Scand J Med Sci Sports 2017: 27: 622–626 ª 2016 John Wiley & Sons A/S. doi: 10.1111/sms.12684 Published by John Wiley & Sons Ltd

Dietary nitrate enhances arterial saturation after dynamic

A. Patrician1, E. Schagatay1,2

1Department of Health Sciences, Mid Sweden University, Ostersund,€ Sweden, 2Swedish Winter Sports Research Centre, Mid Sweden University, Ostersund,€ Sweden Corresponding author: Alexander Patrician, Environmental Physiology Group, Department of Health Sciences, Mid Sweden University, Kunskapens vag€ 8, SE 831 25 Ostersund,€ Sweden. Tel.: +46 702229842, Fax: +46 63165740, E-mail: [email protected] Accepted for publication 1 March 2016

Breath-hold divers train to minimize their oxygen after 2-min countdown and without any warm-up , consumption to improve their apneic performance. Dietary hyperventilation, or lung packing. SaO2 and heart rate nitrate has been shown to reduce the oxygen cost in a were measured via pulse oximetry for 90 s before and after variety of situations, and our aim was to study its effect on each dive. Mean SaO2 nadir values after the dives were arterial oxygen saturation (SaO2) after 83.4 Æ 10.8% with BR and 78.3 Æ 11.0% with PL (DYN) performance. Fourteen healthy male apnea divers (P < 0.05). At 20-s post-dive, mean SaO2 was (aged 33 Æ 11 years) received either 70 mL of 86.3 Æ 10.6% with BR and 79.4 Æ 10.2% with PL concentrated nitrate-rich beetroot juice (BR) or placebo (P < 0.05). In conclusion, BR juice was found to elevate (PL) on different days. At 2.5 h after ingesting the juice, SaO2 after 75-m DYN. These results suggest an oxygen they were asked to perform 2 3 75 m DYN dives in a pool conserving effect of dietary nitrate supplementation, which with 4.5-min recovery between dives. Each dive started likely has a positive effect on maximal apnea performance.

À Interest in dietary nitrate (NO3 ) has increased over apnea), horizontal distance (dynamic apnea) with the past decade because acute ingestion has been (DYN) or without fins, or depth. Performance shown to improve sports performance in a number depends upon a number of physiological factors, but of different conditions by reducing the oxygen (O2) achieving a minimal rate of O2 consumption cost and enhancing tolerance to exercise of healthy (VO2min) is essential for success in all disciplines subjects in normoxia (Larsen et al., 2007; Bailey (Ferretti, 2001; Foster & Sheel, 2005; Schagatay, et al., 2009; Lansley et al., 2011; Breese et al., 2013; 2009, 2010, 2011). Kelly et al., 2014), in (Vanhatalo et al., During apnea, the results have however been con- 2011; Masschelein et al., 2012; Kelly et al., 2014) flicting whether NOÀ ingestion enhances perfor- À 3 and in patients with peripheral artery disease (Ken- mance. Acute NO3 ingestion via beetroot juice was jale et al., 2011). found to increase arterial O2 saturation (SaO2) after The mechanism responsible for the O2 conserving of a fixed duration, it caused greater À effect of NO3 is not clear. An improvement in mito- driving bradycardia during maximal apneas, and it chondrial efficiency is a potential explanation for a also prolonged maximal apnea duration by 11%, lower O2 cost of submaximal exercise (Larsen et al., compared to placebo (Engan et al., 2012). However, 2011). A lower adenosine triphosphate (ATP) a study by Schiffer et al. (2013) reported that NOÀ À 3 requirement for the same power output is another supplementation via potassium NO3 reduced static explanation (Bailey et al., 2010) that could lower O2 apneic duration and enhanced desaturation during cost even if mitochondrial efficiency is unchanged maximal duration static apneas. Yet in the same (Whitfield et al., 2015). study, after simulated dynamic apnea by stationary Sports involving apneic diving rely completely on cycling, a trend for increased SaO2 after maximal À stored O2, and therefore provide a unique model to duration apneas was reported with NO3 supplemen- study O2 consumption. Competitive breath-hold div- tation (Schiffer et al., 2013). ing is a growing sport, with disciplines that aim for Dynamic apnea performance relies on factors also maximal performance in underwater time (static important during static apnea – total body storage of

