Measurement of Tetrahydrobiopterin in Animal Tissue Samples by HPLC with Electrochemical Detection—Protocol Optimization and Pitfalls

Abstract

Tetrahydrobiopterin (BH4) is an essential cofactor of all nitric oxide synthase isoforms, thus determination of BH4 levels can provide important mechanistic insight into diseases. We established a protocol for high-performance liquid chromatography/electrochemical detection (HPLC/ECD)-based determination of BH4 in tissue samples. We first determined the optimal storage and work-up conditions for authentic BH4 and its oxidation product dihydrobiopterin (BH2) under various conditions (pH, temperature, presence of antioxidants, metal chelators, and storage time). We then applied optimized protocols for detection of BH4 in tissues of septic (induced by lipopolysaccharide [LPS]) rats. BH4 standards in HCl are stabilized by addition of 1,4-dithioerythritol (DTE) and diethylenetriaminepentaacetic acid (DTPA), while HCl was sufficient for BH2 standard stabilization. Overnight storage of BH4 standard solutions at room temperature in HCl without antioxidants caused complete loss of BH4 and the formation of BH2. We further optimized the protocol to separate ascorbate and the BH4 tissue sample and found a significant increase in BH4 in the heart and kidney as well as higher BH4 levels by trend in the brain of septic rats compared to control rats. These findings correspond to reports on augmented nitric oxide and BH4 levels in both animals and patients with septic shock.

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

“…In general, the process of H 4 Bip autoxidation has a chain radical character [ 114 ] and the influence of H 4 Bip autoxidation on physiological processes can hardly be overestimated. For this reason, precise determination of H 4 Bip is much needed both in aqueous solutions and biological fluids [ 123 ].…”

Dispel some mist on circulating biopterins: measurement, physiological interval and pathophysiological implication

“…As for issue-i, about half of the works in Table 1 introduced antioxidants in time [20][21][22][23][24][25][26] , while others not [9][10][11][12][13][14][15][16][17][18] . However, according to the previous experience, BH4 was rather fragile to form BH2, B and other unknown compounds through non-enzymatic auto-oxidation [20,[27][28][29] .…”