Antioxidant Pretreatment and Reduced Arterial Endothelial Dysfunction After Diving

Aviation, Space, and Environmental Medicine x Vol. 78, No. 12 x December 2007 1

RESEARCH ARTICLE

Antioxidant Pretreatment and Reduced Arterial Endothelial Dysfunction After Diving

Ante Obad , Zoran Valic , Ivan Palada , Alf O. Brubakk , Darko Modun , and Ž eljko Duji ć

O BAD A, V ALIC Z, P ALADA I, B RUBAKK AO, M ODUN D, D UJIC Z.

Antioxidant pretreatment and reduced arterial endothelial dysfunc-tion after diving. Aviat Space Environ Med 2007; 78:1 – 7.

Introduction: We have recently shown that a single air dive leads to acute arterial vasodilation and impairment of endothelium-dependent vasodilatation in humans. Additionally we have found that predive antioxidants at the upper recommended daily allowance partially prevented some of the negative effects of the dive. in this study we prospectively evaluated the effect of long-term antioxidants at a lower RDA dose on arterial endothelial function. Methods: Eight professional male divers performed an open sea air dive to 30 msw. Brachial artery fl ow-mediated dilation (FMD) was assessed before and after diving. Results: The fi rst dive, without antioxidants, caused signifi cant brachial arterial diameter increase from 3.85 6 0.55 to 4.04 6 0.5 mm and a signifi cant reduction of FMD from 7.6 6 2.7 to 2.8 6 2.1%. The second dive, with antioxidants, showed unchanged arterial diameter and signifi cant reduction of FMD from 8.11 6 2.4 to 6.8 6 1.4%. The FMD reduction was signifi cantly less with antioxidants. Vascular smooth muscle function, assessed by nitroglycerine (endothelium-independent dilation), was unaffected by diving. Discussion: This study shows that long-term antioxidant treatment at a lower RDA dose ending 3 – 4 h be-fore a dive reduces the endothelial dysfunction in divers. Since the scuba dive was of a similar depth and duration to those practiced by numerous recreational divers, this study raises the possibility of routine predive supplementation with antioxidants. Keywords: diving , endothelial function , gas bubbles , antioxidants .

DURING DIVING WITH compressed gas, inert gas is taken up in the tissue depending on the dive’s depth

and duration. During and after the decompression phase of the dive, gas vascular bubbles are regularly seen in the venous vasculature and eventually lodge in the pulmo-nary circulation. Recently we found that a simulated air dive, producing low bubble loads, caused an increase in the brachial artery resting diameter and a reduction in arterial endothelial function, indicating acute endothe-lial dysfunction ( 5 ). However, the physiological stress of open sea versus dry diving is greater due to additional factors besides hyperoxia and gas bubbles, such as im-mersion, exercise, cold temperature, and hemoconcen-tration. We have recently reported that a single air dive in the open sea is associated with acute depression of lung respiratory function and cardiac output ( 12 ) and that fi eld dives cause a considerably higher number of bubbles than chamber dives ( 13,36 ).

Flow-mediated dilation (FMD) is considered to be a reliable method for evaluation of endothelial function

and its reduction is a sign of an impairment of nitric ox-ide (NO) bioavailability ( 8 ). A recent study has shown that increased vascular production of reactive oxygen species (ROS), such as superoxide anions, contributes to impaired endothelium dependent derived NO pro-duction in atherosclerosis ( 37 ). This effect may be medi-ated by oxidative quenching of endothelium-derived NO by ROS generated intra-arterially under hyperoxic conditions ( 43 ). The hyperoxia during scuba diving is as-sociated with oxidative stress and antioxidant enzyme adaptations in order to alleviate oxidative damage ( 19 ). NO plays a critical role not only in regulation of vasomo-tor function, but also in platelet activity and leukocyte adhesion to the vascular wall ( 23 ). NO is synthesized by the NO synthase (NOS) using the amino acid l-arginine as a substrate. Three NOS isoforms have been identifi ed: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). The catalytic activity of all three NOS isoforms is dependent on the availability of its cofactor tetrahydro-bipterine (BH 4 ) ( 51 ). Ascorbic acid scavenges superoxide anions ( 26 ), inhibits low density lipoprotein oxidation ( 20 ), prevents hyperoxia-mediated vasoconstriction in healthy subjects ( 29 ), and protects BH 4 from oxidation ( 27 ). In addition, ascorbic acid (vitamin C) serves a func-tion in reducing oxidized vitamin E and glutathione, thereby regenerating these powerful antioxidants to scavenge ROS and interfere with lipid peroxidation ( 45 ). All these actions would provide optimal conditions for cellular NO synthesis.

