Tetrahydrobiopterin as a key factor in the pathophysiology of diving

MSc Ronja Hesthammer started her work at Norwegian Centre for Diving Medicine, Dept. of Occupational Medicine, Haukeland University Hospital 1.09.2014. Training: Basic Course in Medical and Health Related Research (MEDMET 1, UoB) completed winter 2015, course in Hyperbaric Physiology (MEDT 8013, NTNU) spring 2015, Medical Statistics Part II (KLMED 8005, NTNU) was completed spring 2016. A 5 days writing course for PhD-students was completed autumn 2017. The PhD-student has given presentations at the annually conference of the European Underwater and Baromedical Society in Geneva, 13-16.09.2016, at a meeting of the Norwegian Baromedical Society in Bergen 30.09.2016, at the contact meeting of Norwegian Biochemical Society at Hafjell, 18-21.01.2018, and at the 10th International Conference on the Biology, Chemistry and Therapeutic Applications of Nitric Oxide, Oxford, UK, 16. ? 20.09.2018. Research work: The PhD-student has had training in sterile work at the hyperbaric cell culture laboratory, analytical methods at the Dept. of Clinical Science, and methods in respiratory physiology at the Dept. of Thoracic Medicine. Determination of the co-factor tetrahydrobiopterin (BH4) and the oxidised products dihydrobiopterin (BH2) and biopterin by use of high-pressure liquid chromatography and mass spectrometry requires considerable experience for interpretation of data. She has also established a method for activity determination of the enzyme that generates the signal molecule nitric oxide (NO) in blood vessel cells using radioactive labelled arginine. After training, the PhD-student has been working independently with all the methods. A human study exposed 15 healthy subjects to 100 % O2. NO in exhaled breath (FENO) and BH4 and phenylalanine (Phe) in blood samples were determined prior to and after the exposure. The Regional Committee for Ethics approved the study. A pilot study on patients at hyperbaric oxygen treatment was not carried out due to few available patients. The optimal time for measurements after exposure was chosen based upon previous data and new data from three healthy subjects exposed to 100 % O2 for 90 min. The results indicated that BH4 was declining 60 min. after exposure, and this was chosen as a standard time point for measurements. In some cases NO was determined at both 30 and 60 min. The results showed a transient decrease of FENO and return to basal level within one hour after exposure. Since the decrease in NO occurred earlier than expected, a supplementary study was carried out comprising 13 new subjects and determination of NO and BH4 immediately after exposure. Combining all data demonstrated a decrease in BH4 and FENO of 14 and 12 %, respectively, thus supporting the hypothesis that high O2-level oxidises BH4 and impairs NO-generation. The results are prepared for a scientific paper and submitted for publication. The remaining research work used a cell model of human blood vessel cells exposed to elevated O2 level and high ambient pressure. Both the content of BH4 and generation of NO were determined in the cells after exposure. The amount of BH4 decreased with increasing O2-concentration, probably due to oxidation of BH4. A corresponding increase in the BH2 levels was not observed, suggesting formation of products other than BH2. Generation of NO was not affected to the same extent. Simulated diving at high ambient pressure had little or no additional effect on BH4, but decreased the NO- generation similar to hyperoxia. Ascorbic acid (Vitamin C) was added to the culture media to ensure a sufficient level of BH4. Omitting ascorbic acid resulted in an 86 % decrease in BH4-level, and even lower after hyperoxic exposure, while the NO-generation was hardly affected. Depleting the cells of the antioxidant glutathione (GSH) to a level less than 2 % of control had no or negligible effect neither on BH4 nor the NO-generation. This suggests that antioxidants other than GSH are important to prevent oxidation of BH4. Data from all cell experiments were utilised to explore the relation between the intracellular concentration of BH4 and the NO-generation, but no correlation could be found. This indicates that the level of BH4 required to maintain the cellular NO-generation is lower than previously assumed and that depletion of BH4 due to hyperoxia or lack of antioxidant does not imply a concordant decrease in NO-generation. The results indicate a complex relation between BH4-level and NO-generation in human blood vessel cells. Two scientific papers were prepared based upon the data from the cell culture experiments and were accepted for publication in Undersea and Hyperbaric Medicine and are included in the thesis "Tetrahydrobiopterin and nitric oxide synthesis after exposure to hyperoxia and simulated diving. An experimental study in human endothelial cells and healthy humans". The thesis were submitted to the University of Bergen 27.06.2019 to be evaluated for a PhD degree.

Hovedmålsettingen med prosjektet har vært å undersøke biokjemiske mekanismer som kan ha betydning for utvikling av trykkfallsyke ved dykking. Resultatene tyder på at den biokjemiske kofaktoren tetrahydrobiopterin som er nødvendig for produksjonen av signalmolekylet nitrogenoksid delvis ødelegges under betingelser som tilsvarer det man har ved dykking, men at det er mindre sannsynlig at det medfører de negative konsekvensene for sirkulasjonssystemet man kunne frykte. Det gjenstår imidlertid noe arbeid for å bekrefte at funnene som er gjort med isolerte blodkarceller også gjelder for det vaskulære systemet hos mennesker. Resultatene bidrar til å øke kunnskapsnivået innen dykkefysiologi og hever kompetansenivået hos de involverte fagmiljøene. Det bidrar også til økt forståelse av biokjemiske signalveier i det vaskulære systemet generelt, og er derfor nyttig kunnskap også for dem som arbeider med funksjonalitet til blodkar uavhengig av dykking.

Saturation diving is an efficient intervention method in the offshore petroleum industry, but an occupational activity under extreme environmental conditions. The main objective of the present project is to increase the knowledge of health effects of divi ng and how adverse effects can be prevented. Small signal molecules like nitric oxide (NO) play an important role in the regulation of cardiovascular function and small vessel functionality. The generation of NO in blood vessels and the dependence of crit ical cofactors like tetrahydrobiopterin (BH4) are assumed to be key points in several disease states of the circulatory system. The functionality of blood vessels is important to avoid injury during decompression in diving. BH4 is prone to oxidation and r ecent findings have indicated that high oxygen level decreases both BH4 and NO generation. Since elevated oxygen level is an inevitable part of diving the present project aims to elucidate the role of the signalling molecule NO in relation to diving, with particular focus on BH4. Revealing the underlying mechanisms of blood vessel functionality and decompression injury is a prerequisite for remedial actions, e.g. pharmacological or physiological intervention to protect BH4 from oxidation and maintain the cofactor level, providing divers with an improved resistance against their extreme working environment. Both isolated human blood vessel cells and human volunteers will be used to investigate the effect of high oxygen level and the influence of antioxida nts on the cofactor BH4 and NO-generation. The human study may reveal whether our previous observation of decreased NO concentration in exhaled gas after exposure to high oxygen concentration is related to oxidation of BH4 by determination of the cofactor in blood. The human study may thus extend and possibly confirm the findings from the cell model. Graduation of a PhD student also fulfils the important aim of scientific recruitment.