Table of Contents    
Year : 2019  |  Volume : 10  |  Issue : 3  |  Page : 43-47  

Effects of nitrox II during a single decompression dive on endothelial nitric oxide synthase expression and flow-mediated dilation among trained male divers

Department of Community Medicine, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Date of Web Publication14-Jan-2020

Correspondence Address:
Ika Rahma Mustika Hati
Faculty of Medicine, University of Indonesia, Pegangsaan Timur No# 16, Jakarta 10320
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jnsbm.JNSBM_88_19

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Background: Nitrox II, associated with higher oxygen partial pressure, induces an increase in reactive oxygen species, which can interfere with endothelial nitric oxide synthases (eNOSs), causing endothelial dysfunction and lower flow-mediated dilation (FMD). This study aimed to determine the influence of nitrox II on eNOS expression and FMD as markers of endothelial function. Materials and Methods: A double-blind approach was used with 39 trained male divers who were divided using randomized block design into a control group (the air group) that used compressed air or an intervention group that used nitrox II (the nitrox II group). Both the groups underwent a single decompression dive to 28 m sea water (msw) with a bottom time of 50 min in a hyperbaric chamber. Pre- and postdive eNOS expression was measured by quantitative enzyme-linked immunosorbent assay and FMD by laser Doppler flowmetry on the regio brachii. Results: eNOS (P = 0.029) and FMD (P = 0.001) decreased in the air group, whereas eNOS (P = 0.018) and FMD (P = 0,023) increased in the nitrox II group. The average difference in eNOS and FMD in the nitrox II group was greater compared with the air group (P < 0.05). There was no significant correlation between eNOS and FMD. Conclusion: Nitrox II prevents endothelial dysfunction during a single decompression dive to 28 msw with a bottom time of 50 min, as indicated by divers' increased FMD from increased eNOS expression.

Keywords: Endothelium, endothelial nitric oxide synthase, nitrox

How to cite this article:
Mustika Hati IR, Suryokusumo G, Roestam AW. Effects of nitrox II during a single decompression dive on endothelial nitric oxide synthase expression and flow-mediated dilation among trained male divers. J Nat Sc Biol Med 2019;10, Suppl S1:43-7

How to cite this URL:
Mustika Hati IR, Suryokusumo G, Roestam AW. Effects of nitrox II during a single decompression dive on endothelial nitric oxide synthase expression and flow-mediated dilation among trained male divers. J Nat Sc Biol Med [serial online] 2019 [cited 2021 Jan 19];10, Suppl S1:43-7. Available from:

   Introduction Top

Nitrox II contains an oxygen composition of 36% that according to mathematical calculations reduces venous gas bubbles because of its lower nitrogen fraction.[1] Souday et al. demonstrated that nitrox II reduces the embolism of venous gas bubbles after decompression.[2] However, Marinovic et al. found that nitrox during diving affects systemic vascular function more profoundly compared with diving with air, by reducing the flow-mediated dilation (FMD) response and thus indicating endothelial dysfunction.[3],[4],[5] Although the positive effects of nitrox II include reducing venous gas bubbles, diving with nitrox II is also associated with higher exposure to oxygen partial pressure, inducing an increase in reactive oxygen species (ROS) that can reduce NO bioavailability and cause direct mechanical damage to the endothelium during decompression.[3],[4],[6],[7] The question is whether the effects of nitrox II reduction of venous gas bubbles reported by Souday et al. are related to NO production, which is characterized by changes in postdive endothelial nitric oxide synthase (eNOS) expression when using nitrox II. Whether these changes in eNOS expression are significant, as indicated by FMD, also remains an open question. Thus, this study aimed to determine the influence of nitrox II on eNOS expression and FMD as markers of endothelial dysfunction.

   Materials and Methods Top

Study sample

The target population was trained male divers in East Java, Indonesia. The inclusion criteria were having a diving certificate, being in good general health, and having never experienced decompression sickness (DCS). The exclusion criteria were infection of the ear, nose, or throat, hypertension, or diabetes mellitus. Using block randomization, forty participants were equally divided into two groups: the air group and the nitrox II group. The study was approved by the Ethics Committee of the Faculty of Medicine, University of Indonesia, and each participant signed informed consent. The study took place at the Maritime Health Institution of the Indonesian Navy, LAKESLA, in Surabaya, Indonesia.

