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.

Keywords: HPLC with electrochemical detection; oxidative stress; sepsis; tetrahydrobiopterin.

Figure 1

Scheme with work-up conditions for harvesting and processing of animal tissues. Created with Biorender.com (last accessed 14 June 2022).

Figure 2

(A) Chromatograms of 100 µM BH₄ and BH₂ standards represented at oxidation potentials of 150, 280 and 600 mV for BH₄ (retention time 5.4 min) and at the oxidation potential of 600 mV for BH₂ (retention time 9.4 min). (B) Standard curves for BH₄ and BH₂ solutions, representing a linear range (25–150 and 25–100 µM concentration) with a linear regression correlation coefficient R² of >0.98 for both standards. Data are mean ± SD of n = 4 independent measurements.

Figure 3

(A) Detected BH₄ peak areas in solutions of BH₄ (100 µM) standards (quantified as the sum of 150 mV and 280 mV oxidation potentials, eluted at 5.4 min) upon storage under indicated conditions and time. (B) Peak areas of BH₂ detected (quantified at 600 mV oxidation potential, retention time 9.4 min) in the same samples. (C) Detected BH₂ peak areas in solutions of BH₂ (100 µM) standards (quantified at 600 mV oxidation potential, eluted at 9.4 min) upon storage under different conditions. Solvent conditions were either hydrochloric acid (HCl, 100 µM) or HCl (100 µM) with dithioerythritol (DTE, 1 mM) and diethylenetriaminepentaacetic acid (DTPA, 1 mM). Data are mean ± SD of the number of indicated independent measurements. Note: * means significantly different to fresh standard in HCl; § means significantly different to fresh standard in HCl with DTE/DTPA.

Figure 4

(A) Representative chromatograms showing the changes in peak intensities due to BH₄, BH₂ and unidentified product (marked with “?” symbol, retention time approximately 11 min) in BH₄ (100 µM) solutions incubated in the presence or absence of H₂O₂ (1 mM). (B) Concentrations of BH₄ standard and formed BH₂ product (quantified as the sum of 150 mV and 280 mV oxidation potentials for BH₄, and only 600 mV for BH₂) were either measured in freshly prepared samples in 100 µM HCl or after incubation overnight at 4 °C or after incubation overnight at 4 °C in the presence of 1 mM H₂O₂ in 0.1 mM HCl. Data are mean ± SD of the number of indicated independent measurements. Note: * means significantly different to fresh BH₄; § means significantly different to overnight BH₄.

Figure 5

(A) Representative chromatograms (detected at 280 mV oxidation potential) and determined apparent BH₄ concentrations for aorta of control and diabetic (STZ) rats. Peak areas detected at the retention time of 5.4 min were converted to a BH₄ concentration using the BH₄ standard curve and was finally normalized to mg protein as estimated by Lowry method in the tissue homogenate supernatant. Data are mean ± SD of the number of indicated independent measurements. Note: * means significantly different to control rats. (B) Representative chromatograms showing co-elution of BH₄ and ascorbate standards using the initial HPLC method (protocol A). (C) Representative chromatograms show clear separation of BH₄ and ascorbate standards using the optimized HPLC method (protocol B).

Figure 6

(A) Chromatograms of 25 µM BH₄ and BH₂ standards represented at oxidation potentials of 0, 150 and 280 for BH₄ (retention time 4.56 min) and at the oxidation potential of 280 mV for BH₂ (retention time 6.92 min). (B) Standard curves for BH₄ and BH₂ solutions, representing a linear range (BH₄: 0.3–125 µM, 13 concentration values; BH₂: 0.1–200 µM, 15 concentration values) with a linear regression correlation coefficient R² of 0.99 for both standards. Data are mean ± SD of n = 3 independent measurements per data point.

Figure 7

(A) Representative chromatograms (measured at 0 mV potential) of BH₄ in the brain, heart and kidney of control and LPS-treated septic rats. (B) Concentration levels in these tissues as calculated from BH₄ standard curve. Data are mean ± SD of the number of indicated independent measurements. Note: * means significantly different to control rats. *, p-value is < 0.05; **, p-value is < 0.01 to indicate significance against the control. (C) Representative chromatograms (measured at 0 mV potential) of BH₄ in the heart and brain of control rats with and without spiking with authentic BH₄ standard.