Four subjects were investigated together during 1 d. They showed up in a postabsorptive state at 7:30 a.m. (last meal during the preceding evening at 8:00 p.m.). After briefing, a flexible catheter was introduced into an arteria radialis using local anesthesia (1% lidocaine) and secured with watertight tape. After positioning of pulse watches for measurement of HR and calculation of HRV, the subjects remained seated for at least 12 min (prediving control period) until the first blood sample was drawn while breathing air (sample 1).
Then, the subjects entered the diving tower (air temperature = 28°C–30°C) and were connected to the BIBS with an overboard dumping system (Haux, Karlsbad, Germany), supporting them with one of the different inspiratory gases (O2, air, or Heliox). Subsequently, they started the first submersion (without a diving suit, with a water temperature of 28°C, which was described as agreeable) in an upright position (their feet were positioned approximately 2 m below the surface, and their body center was positioned 1 m below the surface) holding only the hand with the cannulated artery out of the water. During the first submersion, blood was sampled (samples 2 and 3) after 1 min and after 11 min. Concomitantly, HR was monitored for 4 min. Thereafter, the subjects left the water but remained standing at the top of the diving tower breathing air without the BIBS (prescribed by safety regulations) for an 11-min break.
The differences in ambient pressure among the three experiments began during this break. In experiment 1, the subjects used again the regulators inspiring pure oxygen after the break and entered the water for the second submersion in atmospheric pressure. Blood samples 4 and 5 were drawn after 1 and 11 min during the second submersion; the samples were transferred outside of the diving tower and immediately processed. Thereafter, the subjects left the water, returned to air breathing without the BIBS and remained seated until the last blood sample (sample 6) was drawn 11 min later (postdiving control period).
In experiment 2 (breathing gas air), the chamber pressure rose to 5.2 barabsolute within 2 min during the 11-min break. The submersion was repeated with otherwise equal conditions like in experiment 1 (blood samples 4 and 5). Samples were transferred outside of the diving tower to atmospheric pressure through a hand lock and immediately processed. Then, the subjects ascended with the head out of water undergoing decompression stops (Fig. 2). After leaving the water, they remained seated for 12 min until the last blood sample (sample 6, postdiving control period) was drawn.
Experiment 3 was identical with experiment 2 except for the breathing gas Heliox until the end of the second submersion. At this moment, the divers got out of the water and were decompressed with oxygen breathing in the dry section of the diving tower in a sitting position. Because of technical restrictions, decompression with O2 in water as usual was not possible. Thereafter, they switched to environmental-air breathing and remained seated for 12 min until taking sample 6 (postdiving control period).
Because of the different decompression methods, the conditions during the postdiving control period could not be fully standardized. Thus, the time until the last blood sampling was delayed in experiments 2 and 3 by 31 and 45 min, respectively, compared with experiment 1. A final neurological check (e.g., leg and finger coordination, balance check) was performed in experiments 2 and 3 (prescribed by safety regulations of DLRG Berlin).
All blood samples (15 mL each) were immediately processed. For catecholamine determination (adrenaline, noradrenaline, dopamine), 5 mL was transferred to a chilled Vacutainer tube (Amersham, Braunschweig, Germany) containing 5 mg of glutathione and 9 mg of EDTA, put on ice, and centrifuged within 3 min (−4°C to −6°C, 10 min, 1700 rpm). Two aliquots of plasma (1.1 mL each) were immediately frozen in liquid nitrogen and stored at −80° until further processing. For measurement of ACTH and cortisol, 4 mL of blood each was pipetted into prechilled EDTA tubes. Centrifugation and storage were equal to the procedures used for catecholamine determination.
After separation from catecholamine sulfates by solid-phase extraction with activated aluminum oxide, the catecholamine concentrations were measured according to the method of Strobel and Weicker (28) by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection (ESA Coulochem II; ESA, Inc., Chelmsford, MA). For this measurement, catecholamine 3-sulfate isomers served as internal standards. The detection limits (signal-to-noise ratio greater than 3) in plasma samples are about 80 pmol·L−1 for each analyte. The intra-assay and interassay coefficients of variation are less than 4.0% and 10.7%. The calibration curves for all catecholamine sulfates are linear (r > 0.96, P < 0.001) over the respective concentration ranges of 0.1–100 and 5–1000 nmol·L−1.
ACTH and cortisol.
