Sport- and sample-specific features of trace elements in adolescent female field hockey players and fencers

Accepted Manuscript Title: Sport- and sample-specific features of trace elements in adolescent female field hockey players and fencers Author: Alexey A. Nabatov Natalya A. Troegubova Ruslan R. Gilmutdinov Andrey P. Sereda Alexander S. Samoilov Natalya V. Rylova PII: DOI: Reference:

S0946-672X(16)30165-1 http://dx.doi.org/doi:10.1016/j.jtemb.2016.11.002 JTEMB 25848

To appear in: Received date: Revised date: Accepted date:

18-7-2016 19-10-2016 2-11-2016

Please cite this article as: Nabatov Alexey A, Troegubova Natalya A, Gilmutdinov Ruslan R, Sereda Andrey P, Samoilov Alexander S, Rylova Natalya V.Sportand sample-specific features of trace elements in adolescent female field hockey players and fencers.Journal of Trace Elements in Medicine and Biology http://dx.doi.org/10.1016/j.jtemb.2016.11.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Journal of Trace Elements in Medicine and Biology

Sport- and sample-specific features of trace elements in adolescent female field hockey players and fencers

Alexey A. Nabatov, PhD1,2, Natalya A. Troegubova, PhD, MD2, Ruslan R. Gilmutdinov PhD3, Andrey P. Sereda, PhD, MD4, Alexander S. Samoilov, PhD, MD5, and Natalya V. Rylova, PhD, MD.2* 1Science

Center, Volga Region State Academy of Physical Culture, Sport and Tourism,

33 Universiade Village, Kazan, 420138 Russia; 2Kazan

3State

State Medical University, 49 Butlerova str., Kazan, 420012 Russia

enterprise “Research Institute Geolnerud”, 4 Zinina str., Kazan, 420097 Russia.

4Federal

Scientific Clinical Center for Sport Medicine and Rehabilitation; 5 B.

Dorogomilovskaya str., Moscow, 123182 Russia 5Burnazyan

Federal Medical Center of Biophysics, 46/8 Zhivopisnaya str., Moscow,

123182 Russia

*Corresponding author: Rylova N.V., Kazan State Medical University, 49 Butlerova str., Kazan, 420012 Russia; Tel./Fax. +7(843)-2373171; E-mail: [email protected]

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Abstract Active physical exercises and growth are associated with mineral imbalances in young athletes. The purpose of this study was to examine the impact of sport-related factors on tissue mineral status in adolescent female athletes. Saliva and hair samples were used for the analysis of immediate and more permanent tissue mineral status, respectively. Samples taken from a control non-athletic female group and two groups of female athletes (field hockey and fencing) were analyzed for seven essential minerals: calcium, chromium, iron, potassium, magnesium, selenium and zinc. Inductivelycoupled plasma mass spectrometry was used for the quantification of elements having very low concentration range in samples (Se, Cr and Zn) whereas inductively coupled plasma optical emission spectrometry was used for quantification of more ubiquitous elements (Mg, К, Са, Fe). The obtained results for athletic groups were compared with control. Female athletes had increased levels of selenium in both saliva and hair as well as chromium in saliva. Field hockey players had the higher level of zinc in hair whereas fencers had the lower levels of salivary calcium. Strong negative correlation between potassium levels in saliva and hair was identified. Iron and magnesium did not differ between the studied groups. In conclusion, novel sport-specific features of chromium tissue levels in female athletes were found. The studied sport disciplines have different impact on the distribution of osteoporosis-related minerals (calcium and zinc). Our finding can help in the development of osteoporosis preventive trainings and in the proper nutrient supplementation to correct mineral imbalances in female athletes.

Abbreviations: BMI, body mass index; ICP inductively coupled plasma

Key words: trace elements; physical exercises; growth; female athletes.

1. Introduction

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The physical growth of young athletes suggests the increased needs in trace elements. Physical and emotional stresses, associated with competitive sport, can change mineral utilization and absorption [1-3]. Multiple sport disciplines are also different in trainings and mineral losses. Underestimation of these factors can bring to the known negative outcomes for the health of athletes [4]. The mineral levels in blood are strictly regulated by hormones and often do not reflect conditions in tissues, e.g. calcium in osteoporosis. Blood plays an active role in the redistribution of minerals between tissues according to the certain hierarchy (e.g. calcium from pregnant or breast feeding women bones to supply fetus/baby). Although women substantially change their levels of calcium regulating hormones during the menstrual cycle, the calcium serum level remains unchanged [5, 6]. Thus, the blood mineral concentration does not reflect the situation in tissues. A number of studies also suggest that trace element-associated effects are gender dependent and the gender differences in tissue trace elements are more pronounced for athletes [7-10]. The higher prevalence of osteoporosis makes the non-blood-based mineral testing of special importance for women. The level of certain trainings (e.g. endurance) can result in both prevention and induction of female osteoporosis [11-15]. Osteoporosis prevention among women suggests reaching the genetic potential of peak bone mass in childhood and adolescence [16] [17]. This makes tissue mineral studies in young female athletes of special importance in the osteoporosis-related changes research. The growing number of studies suggests that scalp hair and saliva can be used to detect more permanent and real time changes in tissue trace elements, respectively [18-23]. Being a filtrate from blood, saliva loses its resemblance to blood plasma due to tissue specific reabsorption. Due to its specific mineral content, saliva can prevent tooth 3

