Nitric oxide in exhaled gas and tetrahydrobiopterin in plasma after exposure to hyperoxia.

The fraction of nitric oxide in exhaled gas (FENO) is decreased after exposure to hyperoxia in vivo, although the mechanisms for this decrease is not clear. A key co-factor for nitric oxide synthase (NOS), tetrahydrobiopterin (BH4), has been shown to be oxidized in vitro when exposed to hyperoxia. We hypothesized that the decrease of FENO is due to decreased enzymatic generation of NO due to oxidation of BH4. The present study was performed to investigate the relationship between levels of FENO and plasma BH4 following hyperoxic exposure in humans. Two groups of healthy subjects were exposed to 100% oxygen for 90 minutes. FENO was measured before and 10 minutes (n = 13) or 60 minutes (n = 14) after the exposure. Blood samples were collected at the same time points for quantification of biopterin levels (BH4, BH2 and B) using LC-MS/MS. Each subject was his or her own control, breathing air for 90 minutes on a separate day. Hyperoxia resulted in a 28.6 % decrease in FENO 10 minutes after exposure (p < 0.001), confirming previous findings. Moreover, hyperoxia also caused a 14.2% decrease in plasma BH4 (p = 0.012). No significant differences were observed in the group measured 60 minutes after exposure. No significant correlation was found between the changes in FENO and BH4 after the hyperoxic exposure (r = 0.052, p = 0.795), this might be due to the recovery of BH4 being faster than the recovery of FENO.

Hyperoxia and lack of ascorbic acid deplete tetrahydrobiopterin without affecting NO generation in endothelial cells.

Nitric oxide (NO) may protect against gas bubble formation and risk of decompression sickness. We have previously shown that the crucial co-factor tetrahydrobiopterin (BH4) is oxidized in a dose-dependent manner when exposed to hyperoxia similar to diving conditions but with minor effects on the NO production by nitric oxide synthase. By manipulating the intracellular redox state, we further investigated the relationship between BH4 levels and production of NO in human endothelial cells (HUVECs). HUVECs were cultured with and without ascorbic acid (AA) and the glutathione (GSH) synthesis inhibitor buthionine sulfoximine, prior to hyperoxic exposure. The levels of biopterins and GSH were determined in cell lysates while the production of NO was determined in intact cells. Omitting AA resulted in a 91% decrease in BH4 levels (0.49 ± 0.08 to 0.04 ± 0.01 pmol/106 cells, p⟨0.001) at 20 kPa oxygen (O2), and 88% decrease (0.24 ± 0.03 to 0.03 ± 0.01 pmol/106 cells, p=0.01) after exposure to 60 kPa O2. The NO generation was decreased by 23% (74.5 ± 2.2 to 57.3 ± 5.6 pmol/min/mg protein, p⟨0.001) at 20 kPa O2, but no significant change was observed at 60 kPa O2. GSH depletion had no effects on the NO generation. No correlation was found between NO generation and the corresponding intracellular BH4 concentration (p=0.675, r=-0.055) or the BH4 to BH2 ratio (p=0.983, r=0.003), determined across 18 in vitro experiments. Decreased BH4 in HUVECs, due to hyperoxia or lack of ascorbic acid, does not imply corresponding decreases in NO generation.

Decrease of tetrahydrobiopterin and NO generation in endothelial cells exposed to simulated diving.

Purpose: Nitric oxide (NO) has been shown to protect against bubble formation and the risk of decompression sickness. We hypothesize that oxidation of tetrahydrobiopterin (BH4) leads to a decreased production of NO during simulated diving. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to hyperoxia or simulated diving for 24 hours. The levels of biopterins (BH4, BH2 and B) were determined by LC-MS/MS, and the production of NO by monitoring the conversion of L-arginine to L-citrulline. Results: Exposure to hyperoxia decreased BH4 in a dose-dependent manner; by 48 ± 15% following exposure to 40 kPa O2 (P⟨0.001 vs. control at 20 kPa O2), and 70 ± 16% following exposure to 60 kPa O2. Exposure to 40 kPa O2 decreased NO production by 25 ± 9%, but there was no further decrease when increasing oxygen exposure to 60 kPa (25 ± 10%). No additional effects of simulated diving were observed, indicating no additive or synergistic effects of hyperbaria and hyperoxia on the BH4 level or NO generation. Conclusion: NO generation in intact human endothelial cells was decreased by simulated diving, as well as by hyperoxic exposure, while BH4 levels seem to be affected only by hyperoxia. Hence, the results suggest that BH4 is not the sole determinant of NO generation in HUVEC.