Study protocol

Divers performed a single decompression with 50 min of bottom time at 28 m sea water (msw) in a hyperbaric chamber [Figure 1]. The air group used compressed air (21% oxygen and 79% nitrogen) and the nitrox II group used nitrox II (36% oxygen, 64% nitrogen) through mask. Participants were disallowed exercise predive, had no diving activities for 24 h before the study dive, and fasted for 8–10 h before blood sampling [Figure 1]. Pre- and postdive, a 6-ml blood sample was collected from the brachial vein. Blood samples were allocated into ethylenediaminetetraacetic acid (EDTA) tubes, stored at 2°C–8°C for transport to an accredited laboratory, and examined using enzyme-linked immunosorbent assay (ELISA) quantitative techniques to measure eNOS levels. FMD was assessed before and after blood sampling and measured at the brachii region using laser Doppler flowmetry (LDF, PeriFlu × 5000) at room temperature (25°C), with the participant sitting upright with their right arm in the supine position.
Figure 1: Study design with timeline measurement (above) and dive protocol (below)

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Hyperbaric chamber

All divers performed the simulation dive in a hyperbaric chamber (Aquaservice, London) made of steel, aluminum, and acrylic that can withstand >5 atmosphere absolute of pressure. The multicompartment chamber has a 12-person seating capacity (ten in the main chamber and two in the entry chamber). A researcher with training and competency certification as a chamber operator controlled the hyperbaric chamber.

Flow-mediated dilation

In this study, FMD was assessed in the forearm using LDF to measure total perfusion of microcirculation capillary perfusion in included arterioles, venules, and shunting of blood vessels.[8],[9] The laser Doppler flowmeter used (PeriFlux 5000, Perimed, Sweden) can be operated at 5°C–35°C. This technique uses laser beam emission carried by fiber-optic probes on the cuff. The probe is placed on the skin surface, and the laser beam penetrates the skin surface, with results shown directly on the appliance monitor and computer screen connected to the PeriFlux 5000. Magnitude was expressed in perfusion units (PUs), where 1 PU is equal to 10 mV (1 volt = 10 ml/100 g/min). Calibration was initiated when starting the instrument. After cleaning the brachial region and positioning the arm, the probe was placed on the cuff of the electrode and attached to the skin surface on the lateral side of the right arm, about one finger width below the fold of the brachial region. Examination was carried out at room temperature (25°C). Recordings were made for at least 150 s after the graphic display on the screen appeared to be stable and plateaued (i.e., there was no appearance of significant wave differences). The recording was then computer analyzed for the average PU unit of the FMD assessment.

Endothelial nitric oxide synthase expression quantification

Blood samples were collected in a special pyrogen- and endotoxin-free tube containing EDTA, at 2°C-8°C for 30 min; 3 mL of blood was placed in a centrifuge tube and frozen and then centrifuged at 3000 rpm for 10 min. Separated serum was stored in vials, aliquoted, and labeled with a participant code and sample retrieval date and time. Serum samples were stored at –20°C. We used the DuoSet Human eNOS ELISA Kit reagent (R&D Systems) to quantify eNOS using competitive enzyme immunoassay techniques utilizing a monoclonal anti-eNOS antibody and an eNOS-HRP conjugate. Reading eNOS levels on a microplated substrate was performed gradually, from wavelength 450 nm to 540 or 570 nm using a Bio-Rad microplate reader 680. eNOS levels are reported in pg/mL.

Statistical analysis

Data are expressed as mean ± standard deviation. Normality was assessed using the one-sided Shapiro–Wilk test. We used nonparametric tests to analyze nonnormally distributed data. In the air group, both eNOS and FMD predive were nonnormally distributed, whereas both eNOS and FMD postdive were normal. In the nitrox II group, all measures of eNOS and FMD were nonnormally distributed. The Wilcoxon test was used to test within-group differences in eNOS and FMD expression pre- and postdive. The Mann–Whitney test was used to compare the between-group postdive eNOS and FMD values and to investigate the average between-group eNOS differences. Independent t-tests were used to investigate the average differences in FMD. Pearson correlation was used to investigate the linear relationships between eNOS and FMD in the air group, and Spearman correlation was used for the nitrox II group. All tests were two-tailed, and P < 0.05 was considered statistically significant. All analyses were performed using IBM SPSS Statistic for Windows, Version 20.0. Armonk, New York, United States.