Recently we reported that a single air dive to 30 msw for 30 min was associated with modest reductions in

From the Department of Physiology (A. Obad, Z. Valic, I. Palada, Z. Dujic) and the Department of Pharmacology (D. Modun), Univer-sity of Split School of Medicine, Split, Croatia; and the Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway (A. O. Brubakk).

This manuscript was received for review in December 2006 . It was ac -cepted for publication in September 2007 .

Address reprint requests to: Zeljko Dujic, M.D., Ph.D., Department of Physiology, University of Split School of Medicine, Soltanska 2, 21000 Split, Croatia; [email protected] .

Reprint & Copyright © by Aerospace Medical Association, Alexan-dria, VA.

DOI: 10.3357/ASEM.2039.2007

2 Aviation, Space, and Environmental Medicine x Vol. 78, No. 12 x December 2007

ANTIOXIDANTS & DIVING — OBAD ET AL.

cardiovascular and blood vessel function for several days after the dive ( 36 ). However, pre-dive ingestion of a high dose of vitamins C (2 g) and E (400 IU) in a ran-domized placebo-controlled study design partially re-versed the endothelial dysfunction after the dive, while changes in pulmonary artery and heart function were unaffected. Antioxidant vitamins were also shown to at-tenuate endothelial dysfunction after other acute insults such as a high-fat meal ( 38 ) and short-term cigarette smoking ( 44 ). Endothelial dysfunction is a condition as-sociated with increased expression of adhesion mole-cules, increased synthesis of proinfl ammatory factors, increased oxidative stress, and abnormal modulation of vascular tone ( 11 ). It occurs early in the process of ath-erogenesis and is involved in the formation, progres-sion, and complications of atherosclerotic plaque ( 42 ). Atherosclerosis is considered to be an infl ammatory dis-ease associated with free oxygen radical formation ( 42 ). However, the endothelium also contributes to the vas-cular state of infl ammation since it is one of the sources of ROS formation ( 21 ). This study was designed to in-vestigate the effect of 4 wk of low dose antioxidant treat-ment with vitamins on arterial endothelial function in divers after a single open sea air dive.

METHODS

Study Population

The study was carried out on eight nonsmoking Croatian male experienced Navy divers 35.6 6 3.6 yr of age, with an average body mass index of 25.9 6 2.4 kg z m 2 2 , height 1.81 6 0.05 m, and body fat index 17.1 6 3.0 (body fat/kg %). All experimental procedures were con-ducted in accordance with the Declaration of Helsinki, and were approved by the Ethics Committee of the University of Split, School of Medicine. The procedures were explained in detail to all subjects with the possibil-ity to withdraw at any point with no consequences. Informed written consent was obtained from all subjects prior to the onset of the study.

Timeline of Measurements

The divers performed two dives, the fi rst one as control and the second following 4 wk of antioxidant treatment. The subjects were investigated by ultrasound 30 min be-fore the dive and approximately 40 min after surfacing. Mean maximal oxygen uptake ( 2max ) and maximum heart rate (HR max ) were determined in all divers 2 wk prior to the experiments with cycle ergometry (Marquette Hellige Medical Systems 900 ERG, Milwaukee, WI).

Location of the Study

The study was performed at the military base of the Croatian Navy Forces over a 4-wk period. The diving site was located in the vicinity of the base, where the divers were transported by powerboat. Sea temperature was 14°C for both pre- and post-antioxidants dives, and outside temperature varied between 15 – 18°C.

Dive Protocol

The divers performed two dives, the fi rst one as con-trol and the second following 4 wk use of vitamin C and E. All divers were asked not to change their diet during the experimental period. All dives were performed in the morning by divers equipped with wet suits in accor-dance with the U.S. Navy diving manual ( 49 ). Depth of the dive was set to 30 m with a descent rate of 10 m z min 2 1 . Each pair of divers was supplied with a dive computer (Mosquito, Suunto, Finland). The divers were told to swim on the bottom over a distance of 500 m, at a speed requiring approximately 30% of their 2max . After 30 min at the bottom, the divers ascended to de-compression depth at a rate of 9 m z min 2 1 , with a de-compression stop at 3 m for 3 min. During the decom-pression period divers were told not to perform any exercise, as we have recently shown that exercise during decompression signifi cantly reduces bubble grade ( 14 ). This protocol was chosen because it produces a signifi -cant amount of venous bubbles ( 15 ). Heart rate (HR) was continuously monitored in all divers during diving with a Polar S810i HR monitor (Polar Electro Oy, Kempele, Finland ).

