Simplified HPLC methodology for quantifying biological pterins by selective oxidation.

Tetrahydrobiopterin (BH4) has become a potential therapeutic tool to treat cardiovascular diseases, since it is an essential cofactor of nitric oxide synthase. In order to quantify the amount of BH4 and its related biopterins, a procedure that involves differential oxidation is currently used, which measures biopterin (the product of the oxidation of BH4 and BH2) at two different pH conditions to calculate the quantity of BH2 and BH4, using high performance liquid chromatography (HPLC). In this work, a method was established in order to quantify BH4 and BH2 by adapting previously described procedures. Several chromatographic conditions were evaluated to define the most convenient methodology. Four types of mobile phases and two different analytical columns were used for HPLC. Additionally, calibration curves were made in acid and basic pH compatible with the differential oxidation method. Each method was suitable for quantification purposes, but the choice was based on an economic factor. The selected condition was a mobile phase of 95% water/5% methanol using a C18 column at 35°C at a flow rate of 0.9mL/min. Then, it was calculated the recovery rate, which was about 80% using the chosen method. The aim of this work was to establish a simplified method of differential oxidation, compatible with matrixes such as cardiac tissue in order to facilitate the assessment of the BH4/BH2 ratio in biological samples.

Microcystbiopterins A-E, five O-methylated biopterin glycosides from two Microcystis spp. bloom biomasses.

Five previously undescribed biopterin glycosides, microcystbiopterin A-E, were isolated from the extracts of two bloom materials of Microcystis spp. collected from a fishpond (IL-337) and Lake Kinneret (IL-347), Israel. The structure of the pterins was established by interpretation of their UV, CD, 1D and 2D NMR spectra and HR mass measurements. Microcystbiopterin D is the first heptose containing pterin glycoside to be reported in the literature. Their antimicrobial and cytotoxic properties were evaluated but all were found not active in both assays.

Synthesis of biopterin from dihydroneopterin triphosphate by rat tissues.

High performance liquid chromatography procedure for the analysis of pterins of biopterin synthesis from dihydroneopterin triphosphate via sepiapterin in rat tissues has been described. Sepiapterin-synthesizing enzyme 1, which catalyzes in the presence of Mg2+ the conversion of dihydroneopterin triphosphate to an intermediate designated compound X was assayed by determining pterin which is formed from compound X under acidic conditions. Sepiapterin- and biopterin-synthesizing activity were also assayed by determining sepiapterin and biopterin, respectively. Analytical results revealed the presence of these activities in most rat tissues examined and high levels were found in kidney, pineal gland and liver. Activities were also detectable in peripheral erythrocytes.

Estrogen modifies stress response of catecholamine biosynthetic enzyme genes and cardiovascular system in ovariectomized female rats.

Estrogen is likely involved in the gender specific differences in coping with stress. Activation of catecholamine (CA) biosynthetic enzyme gene expression in central and peripheral CA systems plays a key role in response to stress and in regulation of the cardiovascular system. Here we examined whether estradiol can modulate response of hypothalamic-pituitary-adrenal axis (HPA), gene expression of enzymes related to CA biosynthesis in several noradrenergic locations, tetrahydrobiopterin (BH4) concentration and blood pressure (BP) in response to immobilization stress (IMO) of ovariectomized female rats. Rats were injected with 25 mug/kg estradiol benzoate (EB) or sesame oil once daily for 16 days and subsequently exposed to two hours of IMO. The IMO triggered elevation in plasma ACTH was lessened in EB-pretreated animals. However, estradiol did not alter the IMO-elicited rise of tyrosine hydroxylase mRNA levels in adrenal medulla (AM) and in the nucleus of solitary track (NTS) compared with controls. The response of GTP cyclohydrolase I (GTPCH) mRNA in AM to IMO was also similar in both groups. Several responses to IMO in EB-treated rats were reversed. Instead of IMO-elicited elevation in dopamine beta-hydroxylase mRNA levels in the locus coeruleus, GTPCH mRNA and BH4 levels in the NTS, they were reduced by IMO. In a parallel experiment, BP was monitored during restraint stress. The elevation of BP in response to single or repeated restraint stress was sustained during 2 h in controls and reduced after 70 min stress in EB treated rats. One month after withdrawal of EB treatment, the BP response to restraint was similar to that of rats which never received EB. The results demonstrate that estrogen can modulate responses to stress affecting HPA axis, CA biosynthesis, in central and peripheral noradrenergic systems, and BP.

