensi- tion of oxidized biopterin (BH2) was observed in the tive pathway examined organs after administration of sepiapterin The possible involvement of DHFR was examined and 7,8BH2 as was expected. For example, 7,8BH2 for accumulation of BH4 in the liver, kidney, intestinal administration resulted in a 10.7-fold (13.3 M 1.2 nmol mucosa, and blood following administration of the /g) increase in tissue BH2 in the kidney, and sepiapterin pterins. Mice were pretreated with MTX (10 mg/kg, treatment caused a 15.0-fold (18.6 M 1.9 nmol/g) p.o.) for 90 min; then 6RBH4, 7,8BH2, or sepiapterin increase in this organ, but neither brought about an was given orally, and 30 min later, the tissue distribution increase in BH4. Interestingly, MTX inhibited an of biopterin was measured with special regard to fully increase in BH4, even after 6RBH4 administration. In reduced biopterin BH4 (Fig. 3). Sepiapterin administra- the case of the liver, 6RBH4 administration without tion without MTX treatment raised tissue BH4 up to MTX doubled the tissue BH4 level (12.5 M 1.4 nmol/g), 30.4 M 0.61 nmol/g (liver, 4.2-fold over endogenous and although there was no increase in BH4 (6.41 M 0.70 BH4; Fig. 3a), 13.0 M 1.44 nmol/g (kidney, 10.5-fold; vs endogenous 7.20 M 0.28 nmol/g) with MTX, tissue Fig. 3b), 38.9 M 15.3 nmol/g (intestinal mucosa, 24.5- BH2 rose considerably. The observed increase in BH4 fold; Fig. 3c), and 0.77 M 0.15 nmol/g (blood, 3.5-fold; after 6RBH4 administration without MTX treatment Fig. 3d). Furthermore, administration of 7,8BH2 led was, therefore, presumed to be derived from 7,8BH2 to similar results (liver, 14.4 M 2.8 nmol/g; kidney, that was reduced back to BH4 by the action of DHFR. 10.8 M 3.4 nmol/g; intestinal mucosa, 5.97 M 1.84 nmol The kidney and the intestinal mucosa both contain /g; blood, 0.66 M 0.09 nmol/g), but elevation of the relatively low levels of endogenous BH4. Without MTX, tissue BH4 level was lower than with sepiapterin. administration of 6RBH4 strongly increased tissue BH4

Fig. 3. Methotrexate (MTX) inhibition of BH4 accumulation in organs administered with 6RBH4, 7,8BH2, and sepiapterin. MTX (10 mg/kg) was given 90 min before oral administration of the pterins. Pterins were administered at the same dose of 10 mg/kg body weight. Organs were dissected out at 30 min after the pterins were administered. Tissue biopterin levels were measured in liver (a), kidney (b), intestinal mucosa (c), and blood (d). The mucosa was collected from the proximal 1/3 of the small intestine. The height of bars represents the total biopterin. BH2 (closed columns) and BH4 (open columns) were calculated as described in Materials and Methods. Data are mean M S.E.M. values (n 5). *P0.05, **P0.01, in comparison between with and without MTX. Tetrahydrobiopterin Uptake in Mice 129 in these tissues, and with MTX, this increase was only The raised BH2 level in the blood did not disappear reduced by half, suggesting the MTX-resistant increase during our experimental period as was the case with was due to direct accumulation in the form of BH4 kidney. Since blood biopterin might be in equilibrium in without having to take the BH2 route. With MTX tissues throughout the body, the BH2 increase might be a administration, a huge increase in BH2 was observed in reflection of its facilitated excretion from tissues where the blood after administration of any of the pterins it might have been salvaged back to BH4 by the action of including 6RBH4. Simultaneously, the BH4 content was DHFR in the absence of MTX. even decreased by MTX. These results suggested that the steady state level of BH4 had been maintained largely The unnatural diastereomer 6SBH4 was converted to by the catalytic action of DHFR. 6RBH4; additional evidence that exogenous BH4 was oxidized to 7,8BH2 and then reduced back by DHFR An MTX-sensitive process was also involved in main- The increase in BH4 after 6RBH4 administration was taining the endogenous BH4 level suggest to be mediated by an enzyme reaction catalyzed It was noted that the DHFR-catalyzed conversion of by DHFR which would