Diabetic rats exhibit decrease in plasma arginine, NO synthesis and tetrahydrobiopterin in endothelial cells (EC). Treatment with L-arginine may be beneficial for enhancing NO synthesis in diseases associated with endothelial dysfunction. However, little is known about the mechanism responsible for the stimulatory effect of arginine on endothelial NO synthesis. We hypothesized that dietary arginine supplementation increases BH4 for NO synthesis in EC of diabetic rats, thereby preventing endothelial dysfunction. In experiment I, streptozotocin (STZ) induced-diabetic male Sprague Dawley (SD) rats (a model of type-I diabetes) were individually pair-fed a casein-based diet on the basis of feed intake (per kg body weight) of non-diabetic SD rats. Addition of arginine-HCl or alanine to drinking water for the rats were adjusted daily to ensure isonitrogenous provision per kg body weight. In non-diabetic rats, arginine supplementation increased plasma arginine (144%), plasma insulin (44%), EC arginine (88%), EC BH4 (106%) and EC NO synthesis (80%), compared with alanine treatment. In diabetic rats, arginine supplementation reduced body weight loss (36%), and plasma glucose (54%), and increased plasma arginine (110%), plasma insulin (209%), EC arginine (173%), EC BH4 (128%) and EC NO synthesis (125%), compared with alanine treatment. In experiment II, male Zucker diabetic fatty (ZDF) rats (a model of type-II diabetes) were individually pair-fed a Purina 5008 diet on the basis of feed intake by alanine-treated diabetic rats (per kg body wt). Addition of arginine-HCl or alanine to drinking water for the rats was adjusted daily to ensure isonitrogenous provision per kg body weight. Arginine supplementation to ZDF rats did not affect plasma glucose and insulin, reduced epidididmal fat (30%), abdominal fat (43%) and body weight gain (18%), and increased plasma arginine (273%), EC arginine (197%), EC BH4 (120%) and EC NO synthesis (122%), compared with alanine-treated ZDF rats. These results show that dietary L-arginine supplementation increases BH4 and NO synthesis in EC of both STZ-diabetic and ZDF rats. Strikingly, arginine treatment prevented hyperglycemia in STZ-diabetic SD rats and reduced obesity in ZDF rats. Collectively, results demonstrate that oral administration of arginine is beneficial for both type-I and type-II diabetic rats.

Diabetic rats exhibit decrease in plasma arginine, NO synthesis and tetrahydrobiopterin in endothelial cells (EC). Treatment with L-arginine may be beneficial for enhancing NO synthesis in diseases associated with endothelial dysfunction. However, little is known about the mechanism responsible for the stimulatory effect of arginine on endothelial NO synthesis. We hypothesized that dietary arginine supplementation increases BH4 for NO synthesis in EC of diabetic rats, thereby preventing endothelial dysfunction. In experiment I, streptozotocin (STZ) induced-diabetic male Sprague Dawley (SD) rats (a model of type-I diabetes) were individually pair-fed a casein-based diet on the basis of feed intake (per kg body weight) of non-diabetic SD rats. Addition of arginine-HCl or alanine to drinking water for the rats were adjusted daily to ensure isonitrogenous provision per kg body weight. In non-diabetic rats, arginine supplementation increased plasma arginine (144%), plasma insulin (44%), EC arginine (88%), EC BH4 (106%) and EC NO synthesis (80%), compared with alanine treatment. In diabetic rats, arginine supplementation reduced body weight loss (36%), and plasma glucose (54%), and increased plasma arginine (110%), plasma insulin (209%), EC arginine (173%), EC BH4 (128%) and EC NO synthesis (125%), compared with alanine treatment. In experiment II, male Zucker diabetic fatty (ZDF) rats (a model of type-II diabetes) were individually pair-fed a Purina 5008 diet on the basis of feed intake by alanine-treated diabetic rats (per kg body wt). Addition of arginine-HCl or alanine to drinking water for the rats was adjusted daily to ensure isonitrogenous provision per kg body weight. Arginine supplementation to ZDF rats did not affect plasma glucose and insulin, reduced epidididmal fat (30%), abdominal fat (43%) and body weight gain (18%), and increased plasma arginine (273%), EC arginine (197%), EC BH4 (120%) and EC NO synthesis (122%), compared with alanine-treated ZDF rats. These results show that dietary L-arginine supplementation increases BH4 and NO synthesis in EC of both STZ-diabetic and ZDF rats. Strikingly, arginine treatment prevented hyperglycemia in STZ-diabetic SD rats and reduced obesity in ZDF rats. Collectively, results demonstrate that oral administration of arginine is beneficial for both type-I and type-II diabetic rats.