The concentrations of ACTH were determined by the use of a chemiluminescent immunometric assay (Nichols Institute Diagnostics, Bad Nauheim, Germany) and a Lumat LB 9501/16 (Berthold, Bad Wildbach, Germany). The concentrations of cortisol were measured photometrically using an enzyme immune assay (EIAgen Cortisol Kit; BioChem Immunosystems, Bologna, Italy) in an enzyme-linked immunosorbent assay reader (EMax; Molecular Devices, Munich, Germany).
HR and HRV (29,31) were recorded continuously using an HR monitor (Vantage NV; Polar Electro Oy, Kempele, Finland). Because of the limited storage capacity, the recording began when entering the tower and ended after 40 min with blood sample 5. Resting values were measured in the additional experiments. The positioning of the monitors was performed exactly according to the instructions in the manual. HRV was evaluated during 4 min at the time of blood sampling (root mean square successive difference (r-MSSD), pNN50, low frequencies between 0.04 and 0.15 Hz (LF), high frequencies between 0.15 and 0.4 Hz (HF), LF/HF). High r-MSSD, pNN50, and HF indicate increased parasympathetic activity; high LF increased sympathetic activity. LF/HF may be considered to mirror the balance between sympathetic and parasympathetic innervation (29). Despite correct coding and in contrast to statements of the producer concerning underwater use, the pulse watches influenced each other leading to many artifacts. Therefore, only parts of the registration contained exploitable data (values from 8 to 14 subjects per sampling period). It was impossible to repeat all these extensive measurements with different conditions. Thus, we decided to perform the additional shortened experiments (experiment 4) to check if the results were reproducible under frequently used conditions (air breathing, depth down to 40 m). In these experiments, complete data sets were obtained for 10 of 11 subjects.
Statistical evaluation was performed by the use of the software SPSS 11.0 (SPSS, Inc., Chicago, IL). The data are presented as means ± SE. Depending on the number of comparisons, t-tests (if necessary, with Bonferroni correction) or ANOVA was used for significance calculations. The normality of data distribution was checked by the Kolmogorov–Smirnov test. Also, linear regression analysis (Pearson correlation coefficient) was performed if necessary.
Catecholamine concentrations are presented in Figure 3. During the prediving control period, the values were in the range of healthy subjects without physical or psychical stress (21,26–28). The type of breathing gas did not influence the concentration independent of depth. However, at the end of all submersions, [noradrenaline] decreased uniformly (samples 3 and 5). During the postdiving control period, the concentration rose over the values of the prediving control period in experiments 2 and 3. [Adrenaline] changed similarly during the submersions but did not rise in the postdiving control period. [Dopamine] showed no significant variations at all. Similarly, there were no significant changes of ACTH and cortisol concentrations (data not shown, initial values = 102 ± 12 ng·mL−1 and 32 ± 3 pg·mL−1, respectively).
HR and HRV.
There was a nonsignificant tendency for an HR decrease during submersion (e.g., during air breathing from 78 ± 4 bpm during the prediving control period to 69 ± 5 bpm in a depth of 44 m). r-MSSD (Fig. 4) was increased at the end of the first submersion (i.e., under near-atmospheric pressure) in experiments 1, 2, and 3. A similar increase was observed during the second submersion only in experiment 1 (oxygen) in which pressure remained constant. pNN50 behaved equally except for a significant increase also in sample 5 of experiment 3 (Heliox, 44 m). Both LF and HF tended to have higher values during diving, but the changes were not significant (not shown in Fig. 4). The ratio LF/HF, however, decreased in parts of the experiments with high oxygen pressure: in experiment 1, immediately after submersion (oxygen, 2 m), and in experiment 2, only at the end of the second immersion (air, 44 m). In the short experiments (experiment 4), there were not only corresponding changes of r-MSSD (P < 0.02) and pNN50 (P < 0.01) but also significant increases of LF and HF (P < 0.05); a small decrease of LF/HF did not reach significance.
There were weak but significant correlations for the following quantities (135 pairs of values each): r-MSSD, pNN50, and HF correlated negatively with [noradrenaline] (r = −0.29, r = −0.33, r = −0.34, all P < 0.001); LF/HF correlated positively with [adrenaline] (r = 0.32, P < 0.01).