likely convert 7,8BH2 to the BH2 to BH4 seemed to have been involved in maintain- 6R-diastereomer of BH4. For confirmation of the ing BH4 at the endogenous level to a considerable degree involvement of DHFR, we administered 6SBH4 to mice (compare the two leftmost bars in the panels of Fig. 3). intraperitoneally. As shown in Fig. 5, administration of Hence, no pterin supplement was used in examining the 6SBH4 (10 mg/kg, i.p.) resulted in a large increase in effect of MTX on the steady state level of tissue BH4 BH4 (47.1 M 5.2 nmol/g, 30 min) compared to the in liver, kidney, and blood (Fig. 4). The MTX treatment increased the amount of BH2, presented as the total biopterin minus BH4, in all organs tested. The BH2 content in the liver was the least altered by MTX administration among all tissues examined, suggesting that the large pool of liver BH4 might be maintained by de novo biosynthesis which does not include DHFR in the pathway. In contrast, the BH4 level in the kidney dropped to 46.2% within 1 h and the level did not recover even at the end of the experiment (6 h after MTX administration), while the total biopterin level did not show any significant change, indicating that the oxidized biopterin increased instead of being reduced back by the function of DHFR. MTX treatment raised the biopterin Fig. 5. Administration of 6SBH4 increased the levels of 6RBH4. M Mice were injected with 6RBH4 or 6SBH4, i.p. at a dose of 10 mg/kg. concentration in the blood almost 2-fold (0.27 0.02 Tissue extracts after 30 min were applied to a PartiSil-10 SCX vs 0.37 M 0.05 nmol/g) within 1 h. It was interesting column equipped with a UV-detector (254 nm) for separation of that the increase in the blood biopterin was caused 6R- and 6SBH4 (27). The open bar represents 6RBH4 and the dotted bar, 6SBH4. Data are mean M S.E.M. values (n 6). almost exclusively by oxidized biopterin not by BH4.

Fig. 4. Effect of MTX administration (10 mg/kg, p.o.) on the amount of biopterin and its redox state in tissues. The tissue biopterin level was measured in liver (a), kidney (b), and blood (c). Closed circles represent total biopterin and open circles, BH4. Data are mean M S.E.M. values (n 5). *P0.05, **P0.01, compared with 0-time. 130 K Sawabe et al vehicle control (1.24 M 0.09 nmol/g). The major portion Schircks Laboratory). Based on finding from most in of the accumulated BH4 (73%) consisted of the 6R- vitro or in vivo studies, as well as from practical therapy diastereomer which must have been reduced by DHFR of BH4-deficiency, 6RBH4 appeared to be the only after oxidation of exogenous 6SBH4 to 7,8BH2. The choice among agents that elevate the cellular or tissue remainder was in the form of 6SBH4. BH4 level. In 1996, the first scientific evaluation appeared Discussion comparing 6RBH4 and sepiapterin with regard to cellular uptake, and it was revealed that sepiapterin taken up by Phenylketonuria resulting from a hereditary defi- cells in culture elevated the cellular BH4 concentration ciency in phenylalanine hydroxylase causes severe much more effectively than did 6RBH4 (24). Mecha- mental retardation as well as acute nervous disorders. nistic studies on sepiapterin and BH4 incorporation As a malignant variation of phenylketonuria, BH4- have been ongoing using various culture cell lines. In deficiency is known to lead to neuronal disorders related brief, when cells were incubated with BH4, most cells to the lack of effective biosynthesis of catecholamines rejected its uptake, resulting in little increase in the and serotonin. BH4 is a common and essential cofactor cellular BH4 level (36 – 38). However, sepiapterin of tryptophan and tyrosine hydroxylases as well as of more effectively elevated the BH4 level by means of the phenylalanine hydroxylase (1 – 4). Unlike the classic so-called salvage pathway composed of sepiapterin phenylketonuria, marked by a lack of the holoenzyme reductase and DHFR, which are sensitive to N-acetyl- phenylalanine hydroxylase, BH4-deficiency, so-called serotonin and MTX, respectively. In RBL2H3 cells, for malignant phenylketonuria or