Tetrahydro-6-biopterin is associated with tetrahydro-7-biopterin in primary murine mast cells.

Murine bone marrow-derived mast cells proliferate in response to interleukin 3. In addition to 6-biopterin, 7-biopterin was identified in these cells by HPLC analysis of iodine oxidized extracts and by alkaline permanganate oxidation to the 6- and 7-carboxylic acids. 7-Biopterin comprised 31.9 (+/- 7.7)% of the total biopterin. It was absent in cells which were grown with of L-p-chlorophenylalanine, an inhibitor of tryptophan 5-mono-oxygenase. Both 6- and 7-biopterin were present in the cell as their tetrahydro forms. From these data we conclude that 7-biopterin, in contrast to e.g. brain tissue, regularly occurs as a normal metabolite in primary mast cells and that it is generated during hydroxylation of tryptophan.

The major pterin in Tetrahymena pyriformis is 6-(D-threo-1,2,3-trihydroxypropyl)-pterin (D-monapterin) and not 6-(L-threo-1,2-dihydroxypropyl)-pterin (ciliapterin).

The major pterin in Tetrahymena pyriformis, strain W, earlier suggested to be L-threo-biopterin and named ciliapterin [1] is now identified as D-threo-neopterin (D-monapterin). This is the first example of a natural D-monapterin. This compound was characterized by its chromatographic behavior, its fluorescence properties and by its oxidation product with alkaline permanganate. The final identification was obtained by comparison with an authentic material using an exchange ligand chromatography method with D-phenylalanine as chiral modifier and Cu (II) as metal ion. D-monapterin is also present as the major pterin in Tetrahymena pyriformis strains GL and ST, and in Tetrahymena thermophila.

Diagnosis of female genital schistosomiasis by indirect disease markers: determination of eosinophil cationic protein, neopterin and IgA in vaginal fluid and swab eluates.

Based on assumptions about the pathophysiology of egg-related lesions in the lower reproductive tract, putative indirect disease markers were investigated in vaginal fluids from 54 Malawi adolescent girls and women infected with S. haematobium. These women received a careful gynecological examination during which biopsies were taken from the cervix, and, if present, also from suspicious lesions in the vagina and the vulva. If the biopsies, either in wet crushed preparations or in histological sections, contained eggs the patients were considered to have female genital schistosomiasis (FGS; n = 33). The remainder (n = 21) were classified as having urinary schistosomiasis only. Eosinophil cationic protein (ECP), a cytotoxic granule protein of eosinophils, neopterin, a second messenger molecule generated during the activation of macrophages, and IgA as an indicator of local B-cell activation were quantitatively determined in vaginal fluid. To clarify the origin of ECP, this protein was also looked for in histological sections by an immunohistochemical method. In order to explore whether such disease markers can be detected after absorption to a tampon-like material, ECP and IgA were also assessed after elution from a non-porous, polypropylene fibre web impregnated with vaginal fluid. The concentration of ECP in vaginal fluid and the degree of immunohistochemical staining in histological sections were significantly higher in patients with FGS than in women with urinary schistosomiasis only. The amount of ECP detected in histological sections correlated to the number of eggs/mm2 of compressed genital tissue (rho = 0.36, P = 0.02), and the concentration of ECP in vaginal fluid correlated to the concentration of neopterin as well as to that of IgA (rho = 0.52, P = 0.004 and rho = 0.37, P = 0.02, respectively). Median neopterin concentration in vaginal fluid was also higher in the FGS group, but the difference was not statistically significant. ECP could also be detected in eluates from impregnated fibre webs, but the concentration was approximately one power of 10 less than in the original vaginal fluid. These results demonstrate that indicators of immunological mechanisms related to the egg-granuloma might be useful as indirect disease markers for women with FGS if assessed in vaginal washings or swab eluates.