After-Dive Monitoring and Echocardiography Study

Following the dive, the divers were transferred to the base by powerboat. Approximately 20 min after surfac-ing they were placed on the left side in supine position and an echocardiographic investigation was performed with a phase array probe (1.5 – 3.3 MHz) using a Vivid 3 Expert ultrasonic scanner (GE, Milwaukee, WI). High quality images were obtained in all subjects and gas bubbles were seen as high intensity echoes in the right side of the heart and the pulmonary artery. Monitoring was performed every 20 min for 40 min. Images were graded as previously described ( 16 ) and the bubble grades were linearized to bubbles/cm 2 image area as described previously ( 33 ).

Endothelial Function

Endothelial function was determined according to the method of Raitakari and Celermajer ( 41 ) that determines the arterial response to reactive hyperemia and FMD ( 8 ). The subjects were placed in a quiet room with a temper-ature of about 20°C and were resting for 15 min on the bench in a supine position. Participants were tested at the same time of day. Measurements were performed with a 5.7 – 13.3 MHz linear transducer (using the same scanner as above) 40 min post-dive. Brachial artery di-ameter was measured from longitudinal images with lumen-intima interface visualized on both walls. When the images were chosen for analysis, the boundaries for diameter measurement were identifi ed manually with an electronic caliper. In parallel to the imaging of the brachial artery, mean blood velocity (MBV) was ob-tained using the duplex function of the linear array vas-cular probe. The position of the transducer was marked to ensure the same position. Once the basal measure-ments were obtained, arterial occlusion was created by



infl ating a cuff placed on the forearm to 240 mmHg for 5 min. After 5 min infl ation the cuff was defl ated, produc-ing a brief high-fl ow state resulting in artery dilatation due to increased shear stress. Flow and diameter of bra-chial artery were measured at time of cuff defl ation, and every 30 s for the fi rst 3 min, and at the 4th and 5th min. Subjects then rested for 10 min. To assess endothelium-independent response, 0.4 mg nitroglycerine by oral spray (Nitrolingual spray, gliceriltrinitrat, Rhone-Poulenc Rorer, Inc., Collegeville, PA ) was used. FMD was calculated as the percent increase in brachial artery diameter from the resting state to maximal dilatation. Blood fl ow was calculated from the MBV measurements and the vessel diameter, assuming that the vessel was circular. All raw data were saved on hard disk as both still and cine-loop images for later reviewing. According to recent guidelines for FMD ( 39 ), we normalized it to the shear rate, a particular variable which depends on vessel diameter ( 10 ).

Post-Occlusion Reactive Hyperemia

Blood velocity measurements were acquired dur-ing the initial 5 – 7 s after cuff release. Measurements of peak beat and 5-s average fl ow were calculated (vessel cross-sectional area 3 MBV). Peak beat and 5-s aver-age shear rate were determined as (4 3 MBV)/mean diameter ( 10 ).

Normalized FMD

Measurements of FMD were normalized to average shear rate since the amount of dilation has been shown to depend on the resultant hyperemic fl ow stimulus as represented by shear rate (normalized FMD 5 FMD / average shear rate) ( 10 ). Values are expressed as percent per second.

Pharmacological Intervention

The subjects were treated with vitamin C (on average 750 mg z d 2 1 ; initial 2 wk with 500 mg z d 2 1 and last 2 wk with 1000 mg z d 2 1 ; pure powder, Medimon, Split, Croatia) and E (400 IU z d 2 1 ; Twinlab, Hauppauge, NY) for 4 wk. They were instructed to take the last dose on the morning of the return visit, which was 3 – 4 h before the second dive.

Statistical Analysis

Data are given as mean 6 SD. Differences in arterial diameter and response to hyperemia before and after a dive were determined using the Student t -test for paired samples. The limit of signifi cance was set at P 5 0.05. All analyses were performed with Statistica 7.0 software (Statsoft, Inc., Tulsa, OK).