Nitric oxide synthase (NOS) catalyses the production of nitric oxide (NO). A cytochrome P450-like oxygenase, it uses two monooxygenation steps to convert L-arginine (L-arg) first to N -hydroxy-L-arginine (NOHA), a stable intermediate, and then to L-citrulline and NO. Mammalian NOSs are homodimeric enzymes. Each monomer is composed of an oxygenase domain (containing the L-arg binding site, a heme ligated by a cysteine thiolate, and a tetrahydrobiopterin (H4B)) and a reductase domain (binding NADPH, FAD, and FMN). NOS substrates are O2, L-arg, and NADPH. NADPH is the source of electrons required for oxygen activation. H4B is a vital cofactor that aids dimerisation and acts as a reducing/oxidising agent. Controversy still exists as to the final oxygenating species in the NOS mechanism, but the general reaction scheme is known. The ferric heme is reduced to the ferrous state by an electron from the reductase domain. Then oxygen binds to form the oxy-ferrous species. Then H4B donates an electron to form a peroxy-ferric species. It is likely this then forms a compound 1 (Fe(IV)+.=O) species that is the final oxygenating species. This thesis probes the mechanism of NOS to further define the mechanistic intermediates involved. The role of H4B in NO synthesis has been probed in both normal turnover conditions and special case reactions. To elucidate this mechanism further a mutant with a residue capable of stabilising the activated oxygen species was created, G586S, where glycine 586 of nNOS was replaced with a serine. This serine was within hydrogen bonding distance of the oxy-heme. A stabilised intermediate was observed by stopped flow reaction in the presence of H4B, but not aH4B (an inactive pterin analogue). Here single turnover reactions, each following either the reaction of L-arg to NOHA or NOHA to citrulline, were performed on the mutant using an external source of electrons. The reaction products were observed by HPLC. The mutant appears capable of the conversion of NOHA to citrulline, but not L-arg to NOHA. The WT enzyme appears capable of both. The intermediate is observed with either L-arg or NOHA bound, suggesting both reactions proceed via the same active oxygenating species. The inability of the mutant to catalyse the conversion of L-arg to NOHA may be due to protonation of the substrate hindering reaction such that the active oxygenating species decays before reaction can occur. This mutation, in allowing separation of the two monooxygenation steps, deserves further study. H4B binds at the dimer interface of NOS. Here the -systems of the pterins are only 13Å apart. This is within allowed distances for efficient electron transfer. Electron transfer between hemes, via the pterins, would allow a route for the breakdown of a dead end, ferrous-NO, species. Stopped flow monitoring of the decay of the ferrous-heme NO complex with nNOSoxy dimers with varying proportions of the hemes in the ferrous heme-NO complex showed no electron transfer between hemes of the dimer. The rate of decay of the ferrous heme-NO complex in oxygenated buffer is 0.12 s-1 for all conditions tested here. H4B-deficiency leads to several diseases. H4B makes a poor drug due to instability and cost, the search for druggable analogues of it is ongoing. H4B analogues blocked at the 6,7-positions in the dihydropterine-form have been screened here for catalytic activity. Several have shown comparable ability to catalyse NO production in vitro. Structure function analysis of these analogues has revealed the extent extension is tolerated at the C6 and C7 positions of the pterin.

No / Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH4). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10¿6 M 6BH4 to a specific domain 28 amino acids away from the CuA active site of the enzyme. Alternatively it was then shown that 10¿3 M 6BH4 inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH4 (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH4 occurs in the concentration range of 10¿6 M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H2O2 from O2. Moreover, a direct oxidation of 6BH4 to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H2O2 (<0.3 × 10¿3 M), but deactivated at concentrations in the range 0.5¿5.0 × 10¿3 M. In summary, our results confirm a major role for 6BH4 in the regulation of human pigmentation.

Thesis (M. S.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Samuel C. Dudley, Jr., Committee Chair ; Hanjoong Jo, Committee Member ; W. Robert Taylor, Committee Member.

Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 1999. / Includes bibliographical references. Also available on the Internet.