demineralization as well as enhance remineralization playing a role in the protection of enamel and dentin, two bone-like matrixes of teeth [24]. Very low invasiveness of hair and saliva samplings gives additional benefits over blood testing (less traumatic, no need in professional personnel and certified place for sampling, etc.). To study the impact of sport on tissue mineral status, saliva and hair samples from adolescent female athletes and non-athletes were analyzed for minerals by spectral methods with inductively coupled plasma (ICP). We found novel sportassociated chromium changes and clarified relationships between the sport disciplines and tissue distribution of minerals having the strongest impact on bone thickness. Our results can be used for an adequate control and correction of mineral imbalances in females. 2. Materials and Methods 2.1. Participants Forty eight adolescent females, aged from 12 to 17 years, from the city of Kazan, Russia volunteered for this study. The same location of residency allowed us to avoid the regional biases, related to water and food mineral composition. All individuals were reported as healthy without any psychiatric, medical disorders or developmental delays. Participants were also evaluated by a dentist to exclude individuals with gum inflammation or current cavities. The samples from athletes were divided according to sport disciplines: field hockey and fencing, having higher proportion of aerobic and anaerobic trainings, respectively. The control group included 20 age-matched

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individuals with reported sport activity less than four hours per week. The descriptive data on the groups are summarized in Table 1. 2.2. Sampling Hair samples were collected close to the scalp from the occipital region in a sufficient quantity for analysis. The hair sampling was performed for non-bleached or permed hair. Hair samples were washed first in acetone and then three times with deionized water and dried. The hair samples of 100 mg were placed into teflon test tubes in 1ml of the metal-free concentrated nitric acid (15,8 M) (Merc). The test tubes were capped and placed into the microwave digestion system on 115oC for 1 hour. After digestion, all the samples were taken from the microwave oven and cooled to room temperature. The digested samples were put into 50ml polypropylene tubes and brought to the final volume (10 ml) with ultrapure water («MilliPore», Merc). To make saliva samples more reflecting the sport-associated conditions, the sampling from hungry (last meal 8 hours before sampling) athletes took place at the training centers before training. The saliva sampling was not stimulated. Participants gave saliva samples directly into the collecting tubes. The saliva samples (0,5 ml volume) were added to 1 ml of the concentrated nitric acid. The following procedures are identical to ones for hair samples. 2.3. Study variables

The following variables were measured in this study: involvement in sport disciplines, concentration of Zn, Cr, Ca, Fe, K, Se, and Mg in hair and saliva samples. The minerals were chosen due to their highest impact on energetic metabolism, oxidative stress, 5

muscle functioning and bone health, mediating athletic performance [25]. The methods used to evaluate each variable are described below.

2.3. Analytical Methods Mineral concentrations were assessed using the inductively coupled plasma methods. The methods selection was based on the approach described previously by Harrington et al.: inductively-coupled plasma mass spectrometry (ICP-MS) was used for the quantification of elements having very low concentration range in samples whereas inductively coupled plasma optical emission spectrometry (ICP-OES) was used for quantification of more ubiquitous elements [26, 27]. The ICP-MS method was used for quantification of Se, Cr and Zn in the following conditions: ICP RF Power: 1100 W; Nebulizer Gas Flow: 0,89 L/min; Plasma Gas Flow: 15L/min; Sample Flow Rate: 1,5 ml/min; Lens Voltage: 12,25V; Pulse Stage Voltage: 1100 V; Analog Stage Voltage: minus 1950 V; Est. Sample Time: 5 sec; Delay Time: 30 sec; Replicates: 2 («Elan9000», no DRC, PerkinElmer). ICP-OES method was used for Mg, К, Са, Fe quantification under the following conditions: ICP RF Power: 1300W; Nebulizer Gas Flow: 0,8L/min; Auxiliary Gas Flow: 0,2L/min; Plasma Gas Flow: 15L/min; an axial position of the burner: minus 3; Delay Time: 30 sec; Est. Sample Time: 5-20sec, depending on element concentration; Replicates: 2; Sample Flow Rate: 1,5 ml/min («Optima 2000 DV», PerkinElmer). To calibrate the instruments and for quality control, the multi-element standard solution “Quality Control Standard 21” (PerkinElmer) was used. The blank solution results were taken into account in the analysis of samples. To account for the effect of the acid, acidic blank solution was added.