RESULTS

All eight divers ( N 5 8) successfully completed both dives without any symptoms of decompression sick-ness. One participant showed no bubbles during 40 min of rest in either dive, whereas in the other seven this

dive profi le produced a moderate bubble load on both occasions (median bubble grade of 2 on both dives). The average number of venous bubbles per square centime-ter in the right side of the heart during the whole obser-vation period after the fi rst dive was 0.6 6 1.2 and after the second dive 0.9 6 1.3. Bubbles were not observed in the left ventricle, indicating that no large patent foramen ovale were present. 2max for the group was 42.2 6 4.5 ml z kg 2 1 z min 2 1 and the HR max at 2max was 179.5 6 9.1 bpm.

Baseline Mean Brachial Artery Diameter

Mean brachial artery diameter signifi cantly increased from 3.85 6 0.55 mm at baseline to 4.04 6 0.5 mm after the fi rst dive ( Fig. 1 ) ( P 5 0.001), in accordance with our previous fi ndings. Antioxidants alone had no signifi cant effect on baseline diameter. The dive with antioxidants did not cause any additional vasodilation (3.99 6 0.37 vs. 4.01 6 0.38 mm).

Endothelium-dependent and endothelium-independent vas-cular function: FMD of the brachial artery was signifi -cantly reduced from 7.6 6 2.7 to 2.8 6 2.1% ( P 5 0.0008) after the fi rst dive (without antioxidants) ( Fig. 2 ).

Antioxidant treatment for 4 wk did not change pre-dive FMD signifi cantly ( P 5 0.8). The second dive (with antioxidants) did reduce FMD signifi cantly from 8.11 6 2.4 to 6.8 6 1.4% ( P 5 0.044); however, the reduction in FMD was signifi cantly less with antioxidants ( P 5 0.0002) than without. Similar responses were noted for normalized FMD (to shear rate) on the fi rst dive. The re-duction in normalized FMD after the second dive was not signifi cant ( P 5 0.088).

There seemed to be an inverse relationship between bubble formation and the reduction in FMD. Following the control dive, the divers with none or few bubbles (Grade 0 and 1) had a reduction in FMD of 84%, while those with more bubbles had a reduction of 7%. This dif-ference is signifi cant ( P 5 0.03). The same trend was seen after antioxidants, however, this difference was not signifi cant ( P 5 0.1). Vascular smooth muscle function,

Fig. 1. Changes of brachial artery diameter for all divers ( N 5 8) at predive and post-dive for control dive (1,2) and dive after application of antioxidants (3,4). Values are means 6 SD * Signifi cant difference 1 vs. 2 ( P 5 0.001).



assessed by nitroglycerine (endothelium-independent dilation), was normal and unchanged after both dives (12.7 and 12.8%, respectively).

Peak and 5-s Average Reactive Hyperemia and Shear Rate

Peak and 5-s post-occlusion fl ow and shear rates were unchanged before and after both dives (with and with-out antioxidants) ( Table I ) for all divers ( N 5 8). In none of the dives did heart rate change signifi cantly.

DISCUSSION

The main fi nding of this study is that continuous in-take of a low dose of vitamin C ending 3 – 4 h before the dive reduces the endothelial dysfunction after scuba

TABLE I. BRACHIAL POST-OCCLUSION BLOOD FLOW AND SHEAR RATES BEFORE AND AFTER LONG-TERM ANTIOXIDANT TREATMENT.

Control Dive N � 8 Dive After Antioxidants N � 8

Pre Post Pre Post

Peak brachial blood fl ow, (ml z min 2 1 ) 736.3 6 183.0 734.7 6 220.2 827.6 6 175.3 861.4 6 157.1 5-s average blood fl ow, (ml z min 2 1 ) 680.1 6 178.6 705.6 6 213.4 771.6 6 190.1 822.9 6 148.8 Peak shear rate, (s 2 1 ) 119.0 6 20.5 109.0 6 31.2 120.2 6 28.6 120.4 6 30.8 5-s average shear rate, (s 2 1 ) 108.4 6 20.0 101.3 6 30.6 113.0 6 28.4 112.8 6 31.1

Values are means 6 SD.