To our knowledge, this is the first investigation with arterial catecholamine measurements and concomitant HRV determination during scuba diving with different total and individual gas pressures. In previous studies, only venous concentrations were determined (1); therefore, the exchange with various tissues and partly the time delay reduce the information. Because catecholamines are secreted as well as metabolized within a few minutes possibly leading to 10-fold concentration changes (e.g., Kjaer  and Strobel et al. ), immediate measurements in arterial blood as in this study are essential to obtain true information about the actual state of sympathoadrenergic activity during diving. Later determinations might show again control values or even counterregulation (see below). Because of physiological and technical restrictions, it was impossible to standardize fully the postdiving control period. This explains some differences in measured quantities among the experiments in sample 6.
The main result of this study is that submersion per se is no stress for the human sympathoadrenergic as well as adrenocortical system in experienced divers. In contrast, the arterial noradrenaline and adrenaline concentrations hint to a decrease of sympathoadrenergic activity: at the end of the first as well as the second submersion, [noradrenaline] and [adrenaline] were lowered independent of breathing gas and diving depth. Probably, the diving reflex (reduced HR and vasoconstriction caused by face submersion and breath holding) played no essential role for these hormones; otherwise, one would expect an increased noradrenaline secretion from sympathetic nerve endings as a cause of the peripheral vasoconstriction. The significantly increased [noradrenaline] in sample 6 (postdiving control period) only after deep submersion but not after oxygen breathing might be explained by the following mechanism. Immersion and submersion cause a blood shift to the atria; the atrial distension results in a downregulation of the renin–angiotensin–aldosterone system and increased orthostatic lability (e.g., Skipka et al. ). After leaving the water, this lability might be counteracted by an increase of noradrenaline. However, the downregulation is a slow process playing a role only after sufficient time. In our experiments, the last blood sample was taken markedly later in the air and Heliox than in the oxygen experiments because of decompression stops (Fig. 2). Therefore, the differences in [noradrenaline] during the postdiving control period are obviously not caused by different direct effects of the breathing gases during submersion. This is underlined by the fact that all other hormone concentrations in sample 6 did not deviate from the initial values.
Psychological stress, cold, or physical activity may stimulate sympathoadrenergic activity under realistic conditions. Such disturbing factors were intentionally excluded. Only rescue divers with experience in the diving tower participated. Effects of the lower than neutral water temperature should be small because the divers felt comfortable during the short submersions of 15-min duration; they had rather thick cutaneous fat (mean body mass index = 26.2 kg·m−2) and were accustomed to this condition. Physical activity when sitting on land or floating below the water surface is practically nil and hardly different. In the experiments of Anegg et al. (1), however, the increase of catecholamine concentrations in venous blood obviously follows from swimming exercise.
Dopamine outside of the brain is important for intestinal and renal perfusion and blood pressure regulation (9), but its plasma concentration did not change in our experiments. A similar constancy has been found after brief intermittent maximal exercise (3).
Also, [ACTH] and [cortisol] as indicators of the reaction of the hypothalamic–pituitary–adrenal axis to stress showed no changes. This corresponds better to head-out immersion (11) than to hyperbaria (12,19). Obviously, the subjects felt no psychological stress.
HR and HRV.
Because of the problems in obtaining arterial blood samples underwater, data on HRV would be valuable, in particular as the data can be collected noninvasively. Theoretically, one should expect good correlations of some parameters (especially LF and LF/HF) with catecholamine concentrations in our experiments because most interfering factors like differences in age, fitness, and physical or psychical strain (e.g., Aubert et al. , Schipke and Pelzer ) were excluded by measurements in the same accustomed subjects at rest. Only changes in breathing frequency were not controlled, which might affect respiratory cardial arrhythmia and thereby the ratio LF/HF; however, this influence was small in similar experiments (22). However, despite various attempts to improve the recording (e.g., stopping all electromechanical aggregates within the diving tower) also in collaboration with the producer, it was impossible to obtain continuous ECG without artifacts with the used pulse watches even when applying the correction computer program. Therefore, not all registrations could be evaluated. Because a single monitor in the tower registered correctly during a preceding experiment, probably, interference among the four simultaneously used watches occurred underwater. Possibly, reflections by the steel wall of the diving tower might play a role, which does not occur during real diving.
The slight nonsignificant HR decrease during the dives corresponds to observations of other investigators in similar experiments (22). Theoretically, one would expect a reduced HR because of the diving reflex as well as the shift of blood during immersion or submersion from peripheral tissues into the thorax augmenting diastolic heart volume and thus stroke volume. Feedback from arterial pressure receptors consequently should reduce HR. However, also, constant values during snorkeling (17) and even an increase during head-out immersion have been observed, the latter accompanied by a decrease of arterial blood pressure (e.g., Skipka et al. , Ulmer et al. ).
