PhD, Associate Professor I.P. Zaytseva¹
Dr.Med., Professor, Academician of RAS N.A. Aghajanyan²
Dr.Med., Professor A.V. Skalny¹
Dr.Med., Professor A.A. Nikonorov³
PhD, Associate Professor O.N. Zaytsev^4
1P.G. Demidov Yaroslavl State University, Yaroslavl
²Russian Peoples' Friendship University, Moscow
³Orenburg State Medical University, Orenburg
⁴Yaroslavl State Technical University, Yaroslavl

Keywords: macro- and micro elements, whole blood, blood serum, sports, female athletes, physical loads.

Introduction. It is the blood serum and/or blood plasma analysis that are commonly used to profile elemental body statuses in the clinical laboratory diagnostics process. Concentration of some element in the blood serum and/or blood plasma is undoubtedly indicative to some degree of the current situation with the body metabolism [8]. Proper allowance must be made for the fact that elements in the blood serum are present both in free form (in complex with transitional proteins) and within the functional structures that provide for anabolic and catabolic processes. In this context, data obtained through the blood serum/ plasma analysis will be interpreted as fairly indicative of the metabolic interconnections of different body organs and metabolically active compartments [2].

Furthermore, it needs to be remembered in the analytical data interpretation process that it is only the total content of an element as of the sampling moment that is obtained by the analysis. Shortage of elements in the blood serum and plasma may be fixed only in situation of a progressing disease when the body is depleted of some element(s) due to either their increased excretion or insufficient intake – short of the actual body needs. This is the reason why some specific variations in some micro-elemental concentration may not be identified in a timely manner as the variations often fall within the range of the measuring error and partially depend on the protein bonds of the subject elements [8].

Alidzhanova I.E. et al reported experimental data indicative of the concentration of some elements in the blood correlating with their content in bone tissue, liver and body on the whole. In opinion of Iordanskaya F.A. et al [9], blood mineral profiles may be used as indicative of the functional state of the athlete’s body and, therefore, may be viewed as a diagnostic substrate for the mineral/ energy metabolism intensity assessments. Our previous reports confirmed that intensive sport practices may be of significant effect on the macro- and micro-elemental concentrations in whole blood and blood serum [1, 3, 4]. However, the issue of a diagnostic importance of the blood/ blood serum micro-elemental concentration variations with physical workloads of variable intensity still needs to be explored.

Objective of the study was to analyze variations of the levels of macro- and micro-elements in whole blood and blood serum of female athletes exposed to physical workloads of variable intensity in the university sport process; and to identify general regularities of the mineral metabolic process under the variable physical workloads.

Methods and structure of the study. Subject to the study were 43 female university athletes of 18-22 years of age, including 19 athletes (Group 1) engaged in high-intensity sports (volleyball, polyathlon, track and field athletics etc.) and trained at least four times per week; and 10 athletes (Group 2) engaged in moderate-intensity fitness practices 2–3 times per week. Reference Group 3 was composed of 14 university peers engaged in low-intensity physical activities.

Blood sampling was performed at the local medical station. Venous blood (3-5 ml) was sampled from an ulnar vein in morning hours on an empty stomach. The sampled blood was placed in S-Monovette container test tubes of special design for the whole blood sampling. Heparin was applied as an anticoagulant agent in amounts of 0.01 ml per 5 ml of blood. The filled test tubes were softly turned over at least 5 times to prevent micro-clotting effects. The blood samples were stored in a standard refrigerator for 3-5 days at temperatures of 0 to +4°С).

Test samples claimed at least 1.5 ml of blood serum. Blood samples were placed in the S-Monovette container test tubes of special design for blood serum sampling with blood clotting activator gel/ granules free of anticoagulant agent. As soon as the blood was sampled, the container test tube was softly turned over at least 5 times to provide for the full and fast contact of blood with the clotting activator agent. The test tube was then left staying in vertical position for 20–30 minutes to let the clot being formed. Tests were performed on the blood sampling day or the next day.

Every biological liquid sample was subject to sample processing procedure in compliance with [Methodological Guidelines] MUK 4.1.1482-03/ MUK 4.1.1483-03 for “Identifying concentrations of chemical elements in biological media and preparations using Inductively Coupled Plasma Atomic Emission Spectrometry and Inductively Coupled Plasma Mass Spectrometry methods” designed by the Moscow-based Academic Research Organization “Biotic Medicine Centre” and approved by the Ministry of Health of RF in 2003. The whole-blood and blood-serum samples were analysed for macro- and micro-element levels as provided by the relevant medical analytical technology “Disorders of mineral metabolism in human body: identification and correction procedure” (Registration Certificate #ФС-2007/128 of July 09, 2007) using Nexion 300D+NWR213 (made by Perkin Elmer, the USA) Inductively Coupled Argon Plasma Mass Spectrometer (ICP-MS).