Fig. 2. Flow-mediated dilation (FMD) and normalized FMD to shear rate at predive and post-dive for control dive (1,2) and dive after applica-tion of antioxidants (3,4) for all divers ( N 5 8). Values are means 6 SD. Panel A: † Signifi cant difference 1 vs. 2 ( P 5 0.0008); ‡ signifi cant differ-ence for delta 1 – 2 vs. 3 – 4 ( P 5 0.0002); * signifi cant difference 3 vs. 4 ( P 5 0.044). Panel B: * Signifi cant difference 1 vs. 2 ( P 5 0.0003).

diving. This, together with our previous study using a signifi cantly higher dose, shows that antioxidants may have a prophylactic effect on endothelial dysfunction after diving.

In the present study, the last dose of ascorbic acid was given 3 – 4 h before the dive was performed. The effect we have observed may, therefore, be an acute effect of the antioxidant. In fact, Eskurza et al. ( 17 ) showed that acute injection of ascorbic acid increased FMD, while an oral supplement of 500 mg given 72 h before had no ef-fect. Gokce et al. ( 22 ) gave 2 g of ascorbic acid 2 h before testing and found no difference between this regime and 500 mg given in the morning before FMD testing was done. Raitakari et al. ( 40 ) also found no effect of long-term oral treatment, only an acute effect.

Jackson et al. ( 26 ) showed that very high doses, in ex-cess of what is possible to achieve by oral means, of ascorbic acid was required to have a signifi cant antioxi-dant effect. In spite of this, Levine et al. ( 28 ) demon-strated that a dose of 2 g reversed endothelial dysfunc-tion in 2 h. This indicates that other mechanisms than simply an antioxidant effect may play a role. The pres-ent as well as the previous study shows that even quite low doses of vitamin C may prevent acute endothelial dysfunction after diving.

The fi ndings in this study support the hypothesis that oxidative mechanisms play a major role in the changes observed after a single air dive. In the present study, the diameter of the brachial artery increased by 5% as com-pared to 7% in the previous study. A similar increase was seen after breathing 60% oxygen for 80 min, corre-sponding to the oxygen exposure of a dive to 260 kPa for 80 min ( 5 ). In the present study, the divers were exposed to 80-kPa oxygen for 30 min vs. 60-kPa oxygen for 80 min in the previous dive. This gives a “ dose ” of oxygen that is approximately 50% lower in the present dive than in the previous one (195 and 432 oxygen tolerance units, respectively) ( 24 ). This is less than 25 to 50% of the dose that is considered acceptable for 1-d exposure.

Hyperoxia leads to vasoconstriction and this may act as a trigger for an increased NO release (production). Mak et al. ( 29 ) have shown that hyperoxic vasoconstric-tion is mediated by oxidative stress and that hyperoxia impairs acetylcholine-induced vasodilation. These ef-fects of oxygen were prevented by vitamin C, indicating that hyperoxia-derived free radicals impair the activity of endothelium-derived vasoactive factors ( 29 ). Increased NO production has been seen following hyperbaric oxygen administration, but the increase was small when



100-kPa oxygen was used ( 47 ). Furthermore, oxygen ad-ministration induces NO production in pulmonary en-dothelial cells ( 9 ), but seems to have little effect on endo-thelial cells from the systematic circulation ( 53 ). Early vasoconstriction in the cerebral circulation is modulated with eNOS, whereas late hyperoxia-induced vasodila-tion depends upon both eNOS and nNOS ( 2 ). Our pres-ent and previous data ( 5 ) suggest that vasoconstriction induced by hyperoxia lead to vasodilation after exposure and that this effect can be prevented by the use of anti-oxidants.

Scuba diving combines physical activity with hy-perbaria and high oxygen availability, which leads to oxidative stress ( 19 ). Increased production of ROS, like superoxide anion (O 2

2 • ) and hydrogen peroxide (H 2 O 2 ) reduces endothelial function and NO production ( Fig. 3 ). Oxidative stress reduces NO bioavailability, as O 2

2 • and H 2 O 2 oxidize NO to peroxinitrite (ONOO 2 ). Furthermore, O 2

2 • and ONOO 2 can oxidize BH 4 , a co-factor of eNOS, which leads to eNOS uncoupling. Un-coupled eNOS produces O 2

2 • instead of NO, addition-ally increasing the oxidative stress and aggravating the endothelial dysfunction and closing the vicious circle ( 4 ). Endogenous antioxidant defenses, like the enzyme superoxide dismutase (SOD), act against this oxidative stress. Furthermore, at physiological concentrations, NO attenuates oxidative stress, a mechanism that has been implicated in the pathogenesis of hypoxic pulmonary hypertension ( 25 ).