Expression analysis of the aldo-keto reductases involved in the novel biosynthetic pathway of tetrahydrobiopterin in human and mouse tissues.

Tetrahydrobiopterin (BH(4)) acts as a cofactor of the aromatic amino-acid hydroxylases, and its deficiency may result in hyperphenylalaninemia (HPA) and decreased production of the neurotransmitters. BH(4) is synthesized by sepiapterin reductase (SPR) from 6-pyruvoyl-tetrahydropterin (PPH(4)). A patient with SPR deficiency shows no HPA; however, an SPR knockout mouse exhibits HPA. We have reported on the SPR-unrelated novel biosynthetic pathway from PPH(4) to BH(4) (salvage pathway II) in which 3alpha-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert. In this study, we performed the expression analysis of both proteins in humans and wild-type mice. The results of expression analysis indicated that salvage pathway II worked in human liver; however, it did not act in human brain or in mouse liver and brain. For this reason, a patient with SPR deficiency may show progressive neurological deterioration without HPA, and SPR knockout mice may exhibit HPA and abnormal locomotion activity.

Dictyopterin, 6-(D-threo-1,2-dihydroxypropyl)-pterin, a new natural isomer of L-biopterin. Isolation from vegetative cells of Dictyostelium discoideum and identification.

A major pterin was isolated by reverse-phase high-performance liquid chromatography from cellular extract of vegetative cells of Dictyostelium discoideum after perchloric deproteinization and oxidation with acidic iodine. This compound was characterized by its chromatographic behavior, its absorption and fluorescence properties, by its oxidation product with alkaline permanganate, by secondary ion mass spectrometry and by circular dichroism. The final identification was obtained by comparison with authentic materials. It is concluded that the major pterin of D. discoideum is the compound 6-(D-threo-1,2-dihydroxypropyl)-pterin. The name dictyopterin is proposed for this new natural isomer of L-biopterin.

Conversion of 6-substituted tetrahydropterins to 7-isomers via phenylalanine hydroxylase-generated intermediates.

A new variant form of hyperphenylalaninemia has recently been discovered in which the patients characteristically excrete 7-biopterin in their urines in addition to the natural 6-biopterin (Curtius, H. Ch., Kuster, T., Matasovic, A., Blau, N. & Dhondt, J.-L. (1988) Biochem. Biophys. Res. Commun. 153, 715-721). This isomer had not been found previously in humans, and although its origin was not established, preliminary evidence suggested that it might be produced from 6-biopterin. We have now found that 7-biopterin can be formed in vitro from (6R)-tetrahydrobiopterin during the hydroxylation of phenylalanine catalyzed by phenylalanine hydroxylase [L-phenylalanine, tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1]. The resulting 7-biopterin was unequivocally identified by the following criteria: preparative isolation and conversion to 7-hydroxymethylpterin following periodate oxidation and borohydride reduction, quantitative conversion to pterin-7-carboxylic acid after oxidation with permanganate, and liquid chromatography/thermospray mass spectrometry. Addition of 4a-carbinolamine dehydratase, an enzyme involved in the regeneration of tetrahydrobiopterin from the pterin carbinolamine intermediate (also called 4a-hydroxytetrahydrobiopterin) formed in the phenylalanine hydroxylase reaction, greatly decreased the amount of the 7-biopterin formed. This result implies that the in vitro formation of 7-biopterin occurs via the nonenzymatic rearrangement of the unstable substrate of the dehydratase, 4a-hydroxytetrahydrobiopterin, and suggests that this new variant of hyperphenylalaninemia may be caused by a lack of 4a-carbinolamine dehydratase activity. A mechanism for the rearrangement is proposed that predicts that other 6-substituted tetrahydropterin substrates of the aromatic amino acid hydroxylases could also give rise to rearranged products from an opening of the pyrazine ring of the corresponding 4a-hydroxytetrahydropterin intermediate.