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2.4. Statistical analysis The SPSS 14.0 statistical software package was used for statistical analysis. The data were expressed as mean (M) and Standard Deviation (SD). The normality of data distribution was checked using the Shapiro-Wilk test. Comparison between two independent groups of numerical parametric data was performed using Student’s t-test; non-parametric data were assessed using Mann-Whitney U Test. Statistical correlation analysis was performed using the Pearson’s or Spearman’s test for normally and nonnormally distributed data, respectively. P-values of less than 0,05 were considered as significant. 3. Results 3.1. Subject characteristics In this study we analyzed mineral levels of two adolescent female athlete groups representing field hockey and fencing and compared them to the ones of non-athlete group. The age 12-17 years was chosen as critical for bone strength. All three studied groups had similar age, body mass index, height, and body mass parameters (Table 1). 3.3. Concentrations of trace elements in hair The mean K and Se concentrations in hair were significantly increased in athletic groups compare to the non-athletic one. The level of Zn was increased in the field hockey group compare to control. Data of the means per group for the mineral concentrations in hair are given in Table 2.

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3.4. Concentrations of trace elements in saliva In contrast to the level in hair, potassium levels in saliva were significantly lower for athletic groups in comparison to the non-athletic group. Concentrations of Se and Cr in saliva were increased in the athletic groups. The fencers had significantly decreased level of Ca in saliva in comparison to the control group. Data of the means per group for the mineral concentrations in hair are given in Table 3. 3.5. Correlations among trace elements concentration in hair and saliva To assess the level of relationship between the trace element content in hair and saliva, correlation analysis was performed. Positive and negative significant correlations were found for selenium and potassium, respectively (Table 4). 4. Discussion and conclusion Although the growing involvement of young people in recreational sport is a very positive trend for public health, the same trend is seen for competitive and semiprofessional sports, known as health threatening not only due physical traumas but the higher frequency of specific internal diseases as well. The high physical activity in stressful conditions of competitions increases metabolism and turnover of nutrients including minerals. Proper identification and correction of the sport-associated nutrient imbalances should be based on the level, character and environment of trainings [2830]. The increased concentration of selenium was found for both studied female athlete groups in hair and saliva samples. The strongest changes of selenium between

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controls and athletes were found for saliva (more than two fold increase for athletes) whereas the selenium increase in hair of athletes was expressed much less (17-31%). The latter suggests the absence of substantial effect of more frequent hair wash on the hair selenium level that could be expected due to the selenium-containing shampoo use. Antioxidant selenoproteins like glutathione peroxidase and thioredoxin reductase play an important role in maintaining bone homeostasis, differentiation of osteoclasts and protecting against bone loss [31, 32]. The increased selenium levels suggest high levels of tissue antioxidant activities in athletes, which can be a response to the increased reactive oxygen production characteristic for intensive exercises. This result confirms and widens the recently found changes in selenium levels in hair from actively training students [10]. Sport specific trends were also found for potassium: the increased potassium content in hair in athletic groups was associated with the decreased one in saliva. This reverse correlation between potassium levels in saliva and hair can reflect the general mechanism of skin-associated excretion of potassium with sweat, more specific for actively exercising athletic groups than for the control one [33]. The increased potassium excretion with sweat causing the potassium deficit in tissues is a well-known phenomenon causing among others the potassium-related muscle cramps in endurance training athletes. The found reverse correlation can be very helpful in the development of approaches for the assessment of tissue potassium deficit caused by sweating. Sample-dependent differences were found for several minerals in our study. Chromium levels were significantly increased in saliva samples in both athletic groups. Chromium is involved in insulin signal transduction, glucose metabolism, and cellular antioxidative defense. If two first factors mediate energy related processes in the course 9