In a situation of oxidative stress, such as scuba diving, application of antioxidants, like vitamins C and E, could

reduce the oxidative stress and, consequently, preserve the endothelial production of NO ( Fig. 3 ). Vitamin C (ascorbic acid) is the major aqueous phase antioxidant and vitamin E (alpha tocopherol) is the major membrane-bound antioxidant in the body. It was sug-gested that vitamin C and E function together in a cyclic type reaction, where vitamin C regenerates (rereduces) vitamin E. Vitamin C also works well together with antiox-idant enzymes, such as SOD, against the oxidative stress in the organism ( 50 ). Prevention of BH 4 oxidation in the endothelium could be accomplished by intake of antiox-idant vitamins, therefore preserving NO production. In a previous report, application of vitamin C prevented decrease of coronary fl ow velocity and increase in coro-nary resistance in patients with ischemic heart disease after hyperoxic challenge ( 31 ). Furthermore, application of exogenous BH 4 prevented endothelial dysfunction in another model of oxidative stress, ischemia reperfusion injury, in healthy human subjects ( 30 ).

Antioxidants signifi cantly attenuated the reduction in FMD seen after a single dive. This is in line with previ-ous studies. In heavy smokers FMD is found to be re-duced. In one study, adding vitamins C and E to the sub-jects ’ diet for 25 d in similar doses as was used in the present study normalized endothelial function ( 46 ). However, as it was not mentioned in that paper when the last dose was given, this might well have been an acute effect.

FMD is most probably mediated by NO produced by the endothelial cells, as the response can be almost com-pletely abolished by L-NMMA ( 32 ) and oxygen radicals will reduce the vasodilating effects of NO. There are many currently available methods for testing endothe-lial function, such as intracoronary, intra-arterial, im-pedance plethysmographic studies, and venous studies, and measurement of FMD in the brachial artery by ul-trasonography ( 1,52 ). Brachial artery FMD has been shown to correlate with measures of coronary endothe-lial function, which is the gold standard for measure-ment of endothelial function. The main advantage of brachial artery FMD measurement is the non-invasive nature and the ability to repeat multiple tests in the same patient ( 1,52 ). Mak et al. ( 29 ) have shown that hy-peroxia impairs endothelium-dependent vasodilation by a free radical mechanism in healthy humans and hy-perglycemia apparently has the same effect ( 48 ). While hyperoxia plays a major role in reducing FMD after a dive, it is probably not the only effect, as the antioxi-dants were only partly able to eliminate this effect. In addition to reducing NO bioavailability, oxidative stress during diving may also potentially reduce other endothelium-dependent vasodilators, such as prostacy-clin and endothelium-derived hyperpolarizing factor, and inhibit myocyte-endothelial signaling ( 18 ). Further-more, venous bubble formation may conceivably have an effect on arterial endothelial function. We have previ-ously shown in the rabbit that low bubble load will lead to endothelial dysfunction in the pulmonary artery be-tween 1 to 6 h after exposure ( 35 ). In addition, we have shown that venous bubbles may damage the pulmonary

Fig. 3. Endothelial dysfunction caused by oxidative stress. During ox-idative stress, reactive oxygen species, like superoxide anion (O 2

2 • ) and hydrogen peroxide (H 2 O 2 ), reduce the bioavailability of nitric oxide, as they react with NO, producing peroxynitrite, before NO can activate soluble guanil cyclase and induce endothelium – derived vasodilatation. Furthermore, both ONOO 2 and O 2

2 • oxidize tetrahydrobiopterin and uncouple endothelial NO synthase. The uncoupled eNOS produces O 2

2 • instead of NO, closing a vicious circle. Intracellular enzymes, such as superoxide dismutaze, reduce the level of O 2

2 • . Exogenous antioxi-dants, such as vitamins C and E, can help against oxidative stress, by reducing O 2

2 • and H 2 O 2 , assisting superoxide dismutase, or by prevent-ing oxidation of tetrahydrobiopterin and uncoupling of eNOS. Further-more, vitamin C regenerates vitamin E by reducing its oxidized form. BH 4 -eNOS — coupled endothelial nitric oxide synthase; BH 2 -eNOS (uncoupled endothelial nitric oxide synthase); iNOS — inducible nitric oxide synthase; Vit. C — reduced vitamin C; Vit. C • — oxidized vitamin C; Vit. E — reduced vitamin E; Vit. E • — oxidized vitamin E; NO • — nitric oxide; ONOO 2 — peroxynitrite; O 2