Identification of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster.

Using reversed-phase high-performance liquid chromatography with electrochemical detection we have demonstrated the occurrence of 5,6,7,8-tetrahydropterin and 5,6,7,8-tetrahydrobiopterin in Drosophila melanogaster. The former is the first time that has been detected in vivo. The identification has been based on the retention times, hydrodinamic voltagrams and the differential concentration in three strains of Drosophila melanogaster. Compared to the wild type, the Punch2 mutant has diminished levels of both pteridines, whereas Henna-recessive3 lacks completely tetrahydropterin and has increased levels of tetrahydrobiopterin, as expected according to their biochemical lesions.

Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. Activation of intra- and interdomain electron transfer.

In neuronal nitric-oxide synthase (NOS), electron transfer proceeds across domains in a linear sequence from NADPH to flavins to heme, with calmodulin (CaM) triggering the interdomain electron transfer to the heme (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10772). Here, we utilized a neuronal NOS devoid of its bound heme and tetrahydrobiopterin (apo-NOS) to examine whether interdomain electron transfer is responsible for CaM's activation of NO synthesis, substrate-independent NADPH oxidation, and cytochrome c and ferricyanide reduction. Of the four activities, two (cytochrome c and ferricyanide reduction) were similarly stimulated by CaM in apo-NOS when compared with native NOS, indicating that activation occurs by a mechanism not involving flavin-to-heme electron transfer. Further analysis showed that CaM increased the rate of electron transfer from NADPH into the flavin centers by a factor of 20, revealing a direct activation of the NOS reductase domain by CaM. In contrast, CaM's activation of NO synthesis and substrate-independent NADPH oxidation appeared to involve flavin-to-heme electron transfer because these reactions were not activated in apo-NOS and were blocked in native NOS by agents that prevent heme iron reduction. Thus, CaM activates neuronal NOS at two points in the electron transfer sequence: electron transfer into the flavins and interdomain electron transfer between the flavins and heme. Activation at each point is associated with an up-regulation of domain-specific catalytic functions. The dual regulation by CaM is unique and represents a new means by which electron transfer can be controlled in a metalloflavoprotein.

Separation of pineal extracts by gelfiltration. VI. Isolation and identification from sheep pineals of biopterin; comparison of the isolated compound with some synthetic pteridines and the biological activity in in vitro and in vivo bioassays.

Aqueous extracts of sheep pineal bodies were separated on Sephadex G-25. Two low molecular weight Sephadex G-25 fractions, F2 and F3, were ultrafiltrated through the Amicon membrane UM-2. The UM-2 filtrate was subsequently filtrated through the ultramembrane UM-05 and the UM-05 filtrate was separated on Sephadex G-10 columns. After paper electrophoresis, preparative paper chromatography was carried out. The fluorescent band showing a Rf value identical with synthetic 6-biopterin was eluted; gas liquid chromatography and mass spectrometry of the isolated compound were carried out. The mass spectra of the isolated compound were shown to be identical with synthetic 6-biopterin. The results of the Crithidia fasciculata test and thinlayer chromatography study revealed that the isolated compound is identical with 6-L-erythro-biopterin. The activities of the isolated compound and of synthetic biopterin in in vitro and in vivo bioassays are demonstrated.

Purification, cloning, and functional expression of dihydroneopterin triphosphate 2'-epimerase from Escherichia coli.