hyperphenylalaninemia, example, a 100- M sepiapterin feeding elevated the 6 was dramatically reversed by the administration of cellular BH4 (base line level: 30 pmol/10 cells) by 20- BH4. Consequently, supplementation with BH4 has fold or more within 60 min while 6RBH4 raised it by drawn the attention of many clinical specialists. In an merely 1.5 – 2.0-fold at the same concentration. In some attempt to supply the pteridine cofactor, chemically cells such as erythrocytes, however, BH4 appeared to be reduced 5,6,7,8-tetrahydro-6-methylpterin and chemi- incorporated in its reduced form. We were interested in cally reduced tetrahydrobiopterin were examined for whether 6RBH4 was ineffective in elevating the tissue their effects (11, 28). In a classic research study to BH4 level in the whole body as was observed with the explore the biosynthetic pathway of BH4, sepiapterin cellular systems. In this study, administration of sepia- was shown to be efficiently converted to biopterin (23). pterin, 7,8BH2, and even 6RBH4 led to a BH2 surge, as It was long accepted that the first pteridine in BH4 shown in Fig. 2. The emergence of the surge after a biosynthesis from GTP was dihydroneopterin-triphos- 6RBH4 feeding suggested that most of the 6RBH4 given phate, the product of GTP-cyclohydrolase I (29 – 31). was first oxidized and thereafter reduced back to BH4. Sepiapterin and 7,8BH2 were proven to be the inter- The reducing process following putative and systemic mediate precursors of BH4 in a vertebrate system using oxidation of exogenous BH4 was supported first by the bull frog (23). Actually, patients of BH4-deficiency were effective inhibition of BH4 elevation by MTX. The reported to have been relieved from abnormal urinary involvement of a DHFR reaction was further suggested excretion of high phenylalanine and low serotonin by by an experiment in which mice received 6SBH4. Injec- a single administration of sepiapterin (9, 10). In the tion of the mice with 6SBH4 led to a dramatic increase early 1980s, on the other hand, it was feared that since in BH4 in the kidney, 74% of which was in the 6R- this pathway included dihydrofolate reductase as an diastereomer form (Fig. 5). Since the substrate speci- immediate to BH4 production, an MTX supplement ficity of DHFR strictly disallows reduction of quino- might possibly deplete tissues of BH4 (32). Shortly after, noid-BH2, the inevitable oxidation intermediate of however, researchers discovered another biosynthetic biopterin, DHFR reduces 7,8BH2 and provides BH4 pathway from dihydroneopterin-triphosphate to BH4, a with a 6R-configuration (34, 35, 39 – 41) (see Fig. 6). routing other than through 7,8BH2 (for review: (33)). It was clear that the conversion of 7,8BH2 to 6RBH4 Just prior to the finding of that, the de novo pathway of took place inside the cells by the action of the cytosolic BH4 biosynthesis, the naturally occurring C6-diastereo- enzyme DHFR. The precursor 7,8BH2 might have mer of BH4 was determined to be 6RBH4 (34, 35). formed from (6S)quinonoid-BH2 by spontaneous intra- Therefore, 6RBH4 was viewed as being more “natural” molecular rearrangement, and the pterin lost C6- than sepiapterin or 7,8BH2. Within a short while, 6RBH4 chirality. Since dihydropteridine reductase (DHPR) is became commercially available for the therapy of BH4- abundant in most cells, this might take place in some deficient patients (the compound is currently available region outside of the area accessible by the enzyme, from Daiichi Suntory Pharma Co., Ltd., Tokyo, and such as in non-cytosolic cellular compartments and Tetrahydrobiopterin Uptake in Mice 131

Fig. 6. Oxidation and reduction of tetrahydrobiopterin relative to the C6-configuration. 7,8-Dihydrobiopterin does not carry an asymmetric carbon at the C6 position. Oxidation of tetrahydrobiopterin to 7,8-dihydrobiopterin removes C6 chirality. Recycling of quinonoid dihydrobiopterin by dihydropteridine reductase preserves the C6-stereochemical structure, whether 6S or 6R. Reduction of 7,8-dihydrobiopterin by dihydrofolate reductase introduces a 6R-configuration.