of physical trainings, the last one makes some similarity between Cr and Se [34, 35]. The similarity of mediated processes could be reflected in the Se and Cr increased presence in saliva from athletes. The studied groups had no differences in dental care, which was important because chromium containing alloys and teeth brackets could increase chromium levels in saliva [36, 37]. Thus, the newly found higher chromium levels in saliva in athletic groups can be sport related. Some sample–specific differences were also sport-specific ones. Fencing and field hockey belong to different sport types. Fencing is more specific to the development of very precise technical skills and fast short movements whereas field hockey includes substantial endurance and aerobic training, known to prevent osteoporosis development in young adults [38]. Se and Zn are important components of antioxidant enzymes protecting tissues against and reactive oxygen species, associated with active aerobic exercises [39]. At the same time Zn maintains respiratory system, the key component of aerobic training. We found higher levels of zinc in hair and a strong trend for the increased concentration of Zn in saliva of field hockey players. The similarity the found trends between Zn and Se for field hockey players but not for fencers suggests that Zn changes can be specific for the aerobic trainings whereas selenium changes reflect the increased level of general training and stresses. Our results support the findings that moderate endurance component and regular aerobic training increases the level of zinc, which decreases the risk of osteoporosis [40-42]. The increased salivary calcium level is associated with the resistance to caries that resembles osteoporosis in partial decalcification of bone tissue [43]. Our finding on the decreased concentration of salivary Ca in fencers could reflect the current nutritional trend that the diet of young athletes practicing fencing is rich with carbohydrates and 10

insufficient with micronutrients, especially calcium [44]. However, according to the questionnaires analysis, in our study athletic females did not differ from controls in nutrients consumptions. Altogether these data suggest that fencers should pay additional attention to their dental care. The found sport-dependent difference in calcium rather confirms the finding on the bone strengthening effect of sport games with moderate endurance component [13-15]. Iron and magnesium did not differ between the studied groups reflecting the low impact of the studied sport disciplines on their metabolism. In conclusion, the novel sport-associated increase of chromium levels in saliva was identified in young female athletes. Our findings on the sport-specific differences in bone-strength-associated calcium and zinc support the hypothesis that fresh air sport games, associated with the moderate level endurance training, can have a positive impact on bone strength. Our results also provide the basis for nutritional correction of sport-related mineral disbalances. Ethical standards The study was performed in accordance with all applicable regulatory requirements and the principles of the Declaration of Helsinki. The study was approved by the Kazan State Medical University Research Ethics Committee. Written informed consents were obtained from all the study participants or guardians for participants under the age 18 years. Participants were acknowledged that anonymous data would be presented at academic meetings or published in journal articles.

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Conflict of Interest Conflicts of interest: none

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgments We are thankful to Prof. Anatoly Skalny from the Center for Biotic Medicine, Moscow, Russian Federation, for his valuable remarks about this study.

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Table 1. Characteristics of the study participants groups

Groups Number

Sport experience

Age

Height

(years)

(сm)

(years)

Body mass

BMI (kg /сm2)

(kg)

Control

20

-

14,4±1,34

158,6±6,26

52,5±6,7

20,78±1,34

Field hockey

18

6,25±2,5

15,6±0,84

163,9±7,2

56,4±5,93

20,97±1,69

Fencing

10

6,4±1,77

14,8±1,58

162,8±5,37

56,0±5,05

21,0±1,26

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Table 2. Micromineral levels in hair of athletes and control groups (mg/kg)

Mineral

Control

Field hockey

Fencing

Ca

846,1±109,7

1039,4±61,1

698,0±93,8

Cr

1,40±0,39

0,22±0,07

0,44±0,16

Fe

34,80±4,4

23,2±10,3,8

22,6±4,2

K

30,4±6,6

95,9±12,1*

106,2±29,5*

Mg

70,80±8,1

95,4±8,7

73,2±14,0

Zn

85,0±6,1

184,4±15,1**

135,2±16,2

Se

0,41±0,02

0,48±0,01*

0,54±0,02*

where: * and **reflect the level of difference significance to control group less than 0,05 and 0,01, respectively.

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Table 3. Micromineral levels in saliva of athletes and control groups (mg/l)

Mineral

Control

Field hockey

Fencing

Ca

45,6±2,3

50,2±2,4

32,2±3,4*

Cr

0,003±0,0003

0,034±0,004**

0,015±0,001*

Fe

0,049±0,012

0,061±0,026

0,0362±0,009

K

1484,3±81,9

807,7±42,2**

493,6±91,6**

Mg

3,9±0,3

4,9±0,5

2,9±0,7

Zn

0,033±0,006

0,413±0,157

0,029±0,009

Se

5,8±0,3

12,2±1,9**

14,8±1,2**

where: * and **reflect the level of difference significance to control group less than 0,05 and 0,01, respectively.

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Table 4. Correlations between trace elements concentrations in hair and saliva

Minerals

Correlation data R

R2

p

Ca

0,16

2,6%

0,317

Mg

0,08

0,7%

0,607

K

-0,72

52,2%

<0,001

Fe

-0,01

<0,01%

0,958

Zn

0,32

10,1%

0,052

Se

0,44

19,4%

0,004

Cr

-0,27

7,3%

0,091

20