   Results Top

Participant and dive characteristics

Forty divers performed the simulation dive in the hyperbaric chamber; one air group participant was withdrawn after failing to perform the Valsalva maneuver and was removed from the chamber at 6 msw. Thus, 39 participants (19 in the air group and 20 in the nitrox II group) were available for final analyses [Table 1] and [Table 2]. None of the divers developed DCS while participating in the study.
Table 1: Participant characteristics

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Table 2: Marker analyses

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Effect on endothelial nitric oxide synthase expression

The air group showed a significant decrease in eNOS expression postdive (P = 0.029), whereas the nitrox II group had a significant increase in postdive eNOS expression (P = 0.018). Although eNOS expression postdive did not differ significantly between the groups (P = 0.339), the difference in mean eNOS expression between groups was significant (P = 0.001), with the eNOS difference in the nitrox II group being greater compared with the air group [Figure 2].
Figure 2: Effect of diving on endothelial nitric oxide synthase and flow-mediated dilation between compressed air and nitrox II breathing

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Effect on flow-mediated dilation

There was a significant postdive FMD decrease in the air group (P = 0.001) and a significant increase in postdive FMD in the nitrox II group (P = 0.023). Postdive FMD changes differed significantly between the groups (P = 0.012), with the mean change in FMD greater in the nitrox II group compared with the air group (P < 0.000).

Correlation between endothelial nitric oxide synthase expression and flow-mediated dilation

eNOS expression and FMD were not significantly correlated in either the air group (r = 0.417; P = 0.076) or the nitrox II group (r = 0.231; P = 0.327).

   Discussion Top

Effects of diving with nitrox II versus compressed air on endothelial nitric oxide synthase expression

The decrease in postdive eNOS expression in the air group was caused by increased partial pressure of oxygen in the divers at a depth at which oxidative stress increases ROS.[5],[6],[10],[11],[12],[13] ROS oxidizes BH4, a principal cofactor in eNOS synthesis to BH2, resulting in uncoupled eNOS and thus decreased eNOS expression.[10],[11],[12],[14],[15] In contrast, the nitrox II group had a significant increase in eNOS expression. This may have resulted from increased ROS in these divers, possibly stimulating ROS dismutation by superoxide dismutase (SOD) to increase the production of another ROS form, hydrogen peroxide (H2O2), one of the endogenous antioxidants that act as the second messenger to stimulate eNOS expression and activity. The higher stability and diffusion of H2O2 (compared with superoxide anion) participate in the capacity of a ROS to increase eNOS expression and activity throughout the vascular wall. The activity of SOD and H2O2 needs to be proved in further researches. The group of Harrison had previously shown that H2O2 upregulates Nitric Oxide Synthase 3 (NOS3) expression through a calcium/calmodulin-dependent protein kinase II-mediated mechanism, whereas it also acutely activates the eNOS enzyme.[12],[16] Ideally, the formation and scavenging of ROS are well balanced, a condition known as ROS homeostasis. When this balance is disturbed, a specific cellular function can be triggered. In any case, the adaptive response may be beneficial or harmful, depending on the life stage or circumstances under which it occurs (i.e., principle of fundamental flows). We believe that the ROS-generation effects of the higher partial pressure of oxygen during nitrox II breathing can initiate an adaptive process known as the hormetic effect [Figure 3]. Hormesis is an adaptive response to a variety of oxidative and other stresses that render cells and/or organisms resistant to higher (and normally harmful) doses of the same stressing agent.[12],[17]
Figure 3: Dose–response curve depicting characteristics of the oxygen partial pressure hormetic zone (modified from Calabrese, 2006)

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According to the hormesis theory, applying a stimulus will cause a negative or positive response, depending on the intensity of that stimulus.[17],[18] The stimulus of increased partial pressure of oxygen at a depth of 28 msw to 606.48 mmHg results in a negative physiological response, with decreased eNOS in the air group. This means that at a depth of 28 msw, air breathing leads to a negative response because this stimulus is still below the control line; in other words, it has not reached the therapeutic dose. However, a positive eNOS expression response will occur if, at the same depth, the intensity of the partial pressure of oxygen is increased to 1039.68 mmHg by the use of nitrox II. This may happen because the adaptive response is based on having reached an adequate partial pressure of oxygen (i.e., the stimulus) to initiate SOD release, degrading the formed superoxide anions and producing endogenous H2O2 antioxidants that then lead to increased eNOS expression.[12],[16] Nevertheless, the mechanism by which increased eNOS expression may be triggered by increased H2O2 is beyond the scope of the current study and thus requires further investigation.