extracellular spaces; otherwise the enzyme DHPR would In their experiments, in which rats were fed 6RBH4 have reduced quinonoid-BH2 back to BH4 preserving (10 mg/kg, p.o.), they found that tissue BH4 was ele- the 6S-configuration. The formed 7,8BH2 must have vated in the liver (1.5-fold) and kidney (3.4-fold), both been taken up by the cells so that the cytosolic enzyme of which showed a peak rise around 2 h following DHFR created the 6R-diastereomer of BH4 as quantified administration. In the same study in which they fed rats in the kidney 30 min after i.p. injection of 6SBH4. the same dose of radioactive 6RBH4 ((6R)-[6-T]-BH4) Therefore, the major portion of accumulated BH4 in the and performed whole-body autoradiography 2 h after organs we examined might have entered as 7,8BH2 from treatment, virtually no significant radioactivity was other cells, presumably from other organs, via the blood noted in organs such as liver and kidney, but remained stream. A significant portion of the accumulated BH4 only in the gastric contents. Based on these results, remained as the original 6S-diastereomer (26% of the they claimed that 6RBH4 was scarcely absorbed by the increase, Fig. 5). The 6S-isomer might have been pre- digestive tract. In fact, their autoradiogram depicted served inside the cells because either it was maintained almost no deposition of radioactivity; however, our in the tetrahydro-form or was reduced back to BH4 by interpretation is that almost all of the radioactive DHPR immediately after oxidation to quinonoid-BH2. 6RBH4 administered was cumulatively oxidized to non- The C6-chirality is preserved through conversion radioactive 7,8BH2, releasing most of the C6-tritium between quinonoid-BH2 and BH4 (H. Hasegawa, un- within 2 h. Tritium labeling at C6 of 6RBH4 might published results) but is lost when converted to 7,8BH2 have been lost when the [6-T]-BH4 was oxidized to (see Fig. 6). Furthermore, the remaining 6SBH4 could 7,8BH2 while BH4 retrieved by the subsequent reduction have directly moved into the tissue in its original form might not have been visualized by the autoradiography. since the reducing reaction to produce 6SBH4 never All their results were strongly consistent with those of occurs in animals. The other portion of BH4 (74% of this present study showing that most exogenous BH4 was tissue BH4 in the kidney) was the salvaged product of oxidized and delivered throughout the tissues and then 7,8BH2. Our data did not allow us to calculate the reduced back to BH4. Their report further presented a percentage of exogenous BH4 salvaged after oxidation in table without any comment about BH4- and BH2-concen- the entire body. We did make a rough estimation below trations in the blood separate from those in the plasma based on a study published by Hayashi and others (14). over a time course of 24 h after the administration of 132 K Sawabe et al

6RBH4 (100 mg/kg, p.o.). In brief, a peak rise in BH4 Matsumoto for reading this manuscript and to our in both blood and plasma appeared at around 2 h students, Hiroyuki Miura, Atsushi Tanaka, and Yasuko (828.0 ng/ml). The table revealed that a BH2 surge Suetake for their collaboration in the Student Research occurred at around 1 h (76.7 ng/ml, about 36.5-fold Program 2001 – 2003. over the endogenous level) following administration. Although they did not refer to BH4 oxidation, all their References data were consistent with our present results. Interest- ingly, they detected the BH2 surge almost exclusively in 1 Kaufman S. The structure of the phenylalanine-hydroxylation plasma, suggesting either that BH2 was not taken up by cofactor. Proc Natl Acad Sci USA. 1963;50:1085–1093. 