622 Dietary nitrate during dynamic apnea usable O2, tolerance to , and metabolic rate Experimental procedures (Schagatay, 2009), but contains the added challenges Subjects were assigned in a blinded, randomized, cross-over of sustaining propulsive work despite the diving design to receive dietary supplementation with 70 mL of con- response (vasoconstriction and bradycardia) and À centrated NO3 -rich beetroot (BR) or placebo (PL) juice on ~ À central pooling of blood to high priority regions different days. The BR juice contained 5.0 mmol NO3 and ~ À (Scholander et al., 1962; Gooden, 1994; Schagatay, the PL contained 0.003 mmol NO3 (James White Drinks 2010). Ltd., Ipswich, UK). BR is indistinguishable from PL in color, À taste, smell, and texture (Lansley et al., 2011). Subjects were Our objective was to determine if acute NO3 instructed to consume the beverage 2.5 h prior to each test, ingestion would enhance SaO2 after a fixed distance À which were conducted on different days with at least 24 h dive. We therefore studied the effect of dietary NO3 between tests. on SaO2 and heart rate after DYN in a pool. SaO2 Each subject sat resting on a chair at the poolside for 5 min prior to entering the water (Fig. 1). In the water after an addi- after the dive would reflect the O2 consumption, which would likely be predictive also of maximal tional 2-min rest, baseline SaO2 and heart rate were measured via finger pulse oximetry (Nonin Medical Inc., Plymouth, DYN performance. The heart rate recovery response Minnesota, USA) every 10 s for a total of 90 s. Each dive was could reflect the magnitude of vasoconstriction of preceded by a 2-min countdown without any warm-up, hyper- the diving response occurring during the dive. We ventilation, or lung packing, as these maneuvers may affect À diving performance and are difficult to standardize. Warm-up hypothesize that NO3 will decrease the O2 cost and increase the diving response. apneas can pre-stimulate the spleen to release red blood cells that may prolong apneas (Schagatay et al., 2001), hyperventi- lation reduces the level of carbon dioxide (CO2) and urge to Methods breathe (Lin et al., 1974) and may cause -induced cerebral vasoconstriction (Brian, 1998), and lung packing Subjects € increases the O2 stores (Ornhagen et al., 1998). Subjects were 9 Fourteen healthy male subjects (mean Æ SD age then asked to perform 2 75 m DYN in the pool with 4.5- 33 Æ 11 years, height 182 Æ 8 cm and 82 Æ 12 kg) min recovery between the dives. Starting within 10 s after sur- volunteered to participate in this study. They were all experi- facing after each 75 m DYN, SaO2 and heart rate were mea- enced apneic divers with 5.0 Æ 3.8 years of training in the sured continuously until 90 s after each dive. The SaO2 nadir sport with a DYN personal best of 119 Æ 43 m. Most divers was defined as the lowest SaO2 value recorded from each dive in the 90-s post-dive period. Water was were close to their peak training level; eight were tested in con- Æ ° Æ ° nection with a major competition, two were safety divers for 26 1 C and air temperature was 32 2 C, inducing a the competition who trained regularly, and four were tested thermal difference sufficient to enhance the diving response after a training camp. Subjects gave their written informed compared to apnea alone (Schagatay & Holm, 1996). consent after receiving written and oral information about the study procedures and potential risks. The protocol had been Data analysis approved by the local research ethics committee and was conducted in accordance with the Helsinki declaration. All subjects served as their own controls and all but one subject completed all tests; the one subject interrupted the second swim before 75 m. Another three subjects had Methodological considerations incomplete data from one of their 75-m swims. For these No maximal effort DYN dives were included, as they may four subjects, calculations were based on one dive from lead to syncope, which is an unnecessary risk that would each condition, in the same dive order (dive 1 or 2) to require a full-scale safety system with two safety divers in the avoid an order effect. Therefore, 10-s group mean values water. In addition, the maximal performance varies from day- were calculated using the means of two dives in nine sub- to-day due to both physiological and psychological factors, jects and one dive in four subjects in each of the BR and especially in trained athletes (Schagatay, 2010). The authors PL conditions. One subject was excluded from the 10-s considered that the submaximal performance of 75 m would group mean value analysis, but not the SaO2 nadir value comparison, because a complete data from each dive were be more standardized and would produce an O2 debt sufficient À not in the same dive order. Data are presented as enough to conclude whether dietary NO3 had an effect on Æ performance or not. The 75-m dives were performed as part of mean SD, in 14 subjects for the SaO2 nadir values, and their training. Only trained apneists were included because of in 13 subjects for the continuous SaO2 and heart rate val- ues. The lowest individual post-dive SaO2 nadir value was their ability (compared to non-divers) to manage the O2 debt when performing 75-m DYN. A safety diver was present at used for calculating mean SaO2 nadir values. SaO2 rates of the poolside during all events. recovery after surfacing were calculated from SaO2 nadir to 97% SaO2.