Autism spectrum disorder (ASD) affects about 1 in 45 individuals in the United States, yet effective treatments are yet to be defined. There is growing evidence that ASD is associated with abnormalities in several metabolic pathways, including the inter-connected folate, methylation and glutathione pathways. Several treatments that can therapeutically target these pathways have been tested in preliminary clinical trials. The combination of methylcobalamin (mB12) with low-dose folinic acid (LDFA) and sapropterin, a synthetic form of tetrahydrobiopterin (BH4) have been studied in open label trials while high-dose folinic acid has been studied in a double-blind placebo controlled trial. All of these treatments have the potential to positively affect folate, methylation and glutathione pathways. Although the effect of mB12/LDFA and BH4 on methylation and glutathione metabolism have been examined in the open-label studies, these changes have not been compared to controls who received a placebo in order to account for the natural variation in the changes in these pathways. Furthermore, the recent study using high-dose folinic acid (HDFA) did not analyze the change in metabolism resulting from the treatment. Thus, we compared changes in methylation and glutathione metabolism and biomarkers of chronic oxidative stress as a result of these three treatments to individuals receiving placebo. In general, mB12/LDFA treatment had a significant effect on glutathione and cysteine metabolism with a medium effect size while BH4 had a significant effect on methylation and markers of chronic oxidative stress with a large effect size. HDFA treatment did not significantly influence biomarkers of methylation, glutathione or chronic oxidative stress. One caveat was that participants in the mB12/LDFA and BH4 studies had significantly worse markers of glutathione metabolism and chronic oxidative stress at baseline, respectively. Thus, the participants selected in these two clinical trials may have been those with the most severe metabolic abnormalities and most expected to respond to these treatments. Overall this study supports the notion that metabolic abnormalities in individuals with ASD may be amenable to targeted treatments and provide some insight into the mechanism of action of these treatments.

The inflammatory nature of the tumor microenvironment provides a cytokine and chemokine rich proliferative environment. Much of the responsibility of this environment is due to the production of Reactive Oxygen Species (ROS). These studies examined the proliferative rich tumor environment from a new perspective of Nitric Oxide Synthase (NOS) dysregulation. NOS’s have the ability to become uncoupled and generate superoxide in lieu of nitric oxide (NO). A requirement of NOS for the production of NO is the cofactor tetrahydrobiopterin (BH4) and when it is missing NOS becomes uncoupled and turns into a peroxynitrite synthase. Here I demonstrate that NOS is uncoupled in tumor cells due to depleted BH4 levels. This uncoupling leads to decreased NO signaling and increased pro-inflammatory, pro-survival, signaling as a result of the increased generation of ROS/RNS from uncoupled NOS activity. I was able to recouple NOS through exogenous BH4 both in vitro and in vivo, reducing ROS/RNS and reestablishing NO signaling through cGMP protein associated kinase. Reduction of ROS/RNS resulted in the reduced activity of two major constitutively active transcription factors in breast cancer cells, NFκB and STAT3. In MCF-7 and MDA231 cells I found that increased NO-dependent PKG signaling led to tumor cell toxicity mediated by downregulation of β-catenin. Downregulation of β-catenin led to increased protein levels of p21 in MCF-7 and p27 in MDA 231cells, ultimately resulting in cell death. These results suggest that there is potential for BH4 as a therapeutic agent since exogenous dietary BH4 ameliorates chemically induced colitis, and reduced azoxymethane (AOM) induced colon and spontaneously developing mammary carcinogenesis.

Background Tetrahydrobiopterin (BH4)-sensitive phenylketonuria (PKU) can be treated with sapropterin dihydrochloride. We studied metabolic control and health-related quality of life (HRQoL) in PKU patients treated with BH4. Subjects and methods Based on the review of neonatal BH4 test results and mutation analysis in 41 PKU patients, 19 were identified as potentially BH4-sensitive (9 females, 10 males, age 4–18 years). We analyzed phenylalanine (phe) concentrations in dried blood samples, nutrition protocols, and HRQoL questionnaires (KINDL®) beginning from 1 year before, during the first 42 days, and after 3 months of BH4 therapy. Results Eight BH4-sensitive patients increased their phe tolerance (629±476 vs. 2131±1084 mg, p00.006) while maintaining good metabolic control (phe concentration in dried blood 283±145 vs. 304±136 μM, p01.0). Six of them were able to stop dietary protein restriction entirely. BH4- sensitive patients had average HRQoL scores that were comparable to age-matched healthy children. There was no improvement in HRQoL scores after replacing classic dietary treatment with BH4 supply, although personal reports given by the patients and their parents suggest that available questionnaires are inappropriate to detect aspects relevant to inborn metabolic disorders. Discussion BH4 can allow PKU patients to increase their phe consumption significantly or even stop dietary protein restrictions. Unexpectedly, this does not improve HRQoL as assessed with KINDL®, partly due to high scores even before BH4 therapy. Specific questionnaires should be developed for inborn metabolic disorders.

München, Techn. Univ., Diss., 2002.