The measures of HRV change similarly like in comparable studies (5,6,17,22) and show partly a correlation to the sympathoadrenergic hormone levels (r-MSSD, pNN50, LF/HF). Because r-MSSD and pNN50 are indicative only for parasympathetic activity, the increases of these quantities hint to an enforced parasympathetic innervation. The decrease of LF/HF in part of the experiments indicates that the balance between sympathetic and parasympathetic innervation was shifted in favor of the latter. Schipke and Pelzer (22) observed an increase of both LF and HF during 10 min of scuba diving (sitting at a depth of 4 m) and suggested a coactivation of both the sympathicus and the parasympathicus. Our results do not contradict this conclusion because we measured a similar but not significant tendency in the main experiments and even a significant increase of LF and HF in the additional series. However, the decrease of LF/HF in some of our experiments might indicate that the activation of the cardiac sympathicus was relatively smaller compared with the vagus. In any case, because catecholamine concentrations in blood mirror whole-body metabolism of these compounds, they must not be strongly related to parameters of heart innervation. However, if, in dangerous situations, an activity rise in all parts of the sympathoadrenergic system occurs, HRV measures might be useful indicators for beginning panic reactions. The possible importance is supported by results of Flouris and Scott (6), who detected a reduced parasympathetic activation in a not dangerous but psychologically stressful situation during scuba diving.
Disturbing influences like physical activity were absent, and metabolism was scarcely different between the periods of our experiments. In a study of Chouchou et al. (5), scuba diving with low physical activity showed not only no significant change of HR but also an increase of parasympathetic activity measured from HRV compared with quiet sitting on the boat.
The reduction of [adrenaline] and [noradrenaline] in the experiments during scuba diving seems to be caused by orthostatic relief. External hydrostatic pressure counteracts vascular distension because of gravity; therefore, less vasoconstrictive stimuli are necessary than at land. The vertical body position causes similarly like head-out immersion an additional pressure difference (approximately 25 cm H2O) between the gas in the alveoli (corresponding to pressure at the mouthpiece of the scuba apparatus) and the pulmonary vessels and the heart (intravascular pressure equilibrated to water pressure at that depth); this shifts blood into the thorax. Because these intracorporal pressure effects were equal for all experiments, a different change for breathing air, oxygen, or Heliox did not occur. The main reaction during head-out immersion in males seems to be a long-known marked reduction of systolic and diastolic blood pressure as an indicator of reduced peripheral resistance (e.g., Skipka et al. , Ulmer et al. ), but measurements during scuba diving seem not to exist.
The determination of arterial noradrenaline and adrenaline concentrations during scuba diving without exercise and in a nonexciting environment showed a reduction in sympathoadrenergic activity independent from breathing gas and water depth in experienced subjects, whereas the hypothalamic–pituitary–adrenal axis seemed to be unaffected. There was no sign that panic reactions might be facilitated. If catecholamines remain constant in nonmoving inexperienced subjects during a dive, this might already be an indication for stress. Noradrenaline and adrenaline concentration changes were roughly correlated with indices of HRV, which were partly indicative for a parasympathetic stimulation. In case of real or imagined danger, sudden decreases of r-MSSD and pNN50 as well as increases of LF/HF are therefore probably of diagnostic value, at least in unmoving or slowly moving subjects. After technical improvement of the used HR monitors or exclusion of external interfering factors, noninvasive measurement of the activity of the vegetative nervous system might be possible also in groups of divers with little distance among them. This is necessary for a routine application to detect beginning panic reactions.
The authors thank the DLRG for the permission to use the diving tower with all its facilities. They thank all test subjects for their willing cooperation and Mrs. J. Nadol and Mrs. B. Himmelsbach for technical assistance. The local members of the DLRG and medical diving specialists supported the study with enthusiasm. For statistical consulting, the authors thank Mr. Orawa.
The authors disclose the receipt of funding for this work from the National Institutes of Health, Wellcome Trust, Howard Hughes Medical Institute, or any others. F. Weist, G. Strobel, M. Hölzl, and D. Böning disclose professional relationships with companies or manufacturers who will benefit from the results of the present study.
There are no conflicts of interest to report.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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Keywords:©2012The American College of Sports Medicine
HELIOX; HEART RATE VARIABILITY; DIVING SIMULATOR; DIVING EXPERIENCE; PANIC ATTACKS