A set of standard reference blood serum samples from ClinChek Plasma Control lot 129, Recipe, Germany was applied in the laboratory test process to ensure high-efficiency quality control in the analysis of level of the sampled bio-substrates (see Table 1 hereunder). Two standard reference samples were applied in each of the bio-substrate samples testing process: a high-level one designed for increased levels of elements in the bio-substrate; and a low-level standard to test the lower concentrations of elements. This was done to secure high quality and accuracy of the analysis in both of the standard concentration ranges.

Table 1. Standard reference blood serum samples from ClinChek Plasma Control lot 129, Recipe, Germany

In view of the fact that the actual concentration distributions in the samples were found different from the normal distributions, we applied median values in addition to the mean ones. Couple matching of the Groups was performed using the Mann–Whitney U test criterion.

Study results and discussion. Having analyzed the whole-blood samples (of the subject female athletes exposed to variable physical loads in university sports) for 15 macro- and micro-elements, we have found significant concentration variations for only two elements from the heavy metals group (see Table 2 hereunder). Specifically, we found the Cd levels being significantly lower in the low-activity group of the subject athletes. The second indicative element was Pb which level was found to be significantly lower in the moderate-activity group versus both the high- and low-activity groups of the subject athletes.

Blood serum elemental profiles of the subject athletes showed more intergroup differences (see Table 3 hereunder). We found, in particular, modest but meaningful (p = 0.037) drop in the Ca level in the moderate-activity group versus the high-activity athletes group. Furthermore, the high-activity athletes group was tested with notably (2 times plus) higher Co levels versus the two other groups.

These study results were clearly indicative of the women blood serum elemental profiles being more sensitive to physical loads than the whole blood ones. The intensive sport exercises were found to meaningfully increase the Ca and Co levels and evoke the K and Se depletion trends as verified by the blood serum levels profiling tests; whilst the whole blood tests showed only the Pb concentrations significantly increased (in the moderate-activity group), with some Ca decrease and Co growth trends.

The study data gives reasons to conclude that the Ca metabolism is more sensitive to physical loads – that apparently trigger disorders in homeostatic regulation of the concentration of this microelement in the blood; with some redistribution of Ca between blood corpuscles and blood serum towards the latter. Additional indirect evidence of the Ca-related metabolic process disorders is provided by the Pb level variations (Pb is an antagonistic element to Ca) in the whole blood – which is commonly acknowledged as the best bio-substrate for diagnostics of excessive Pb accumulations in the body – for instance, in case of saturnism (lead poisoning) [5, 6].

It is Co (as an important constituent of В₁₂ vitamin) that was found to be the second element most sensitive to physical loads, albeit it shows only one-way sensitivity (responding by increase only) as verified by the whole-blood and blood-serum Co profiling tests. This fact may be interpreted as indicative of Co being mobilized from some depot; or intensively produced in the В₁₂ vitamin synthesis by the intestine; or being taken by the subject athletes as a component of popular vitamin complexes.

It may be pertinent to stress the study finding as to the K and Se depletion trends under intensive physical loads (that may increase the risks of hypokaliemia and hyposelenic disease) that appear to be in agreement with the study data reported by other researchers [2]; and this depletion tend to increase the risks of cardiovascular and immune system disorders.

Table 2. Whole blood macro- and micro-elemental concentrations of the variably active subject female athletes, Me (q25 – q75)*, mcg/ ml

*Me means median, q25 means the low quartile, and q75 means the upper quartile

Table 3.Blood serum macro- and micro-elemental concentrations of the variably active subject female athletes, Me (q25 – q75)*, mcg/ ml

*Me means median, q25 means the low quartile, and q75 means the upper quartile

Conclusion. The study data demonstrates the blood serum elemental profiles being more sensitive to the female athletes’ workloads than that of the whole blood. It was demonstrated that the blood serum concentration variations of calcium, cobalt, potassium and selenium may be used as biomarkers of the workload intensities going beyond the body adaptive capabilities and, hence, as predictors of the growing risk of injuries and cardiovascular and immune system disorders. Therefore, the blood serum elemental profiling procedures may be recommended as beneficial for the professional athletes’ and sport activists’ health assessment and monitoring practices.