2 Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase. The erythrocytes or that any incorporated BH2 had been initial step in norepinephrine biosynthesis. J Biol Chem. 1964; reduced to BH4 in these cells. 239:2910–2917. Based on our preliminary studies on BH4 uptake by 3 Lovenberg W, Jequier E, Sjoerdsma A. Tryptophan hydroxyl- homogeneous cells in culture as mentioned above, we ation: measurement in pineal gland, brainstem, and carcinoid expected that BH4 would be taken up by tissue much tumor. Science. 1967;155:217–219. less efficiently than sepiapterin and 7,8BH2. However, 4 Ichiyama A, Nakamura S, Nishizuka Y, Hayaishi O. Enzymic BH4 was as effective a supplement as 7,8BH2 or sepia- studies on the biosynthesis of serotonin in mammalian brain. pterin (Fig. 1). This was most likely because the admin- J Biol Chem. 1970;245:1699–1709. 5 Kwon NS, Nathan CF, Stuehr DJ. Reduced biopterin as a istered BH4 was first oxidized to BH2, a form relatively easily taken up by the cells and a precursor of the salvage cofactor in the generation of nitrogen oxides by murine macro- phages. J Biol Chem. 1989;264:20496–20501. pathway. Whatever pterins would be chosen for admin- 6 Mayer B, John M, Heinzel B, Werner ER, Wachter H, Schultz istration, the BH2 surge was shown to be accompanied G, et al. Brain nitric oxide synthase is a biopterin- and flavin- by sepiapterin, 7,8BH2, or even 6RBH4. The DHFR- containing multi-functional oxido-reductase. FEBS Lett. 1991; catalyzed reaction was involved in the delivery and 288:187–191. subsequent retrieval of BH2. Furthermore, this anti- 7 Marletta MA. Nitric oxide synthase structure and mechanism. folate-sensitive process was also shown to be involved J Biol Chem. 1993;268:12231–12234. 8 Stuehr DJ. Structure-function aspects in the nitric oxide in maintaining the endogenous BH4 level to a consider- synthases. Annu Rev Pharmacol Toxicol. 1997;37:339–359. able degree under ordinary conditions (Fig. 4). When 9 Curtius HC, Niederwieser A, Viscontini M, Otten A, Schaub J, anti-folate is prescribed to a patient, a chronic increase Scheibenreiter S, et al. Atypical phenylketonuria due to tetra- in blood BH2 could last for a long time. At a high hydrobiopterin deficiency. Diagnosis and treatment with tetra- level, BH2 may bind to NOS in the place of BH4, hydrobiopterin, dihydrobiopterin and sepiapterin. Clin Chim resulting in the production of active oxygens (6, 42 – 45) Acta. 1979;93:251–262. which are potentially detrimental to tissues. Actually, 10 Niederwieser A, Curtius HC, Wang M, Leupold D. Atypical phenylketonuria with defective biopterin metabolism. Mono- the possible relationships between an elevated BH2 therapy with tetrahydrobiopterin or sepiapterin, screening and level and various circulatory disorders have been study of biosynthesis in man. Eur J Pediatr. 1982;138:110–112. suggested in high fructose-induced diabetes model rats 11 Kaufman S, Kapatos G, McInnes RR, Schulman JD, Rizzo WB. (46, 47). In the present study, administration of 6RBH4 Use of tetrahydropterins in the treatment of hyperphenyl- was demonstrated to evoke a BH2 surge and that use of alaninaemia due to defective synthesis of tetrahydrobiopterin: MTX results in a long lasting rise in BH2 levels in blood. evidence that peripherally administered tetrahydropterins enter Since many patients would have to receive long term the brain. Pediatrics. 1982;70:376–380. 12 Kaufman S, Kapatos G, Rizzo WB, Schulman JD, Tamarkin L, treatment with 6RBH4 or with anti-folate reagents, a Van Loon GR. Tetrahydropterin therapy for hyperphenyl- BH2 surge or long lasting rise in BH2 levels should be alaninaemia caused by defective synthesis of tetrahydro- carefully monitored for any deleterious effects on the biopterin. Ann Neurol. 