Enters 2-min 2-min water countdown countdown

75 m 75 m DYN DYN 5 min 5.5 min 4.5 min

Fig. 1. The experimental procedure consisting of 2 9 75 m dynamic apnea with fins (DYN) with 4.5-min recovery between the dives. Line signifies the 90-s measurement of SaO2 and heart rate.

623 Patrician & Schagatay

0.8 Æ 0.6%/s with BR and 0.9 Æ 0.5%/s with PL Statistics (P = 0.340). Wilcoxon matched-pairs signed rank test was used to compare SaO2 and heart rate between BR and PL conditions. Bonfer- roni correction was used for multiple comparisons of up to Heart rate 40 s after surfacing. Statistical significance was accepted at The resting control heart rate was 72 Æ 12 beats/min P ≤ 0.05. with BR and 76 Æ 15 beats/min with PL (P = 0.727). There were no significant differences in Results heart rate between BR and PL conditions after the Arterial O2 saturation dives (Fig. 5).

The resting control SaO2 was 98.2 Æ 0.7% with BR and it was 98.1 Æ 0.5% with PL (P = 0.481). Mean Discussion of individual SaO nadir values were 83.4 Æ 10.8% 2 The principal finding was that SaO after 75-m DYN with BR and 78.3 Æ 11.0% with PL (P = 0.023). 2 was higher after BR ingestion compared to PL. This The reductions from resting control values to SaO 2 was true both for the mean nadir SaO and the SaO nadir were 14.8 Æ 11.0% with BR and 2 2 at the point 20 s after the dive, suggesting that BR 19.8 Æ 11.1% with PL (P = 0.027; Fig. 2). At 20-s reduces the O cost of exercise during dynamic post-dive, mean SaO was 86.3 Æ 10.6% with BR 2 2 apnea. and 79.4 Æ 10.2% with PL (P = 0.019; Figs 3 and 4). There were no differences between BR and PL in the ensuing 10-s intervals across recovery. Mean rate of SaO2 recovery from SaO2 nadir to 97% was

100 *

90 14.8% 19.8% (%)

2 80

SaO 70

60 PL BR

Fig. 2. Mean Æ SD (n = 14) for SaO2 nadir with beetroot (BR) and placebo (PL) juice after 75-m dynamic apnea (DYN). The “open” and “closed” squares, respectively, rep- Fig. 4. Individual mean arterial oxygen saturation (SaO2; resent the mean SaO2 for baseline and after the DYN. = Values inserted are the percent reduction from baseline n 13) between placebo and beetroot juice 20 s after surfac- ing from 75-m dynamic apnea (DYN; *P < 0.05). SaO2 before the DYN to SaO2 nadir values (*P < 0.05).

100 * 125 PL

90 BR 100 (%) 2 80 BR SaO 75 PL Heart Rate (bpm) 70

50 0 102030405060708090 0 102030405060708090 Time after surfacing (s) Time after surfacing (s)

Fig. 3. Mean Æ SD (n = 13) arterial oxygen saturation Fig. 5. Mean Æ SD (n = 13) heart rate with beetroot (BR) (SaO2) with beetroot (BR) and placebo (PL) juice during and placebo (PL) juice during 90 s of recovery after 75-m 90 s of recovery after 75 m dynamic apnea (DYN). Zero dynamic apnea (DYN). Zero (“0”) indicates the moment the (“0”) indicates the moment surfaces (*P < 0.05). diver surfaces.