2 • — superoxide anion; H 2 O 2 — hydrogen peroxide; SOD — superoxide dismutase.



endothelium ( 34 ). Activation of the endothelium in the venous circulation will produce endothelial microparti-cles that can initiate endothelial dysfunction at remote sites ( 6 ). Endothelial microparticles (i.e., small vesicles released from endothelial cells consisting of a plasma membrane and cytosolic material) are only a few microns in size, and could possibly pass the lung fi lter and enter the arterial system. Therefore, changes in arterial endo-thelial function can occur without direct contact with the bubbles. However, the fact that the reduction in FMD was inversely related to bubble formation would indi-cate that this mechanism did not play a signifi cant role in this study. It is worth noting that a positive, but non-signifi cant, relationship between the reduction in FMD and bubble formation was seen in our previous study ( 5 ). As these relationships are based on few data, the observa-tions must be viewed with considerable caution.

The resting diameter of the brachial artery will infl u-ence FMD when the relative change in diameter is used. Thus, we also tried to normalize the change using the method described by de Groot et al. ( 10 ). There is no agreement as to which method is preferred in evaluat-ing endothelial function ( 8 ); in our case this normaliza-tion had the effect of making the reduction in FMD fol-lowing a dive with antioxidants non-signifi cant.

In normal individuals, the maximum diameter is usu-ally found 1 min after the release of the occluding cuff ( 7,32 ). In some of the divers, even before the fi rst dive, the increase was slower, with maximum vasodilatation seen at the end of the 2nd min. The timing of peak hy-peremia was further delayed after the dive. For example, in two divers the maximal diameter was observed at the end of the 4th min. After 4 wk of antioxidants the re-sponse before the second dive was the same as for the fi rst dive. However, the response after the second dive was much faster; e.g., in seven out of eight divers the maximal vasodilation occurred within 90 s after occlu-sion. This indicates that the time to peak response might be an indication for endothelial function.

In order to distinguish between endothelium medi-ated vasodilatation and direct smooth muscle reaction, nitroglycerine is usually given, which will lead to vaso-dilatation even in the absence of the endothelium ( 8 ). As reported in a pilot study of our previous study ( 5 ), all divers in this study showed normal response to admin-istration of nitroglycerine after both dives, indicating that smooth muscle function remained intact following the dives and that the reduced FMD observed was mainly endothelium dependent.

Study Limitations

This was not a placebo-controlled, randomized study, and each diver was studied on two occasions, the sec-ond of which was following 4 wk of treatment with antioxidant vitamins. Although we cannot exclude the possibility that sequence, rather than intervention, ac-counted for the fi ndings, we think that this is unlikely as there were similar resting diameters and FMD responses prior to both dives. Antioxidant therapy alone did not

seem to modify the response. Furthermore, in a random-ized placebo-controlled design with acute vitamin in-take, we recently found that endothelial dysfunction af-ter diving is partially reversed by this treatment ( 36 ), supporting the fi ndings for lower doses of vitamins in this study. However, as antioxidant activity was not monitored, we do not know the dose-response of the ob-served effects. Because we did not directly measure NO or ROS in our subjects in this study, our interpretation that application of antioxidant vitamins prevented the scuba-dive-induced endothelial dysfunction (estimated as reduced FMD values) is inevitably speculative. Other mechanisms could also be involved in this effect; for in-stance, vitamin C prevented endothelin-induced endo-thelial dysfunction ( 3 ).

In summary, this study indicates that long-term treat-ment with vitamins C and E signifi cantly reduces the negative effect of a dive on endothelial function. Based on our previous study with higher doses and evidence from the literature, we believe that this effect is reached by giving oral antioxidants shortly before the dive. Since the long-term deleterious effects of diving on endothe-lial function are presently unknown, future studies are needed to evaluate the importance of these fi ndings.

ACKNOWLEDGMENTS This study was supported by the Croatian Ministry of Science,

Education and Sports, Grants No. 216-2160133-0130 and 216-2160133-0330 and by the Norwegian Petroleum Directorate, Norsk Hydro, Esso Norge, and Statoil under the ‘ Dive contingency contract no 4600002328 ’ with Norwegian Underwater Intervention.

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Antioxidant Pretreatment and Reduced Arterial Endothelial Dysfunction After Diving