1983;14:308–315. body. 13 Smith I, Hyland K, Kendall B. Clinical role of pteridine therapy in tetrahydrobiopterin deficiency. J Inherit Metab Dis. 1985;8: Acknowledgments 39–45. 14 Hayashi T, Ogata A, Takehisa M, Komoriya K, Ohnuma N. This work was supported in part by the Japan Private Studies on metabolism and disposition of sapropterin hydro- School Promotion Foundation and by a Grant-in-Aid chloride (SUN 0588) L-erythro-tetrahydrobiopterin hydro- chloride in rats. Clin Rep. 1992;26:3471–3495. for Advanced Scientific Research on Bioscience/Bio- 15 Blau N, Thony B, Heizmann W, Dhondt JL. Tetrahydrobiopterin technology Areas from the Ministry of Education, deficiency: from phenotype to genotype. Pteridines. 1993;4:1– Culture, Sports, Science, and Technology of Japan. 10. We are grateful for the kind support of Chugai Pharma- 16 Hoshiga M, Hatakeyama K, Watanabe M, Shimada M, Kagami- ceutical Co., Ltd. We are grateful to Dr. Kazuhiko yama H. Autoradiographic distribution of [14C]tetrahydro- Tetrahydrobiopterin Uptake in Mice 133

biopterin and its developmental change in mice. J Pharmacol rat brain in vivo. Proc Natl Acad Sci USA. 1983;80:1546–1550. Exp Ther. 1993;267:971–978. 33 Nichol CA, Smith GK, Duch DS. Biosynthesis and metabolism 17 Brand MP, Hyland K, Engle T, Smith I, Heales SJ. Neuro- of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem. chemical effects following peripheral administration of tetra- 1985;54:729–764. hydropterin derivatives to the hph-1 mouse. J Neurochem. 34 Hasegawa H, Imaizumi S, Ichiyama A, Sugimoto T, Matsuura S, 1996;66:1150–1156. Oka K, et al. Cofactor activity of diastereoisomers of tetra- 18 Canevari L, Land JM, Clark JB, Heales SJ. Stimulation of the hydrobiopterin. In: Kisliuk RL, Brown GM, editors. Chemistry brain NO/cyclic GMP pathway by peripheral administration of and biology of pteridines, Vol. 4. North-Holland: Elsevier; 1979. tetrahydrobiopterin in the hph-1 mouse. J Neurochem. 1999;73: p. 195–200. 2563–2568. 35 Matsuura S, Sugimoto T, Hasegawa H, Imaizumi S, Ichiyama A. 19 Sawabe K, Yamamoto K, Kamata T, Wakasugi KO, Hasegawa Studies on biologically active pteridines III: the absolute H. Sepiapterin administration raises tissue BH4 levels more configuration at the C-6 chiral center of tetrahydropterin efficiently than BH4 supplement in normal mice. In: Milstien S, cofactor and related compounds. J Biochem. 1980;87:951–957. Kapatos G, Levine RA, Shane B, editors. Chemistry and biology 36 Hasegawa H, Yamamoto K, Matsuhashi Y, Miyazawa T, of pteridines and folates. Boston: Kluwer Academic Publishers; Nakanishi N. Ability of RBL2H3 cells to lower environmental 2001. p. 199–204. tetrahydrobiopterin concentration. Pteridines. 2000;11:121–125. 20 Fiege B, Ballhausen D, Kierat L, Leimbacher W, Goriounov D, 37 Hasegawa H, Yamamoto K, Sawabe K, Matsuhashi Y, Oguro K, Schircks B, et al. Plasma tetrahydrobiopterin and its pharmaco- Wakasugi KO, et al. Cells take up BH4, oxidize it and the kinetic following oral administration. Mol Genet Metab. 2004; oxidized biopterin is preferentially released. In: Milstien S, 81:45–51. Kapatos G, Levine RA, Shane B, editors. Chemistry and Biology 21 Duch DS, Smith GK. Biosynthesis and function of tetrahydro- of Pteridines and Folates. Boston: Kluwer Academic Publishers; biopterin. J Nutr Biochem. 1991;2:411–423. 2001. p. 205–210. 