624 Dietary nitrate during dynamic apnea

This finding aligns with results from an apneic usable value about 10–15 s after surfacing. Conse- model without exercise (Engan et al., 2012) and with quently, the focus of the analysis was set on the per- the tendency for O2 conservation observed in iod from 15 s and onwards. Measuring SaO2 from another exercise-apnea model (Schiffer et al., 2013). the hand is favorable in this case, compared to the It also aligns with several studies involving eupneic ear for example, due to the longer circulation time exercise in normoxia (Larsen et al., 2007; Bailey between the lungs, heart, and the finger. A delay of et al., 2009; Lansley et al., 2011; Breese et al., 2013; approximately 15–20 s occurs before the SaO2 nadir Kelly et al., 2014) and in hypoxia (Vanhatalo et al., corresponding to the end of apnea appears in the fin- 2011; Masschelein et al., 2012; Kelly et al., 2014). ger (Andersson & Evaggelidis, 2009), thus we could The result suggests that the total apneic duration likely detect the nadir values. and the total distance covered in DYN would likely In conclusion, BR juice was found to result in a be increased during a maximal attempt, due to higher SaO2 after 75-m DYN. These results suggest À increased remaining O2 stores (Schagatay, 2010). an O2-conserving effect of dietary NO3 supplemen- Engan et al. (2012), who concluded there was an tation, which increases the safety margins of sub- O2 conserving effect during static apnea, used similar maximal dives and potentially has a positive effect NOÀ administration procedures as in this study, and on maximal apnea performance. 3 À did not incorporate warm-up apneas or allow sub- NO3 supplementation could also be applied to jects to hyperventilate (Schagatay et al., 2001; Ras- other sporting activities with limited oxygen avail- mussen et al., 2006). In Schiffer et al. (2013), who ability, e.g., swimming where is restricted À reported the contrary effect, a different NO3 admin- to optimize hydrodynamics, competition apnea, istration was used, and warm-up apneas and hyper- spearfishing, synchronized swimming, and underwa- ventilation were not controlled for, which could have ter team sports such as and contributed to their different results. Varying warm- hockey. Short-term supplementation with beetroot up procedures likely play important roles in the out- juice has also been shown to improve the walking come of physiological responses during static apnea time performance of patients with peripheral arterial performance (Schagatay, 2009) after consuming diet- disease (Kenjale et al., 2011), counteract the decline ary NOÀ. Interesting, however, is the effect during in endothelial function occurring during ascent to 3 À exercise of NO3 supplementation in the study by high altitude environments (Bakker et al., 2015) and Schiffer et al. (2013), where their data show a ten- preserve muscle oxygenation during exercise in acute dency for the same O2-conserving effect as seen in normobaric hypoxia (Masschelein et al., 2012). the study. There was no effect of BR on heart rate in neither the current study nor in Schiffer et al. (2013), while Perspectives Engan et al. (2012) found that the diving bradycar- À The O2-conserving effect of dietary NO3 could likely dia was more pronounced in maximal static apneas. À be beneficial in various sports involving apnea, e.g., NO3 supplementation has been shown to reduce apnea diving, spearfishing, swimming, synchronized the O2 cost during submaximal exercise (Larsen swimming and underwater rugby, and hockey. et al., 2007; Bailey et al., 2009), and during the sub- Effects in apneic-related sports would be expected on maximal effort in the current study, but it did not several levels; by increasing safety margins in sub- appear from the heart rate response that an accu- maximal dives, potentially increasing maximal per- mulated O2 debt occurred with BR. An aspect formance in competition, and by reducing recovery unique to the current study is the inclusion of time between short, repeated dives. Other applica- immersion, particularly facial immersion (de Bruijn À tions of dietary NO3 supplementation could possibly et al., 2009) which has been shown to be an impor- be found in exercise at high altitude where the O2 tant contributor to apneic performance by augment- supply is also limited. ing the diving response (Schuitema & Holm, 1988). A fully activated diving response due to a thermal Key words: Apneic diving, breath-hold, hypoxia, stimulus, could provide beneficial conditions during immersion, arterial desaturation, anaerobic exercise, exercise, when an O2 conserving effect is even more nitrate, sport performance. essential than compared to rest. Therefore, it would be interesting to further investigate the influence of BR on the cardiovascular diving response during Acknowledgements submaximal exercise. We thank the apnea divers for their participation, as well as From the moment the diver surfaces, time is the coaches and organizers of the events for their help in con- required to dry the hand and allow the pulse oxime- ducting the study. The study was supported by the Swedish ters to obtain a reliable reading, resulting in a first National Centre for Research in Sports.

625 Patrician & Schagatay

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