Effects of diving with nitrox II, compared with compressed air, on flow-mediated dilation

The FMD decrease in the air group and increase in the nitrox II group was a vasodilation response highly dependent on NO activity. Decreased FMD in the control group may have been due to oxidative stress-increased ROS causing a decrease in eNOS expression and disrupting NO production. This decrease in NO production then causes vasoconstriction that decreases FMD. Consistent with this, the increase in FMD in the nitrox II group was due to an increase in eNOS expression, which increased NO production. FMD is based on two physiologic principles: (1) blood flow-induced shear stress activates the endothelium to produce NO and (2) NO elicits vascular dilation through relaxation of vascular smooth muscle. With increased endothelial oxidative stress, eNOS is uncoupled, decreasing NO production and bioavailability. Thus, increased endothelial oxidative stress should decrease endothelial reactivity to shear stress, which will negatively impact FMD.[5],[9] Increased eNOS expression, which activates the production of NO, may lead to vascular dilation, which will positively impact FMD.

Lack of correlation between endothelial nitric oxide synthase expression and flow-mediated dilation

In this study, FMD was assessed with LDF, a method for evaluating skin microcirculation based on the assumption that the microcirculation responses reflect systemic arterial endothelial function.[5] Four main characteristics can be attributed to different microvascular activities: resistance, exchange, shunting ability, and capacitance. The vascular smooth muscle cells of the different vessel segments differ in their electrical coupling and their responsiveness to stimuli, partly explaining different zones of influence of diverse dilator and constrictor mechanisms.[19]

The myogenic response (Bayliss effect) is an example of vascular autoregulation. Dilatation of the microvessel leads to ion influx (Na + and Ca 2+) through stretch-sensitive membrane ion channels and thereby to contraction of the vessel smooth muscle cells due to depolarization. The Bayliss effect, which maintains tissue blood flow in the face of different blood pressure levels, can be blocked (e.g., pharmacologically, with calcium antagonists). Local metabolic effects, particularly effective in the terminal arterioles, are elicited primarily by changes in pO2, pCO2, pH, osmolarity, potassium ion concentration, and released catabolites (e.g., adenosine). Finally, shear stress evoked by the movement of blood, which impinges primarily at the endothelial surface, causes NO release. This is a positive feedback mechanism in which dilatation induced locally at terminal arterioles (e.g., by metabolic signals) increases flow and thus shear stress and liberation of NO upstream.[19] Despite the increased eNOS and FMD expression in the nitrox II group and decreased expression of eNOS and FMD in the air group, there was no significant correlation between eNOS and FMD changes within groups. The response of vascular autoregulation to stimulation in different blood vessel segments results in relatively different sensitivities.[19] In this study, we could not be certain of the blood vessel segments in which FMD was measured. However, based on the nonsignificant correlation between eNOS expression and FMD, we can presume that FMD measurements were from the terminal arteriole. In this segment, metabolite and myogenic response stimulation is more dominant compared with NO release. We hypothesize that the metabolite effects primarily consisted of changes in pCO2. Increasing the partial pressure of oxygen will increase oxygen saturation, and oxygen perfusion to the tissues will consequently inhibit the release of CO2 in the periphery. Increased CO2 in the periphery will decrease the L-type Ca channel response, contributing to hyperpolarization, opening the K + ion channel so that K + efflux increases and vasodilation occurs.[19] However, this hypothesis needs further investigation.

   Conclusion Top

Our findings suggest that the effect of nitrox II-reduced venous gas bubbles reported by Souday et al. was related to NO production. Using nitrox II on a single decompression dive to 28 msw prevents endothelial dysfunction by increasing eNOS expression and increasing FMD response. The present study also suggests that there may not be a significant association between eNOS expression and FMD, although this is presumably due to placement of the LDF probes at the terminal arteriole, so that the metabolic response and myogenic effect were more dominant than NO release. Further research is needed to determine the effects of inflammatory agents and endogenous antioxidants on vasoconstriction and endothelial dysfunction during diving, taking into account individual factors such as muscle mass, body fat, and pCO2.


The authors would like to show our gratitude to University of Indonesia and the Indonesian Navy, especially LAKESLA in Surabaya, Indonesia for sharing their wisdom and for providing the study site and equipment.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]


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