Dihydroneopterin triphosphate (H2NTP) 2'-epimerase from Escherichia coli catalyzes the epimerization of H2NTP to dihydromonapterin triphosphate (H2MTP). The enzyme was purified 954-fold to apparent homogeneity by a combination of ammonium sulfate fractionation and column chromatography of Cibacron blue 3GA dye ligand, phenyl-Sepharose CL-4B, methotrexate-agarose, and Superdex 200 HR 10/30 FPLC column. The molecular mass of the epimerase determined on a Superdex column was 82.6 kDa, while the subunit molecular mass determined on SDS-polyacrylamide gel electrophoresis was 13.7 kDa. This implies that the epimerase most probably exists as homohexamer. The 20-amino acid sequence from the N terminus was determined (AQPAAIIRIKNLRLRTFIGI). Based on this sequence, the gene encoding the epimerase was cloned using a simple polymerase chain reaction approach. Translation of the nucleotide sequence of the cloned gene revealed the presence of an open reading frame containing 120 amino acids with a predicted molecular mass of 13,993 Da. The epimerase gene located in a 2.3-kilobase BamHI-EcoRI fragment from Kohara's clone 406 was overexpressed 300-fold, which was confirmed by the prominent increase in the 14-kDa protein band on SDS-polyacrylamide electrophoresis gels. It showed no homology with the sequences of isomerases or other enzymes in GenBank/EMBL data bases.

Guanosine triphosphate cyclohydrolase I assay in human and rat liver using high-performance liquid chromatography of neopterin phosphates and guanine nucleotides.

D-erythro-7,8-Dihydroneopterin triphosphate (NH2TP) formed from guanosine triphosphate (GTP) by GTP cyclohydrolase I (EC 3.5.4.16) in the presence of EDTA was oxidized to neopterin triphosphate (NTP) by iodine, separated from substrate and other compounds by ion-paired reverse-phase HPLC, and quantitated by fluorometric detection at 365/446 nm. Excess GTP at the end of reaction was controlled by simultaneous detection of guanine nucleotides at 254 nm. The method required only 15 mg of liver tissue for the measurement of GTP cyclohydrolase I and is suitable for activity measurement in liver biopsies. The detection limit was 4 pmol of NTP at a signal to noise ratio of 10:1. The activity of GTP-cyclohydrolase I in homogenates of human liver (n = 10) was 45 pmol NH2TP (range 32-60) formed per milligram protein per hour at 37 degrees C. Liver homogenates from Wistar rats (n = 8) formed 47 pmol NH2TP (range 35-61) per milligram protein per hour.

Tetrahydrobiopterin and total biopterin content of neuroblastoma (N1E-115, N2A) and pheochromocytoma (PC-12) clones and the dependence of catecholamine synthesis on tetrahydrobiopterin concentration in PC-12 cells.

Tetrahydrobiopterin content was determined in several clonal cell lines by reversed-phase HPLC and subsequent electrochemical detection. The same chromatography system was used to determine the total biopterin (tetrahydrobiopterin and 7,8-dihydrobiopterin) by fluorescence detection. The catecholamine-producing clones neuroblastoma N1E-115 and pheochromocytoma PC-12 contained 96 and 60 ng tetrahydrobiopterin/mg protein, respectively. The corresponding amount for the neuroblastoma clone N2A was 36 ng/mg protein. The tetrahydrobiopterin content in C-6 glioma cells was below the limit of detection. The total biopterin is about 20% above the tetrahydrobiopterin content. Tetrahydrobiopterin and biopterin from the cells were identified by coelution with standard solutions and by potential-current relationship or emission and excitation spectra, respectively. Addition of 2,4-diamino-6-hydroxypyrimidine, an inhibitor of biopterin synthesis from GTP, to the culture medium of PC-12 cells resulted in a dose-dependent decrease of tetrahydrobiopterin and total biopterin content within 4 h, suggesting that the cells are capable of synthesising the biopterin which was found. A decrease in intracellular tetrahydrobiopterin levels by different concentrations of 2,4-diamino-6-hydroxypyrimidine reduces the cellular production of dihydroxyphenylalanine after inhibition of aromatic L-amino acid decarboxylase, indicating that the concentration of tetrahydrobiopterin might be a limiting factor for catecholamine synthesis in catecholamine-producing cells.

Genetic engineering of Escherichia coli for production of tetrahydrobiopterin.

Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.