22 Thony B, Auerbach G, Blau N. Tetrahydrobiopterin bio- 38 Sawabe K, Yamamoto K, Matsuhashi Y, Miura H, Wakasugi synthesis, regeneration and functions. Biochem J. 2000;347:1– KO, Nakanishi N, et al. Increase in the biopterin pool after 16. tetrahydrobiopterin administration is due to uptake of an oxida- 23 Fukushima T. Biosynthesis of pteridines in the tadpole of the tion product dihydrobiopterin and conversion to the reduced bullfrog, Rana catesbiana. Arch Biochem Biophys. 1970;139: state. In: Blau N, Thony B, editors. Pterins, folates and 361–369. neurotransmitters in molecular medicine. Heilbronn: SPS 24 Yamamoto K, Nagano M, Nakanishi N, Hasegawa H. A compar- Verlagsgesellschaft; 2003. p. 41–47. ison of sepiapterin and tetrahydrobiopterin uptake by RBL2H3 39 Kaufman S. The enzymatic conversion of phenylalanine to Cells. Pteridines. 1996;7:154–156. tyrosine. J Biol Chem. 1957;226:511–524. 25 Yamamoto K, Nakazawa N, Hasegawa H, Nakanishi N. Uptake 40 Kaufman S. Metabolism of the phenylalanine hydroxylation of tetrahydrobiopterin by cells in monolayer cultures of cofactor. J Biol Chem. 1967;242:3934–3943. RBL2H3 and PC12 cell lines. In: Pfleiderer W, Rokos H, 41 Nagai M. Studies on sepiapterin reductase: further character- editors. Chemistry and biology of pteridines and folates. Berlin: ization of the reaction product. Arch Biochem Biophys. Blackwell Science; 1997. p. 759–763. 1968;126: 426–435. 26 Fukushima T, Nixon JC. Analysis of reduced forms of biopterin 42 Heinzel B, John M, Klatt P, Bohme E, Mayer B. Ca2+/cal- in biological tissues and fluids. Anal Biochem. 1980;102:176– modulin-dependent formation of hydrogen peroxide by brain 188. nitric oxide synthase. Biochem J. 1992;281:627–630. 27 Bailey SW, Ayling JE. Separation and properties of the 6- 43 Cosentino F, Katusic ZS. Tetrahydrobiopterin and dysfunction diastereoisomers of l-erythro-tetrahydrobiopterin and their of endothelial nitric oxide synthase in coronary arteries. Circula- reactivities with phenylalanine hydroxylase. J Biol Chem. 1978; tion. 1995;91:139–144. 253:1598–1605. 44 Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, 28 Kapatos G, Kaufman S. Peripherally administered reduced Masada M, et al. Abnormal biopterin metabolism is a major pterins do enter the brain. Science. 1981;212:955–956. cause of impaired endothelium-dependent relaxation through 29 Shiota T, Palumbo M. Enzymatic synthesis of the pteridine nitric oxide/O2 imbalance in insulin-resistant rat aorta. moiety of dihydrofolate from guanine nucleotides. J Biol Chem. Diabetes. 1999;48:2437–2445. 1965;240:4449–4453. 45 Vasquez-Vivar J, Martasek P, Whitsett J, Joseph J, Kalyanara- 30 Shiota T, Palumbo M, Tsai L. A chemically prepared formam- man B. The ratio between tetrahydrobiopterin and oxidized idopyrimidine derivative of guanosine triphosphate as a possible tetrahydrobiopterin analogues controls superoxide release from intermediate in pteridine biosynthesis. J Biol Chem. 1967;242: endothelial nitric oxide synthase: an EPR spin trapping study. 1961–1969. Biochem J. 2002;362:733–739. 31 Burg A, Brown G. The biosynthesis of folic acid. 8. Purification 46 Shinozaki K, Nishio Y, Okamura T, Yoshida Y, Maegawa H, and properties of the enzyme that catalyzes the production of Kojima H, et al. Oral administration of tetrahydrobiopterin formate from carbon atom 8 of guanosine triphosphate. J Biol prevents endothelial dysfunction and vascular oxidative stress in Chem. 1968;243:2349–2358. the aortas of insulin-resistant rats. Circ Res. 2000;87:566–573. 32 Nichol CA, Lee CL, Edelstein MP, Chao JY, Duch DS. Bio- 47 Shinozaki K, Kashiwagi A, Masada M, Okamura T. Stress and synthesis of tetrahydrobiopterin by de novo and salvage path- vascular responses: oxidative stress and endothelial dysfunction ways in adrenal medulla extracts, mammalian cell cultures, and in the insulin-resistant state. J Pharmacol Sci. 2003;91:187–191.