The study was performed under Project #544 within the frame of the base part of the State Order for Research and Development Studies at the premises of Yaroslavl State Technical University named after P.G. Demidov.

References

1. Alidzhanova I.E. Osobennosti elementnogo statusa laboratornykh zhivotnykh pri vozdeystvii razlichnykh vneshnikh faktorov (Element status features of laboratory animals when exposed to various external factors) / I.E. Alidzhanova, S.V. Notova, E.V. Kiyaeva // Tekhnologii zhivykh sistem. – 2009. – V. 6. – № 6. – P. 59-62.
2. Golubkina N.A. Selen v meditsine i ekologii (Selenium in medicine and ecology) / N.A. Golubkina, A.V. Skal'ny, Ya.A. Sokolov, L.F. Shchelkunov. – Moscow: KMK Scientific Press, 2002. – 134 p.
3. Zaytseva I.P. Aminokislotny i vitaminny profili syvorotki krovi studentok vuza, zanimayuschihsya sportom (Serum amino acids and vitamin profiles of female students engaged in sports) / I.P. Zaytseva, E.P. Serebryanskiy, M.G. Skal'naya, D.V. Kapustin // Vestnik vosstanovitel'noy meditsiny (Herald of regenerative medicine). – 2014. – № 2. – P. 62-65.
4. Zaytseva I.P. Vliyanie povyshennoy fizicheskoy nagruzki na soderzhanie makro- i mikroelementov v krovi sportsmenok – studentok VUZa (Macro- and microelements' concentration in the blood of female student-athletes under high-intensity physical load) / I.P. Zaytseva, J.N. Lobanova, A.V. Skal'ny, E.S. Berezkina // Voprosy biologicheskoy, meditsinskoy i farmatsevticheskoy khimii (Questions of Biological, Medical and Pharmaceutical Chemistry). – 2013. – №12. – P. 32–35.
5. Iordanskaya F.A. Diagnosticheskoe i prognosticheskoe znachenie mikroelementov krovi v monitoringe funktsional'noy podgotovlennosti vysokokvalifitsirovannykh sportsmenov: nauchno-metodicheskoe posobie (Diagnostic and prognostic value of blood trace elements in monitoring of functional fitness of elite athletes: scientific-methodical manual) / F.A. Iordanskaya, N.K. Tsepkova, S.V. Kryazheva. – Moscow: Skyprint, 2013. – 112 p.
6. Skal'ny A.V. Fiziologicheskie aspekty primeneniya makro- i mikroelementov v sporte (Physiological aspects of application of macro- and microelements in sport) / A.V. Skal'ny. – Orenburg: OSU PPC, 2005. – 210 p.
7. Toksikologicheskaya khimiya. Analiticheskaya toksikologiya (Toxicological Chemistry. Analytical Toxicology) / S.A. Eremin [et al.]. – Moscow: GEOTAR-Media, 2010. – 752 p.
8. Brаеtter P. Auswahl und Zugänglichkeit von Probenmaterialzur Bestiummung von Spurenelemente / P.Brаеtter// Vitamine, Spurenelemente und Mineralstoffe. Praevention und Therapiemit Mikronaehrstoffen/ Hrsbg.: H. K.Biesalski,J.Koehrle, K. Schuemann. – Stuttgart: Thieme, 2002. – S. 682–687.
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Corresponding author: irisha-zip@yandex.ru

Abstract
Objective of the study was to profile the sport physical loads in the female student-athletes versus the blood/ blood serum elemental compositions. Subject to the study were 43 female student-athletes of 18-22 years of age engaged in variable-intensity sports at Yaroslavl State Technical University named after P.G. Demidov. Nexion 300D+NWR213 (made by Perkin Elmer, the USA) Inductively Coupled Argon Plasma Mass Spectrometer (ICP-MS) was used to make elemental analysis of the bio-substrates. The test results of the two subject bio-substrates were compared to identify non-specific variations of mineral metabolism with the physical loads. The study data demonstrates the blood serum elemental profiles being more sensitive to the female athletes’ workloads than that of the whole blood. It was demonstrated that the blood serum concentration variations of calcium, cobalt, potassium and selenium may be used as biomarkers of the load intensities going beyond the body adaptive capabilities and, hence, as predictors of the growing risk of injuries and cardiovascular and immune system disorders. Therefore, the blood serum elemental profiling procedures may be recommended as beneficial for the professional athletes’ and sport activists’ health assessment and monitoring practices.