Abstract 5455: A Novel Haplotype on GCH1 Gene, Encoding GTP Cyclohydrolase 1, Regulates Vascular Biopterin Synthesis, eNOS Coupling and Vascular Redox in Human Vessels

Background: GTP cyclohydrolase (GTPCH) is a key enzyme in biopterins synthesis, while tetrahydrobiopterin (BH4) is a regulator of eNOS coupling in vascular endothelium. A novel haplotype in GCH1 gene, combining dbSNPs: rs8007267G/A, rs3783641A/T and rs10483639C/G, affects GTPCH activity and biopterins levels in inflammatory cells. We examined the effect of this haplotype on vascular biopterins, eNOS coupling and redox state in human vessels from patients with coronary atherosclerosis. Methods: Samples of saphenous veins (SV) were obtained from 347 patients undergoing CABG. Vasorelaxations of SV to acetylcholine (ACh) and vascular O2- (± eNOS inhibitor LNAME) were determined. Biopterins were measured by HPLC. The haplotypes were defined as X (rs8007267A+ rs3783641T+ rs10483639G) or O (all other haplotypes). Results: The haplotype distribution was OO:245(71%), OX:95(27%) and XX:7(2%). Carriers of the X haplotype had lower plasma (Fig. a ) and vascular (Fig. b ) BH4. The X haplotype was associated with higher vascular O2- (XX+XO: 2.97±0.44 vs OO:1.90±0.10 RLU/Sec/mg, p<0.01), greater LNAME-inhibitable O2- (Fig. c ) suggesting eNOS uncoupling) and lower NO bioavailability (Fig. d ) in human vessels. The X haplotype was also associated with higher plasma ox-LDL (51.0±2.2 in XX+XO vs 44.2±1.4 U/L in OO p<0.05) and lower BH4:total biopterins ratio (43.1±3.2 in XX+XO vs 51.7±2.1% in OO, p<0.05). Conclusions: This novel haplotype on GCH1 gene regulates biopterins biosynthesis in both plasma and vascular endothelium. This haplotype also regulates eNOS coupling, O2- production and NO bioavailability in human vessels, and may play a role in atherogenesis.

Influence of an Aromatic Amino Acid Decarboxylase Inhibitor on GTP Cyclohydrolase I Activity in the Rat Brain

Summary Based on the hypothesis that catecholamine decreases may cause increases in biopterin, we measured the GTP cyclohydrolase I(GTPCH-I) activity and biopterin content in the cerebral cortex, midbrain/diencephalon, cerebeilum, pons/medulla oblongata, striatum , and hippocampus of brains from rats given NSDlOIS, an inhibitor for aromatic amino acid decarboxylase . The catecholamine contents decreased in all the regions tested. The changes in the GTPCH -I activity and bioptcrin content were different among the regions; they decreased in some regions contrary to expectations and increased in other regions. However, either the enzyme activity or biopterin content increased in the midbrain/ diencephalon and pons/ medulla oblongata, in which similar levels of catecholamines, GTPCH -I activity and biopterin were detected. These results suggest that for the midbrain/diencephalon and pons/medulla oblongata the surmise that a decrease in catecholamines causes an increase in the biopterin synthesis may be correct.

Immune Stimulation of Interferon-Gamma- and Biopterin Synthesis with Endotoxin in Animal-Experimental Peritonitis

Summary As a result to a microhial stimulus different defence systems are activated. Bacterial lipopolysaccharids (endotoxins) can lead to activation of the immune system in which phagocytic cells like macrophages. secrete cytokines and inflammatory mediators. Owing to a hyperactivation of immune mechanisms and also to generalized inflammatory processes organic lesions or even multiple failure of organs can evolve due to influences by cytokines and anaphylatoxines. On a standardized peritonitis model with wistar-pragrats. release of endotoxin. interferon-gamma synthesis and release of hiopterin into plasma has heen measured 6 hours after intraperi to neal administration of E.-coli-haemoglobin suspension. In the initial phase of the immune response interferon-gamma - being endogenously induced by endotoxin - stimulates synthesis of pteridines. Due to antagonistic effects to the cytokines (which stimulate the inflammatory processes) interferon-gamma regulatorily intervenes with the inflammatory mechanisms. The production of biopterin serves as a measure of activation of the monocyte/macrophage system. In comparison to a control group. treated with sodium chloride. statistically significant different have been found in the measuring parameters endotoxin. biopterin and interferon-gamma. The significant correlation of all parameters confirm the quick activation of immune mechanisms as response of the immune system to endotoxinaemia in diffuse purulent peritonitis.

Liquid-chromatographic measurement of biopterin and neopterin in serum and urine

We report an improved "high-performance" liquid-chromatographic (HPLC) method for measuring biopterin and neopterin in serum and urine. Specimens are acidified, treated with iodine in 0.2 mol/L trichloroacetic acid, party purified on Bio-Rad MP-50 cation-exchange columns, and analyzed by reversed-phase HPLC with fluorometric detection. The minimal concentration of biopterin detectable is 0.3 micrograms/L in a 50-microL injection. The total CV is less than or equal to 10%. Improvements over other reported methods include the use of a single, simplified sample-preparation step with a Baker-10 SPE System, and a guard column to increase analytical column stability and analyte recovery. The assay is semiautomated to reduce technician time and improve precision. Mean observed values for biopterin and neopterin in sera of normal human adults were 1.64 and 5.52 micrograms/L, respectively. The mean ratio of neopterin to biopterin in acidified adult urine samples was lower than that found in matched nonacidified samples (n = 10). Serum specimens from diagnosed phenylketonuric (PKU) and hyperphenylalaninemic patients were also analyzed for biopterin and neopterin; the findings agreed with reported values for similar patients. One patient, previously identified as an atypical PKU patient, showed serum values of neopterin and biopterin suggestive of a defect in biopterin synthesis.