Vereshchagina S.V., Fokin V.A., Smirnova I.N., Abdulkina N.G. Laboratory markers in predicting the effectiveness of velvet antler medication in athletes

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Publication date: 01.12.2021
DOI: 10.51871/2588-0500_2021_05_04_2
UDC 615.3; 616-092.19

LABORATORY MARKERS IN PREDICTING THE EFFECTIVENESS OF VELVET ANTLER MEDICATION IN ATHLETES

S.V. Vereshchagina^1,2, V.A. Fokin³, I.N. Smirnova², N.G. Abdulkina²

¹Federal State Budgetary Institution "Federal Siberian Scientific and Clinical Center of the Federal Medical and Biological Agency", Krasnoyarsk, Russia

²Federal State Budgetary Institution "Siberian Federal Scientific and Clinical Center of the Federal Medical and Biological Agency", closed city Seversk, Russia

³Federal State Budgetary Educational Institution of Higher Education "Siberian State Medical University" of the Ministry of Health of the Russian Federation, Tomsk, Russia

Key words: athletes, maral velvet antlers, efficiency prediction.  

Annotation. The purpose of the study is to develop models for predicting the effectiveness of velvet antler medication in athletes of winter cyclic sports during the preparatory period of the annual training cycle. In order to justify the use of laboratory markers, a correlation analysis of the relationships between them and indicators of physical performance was carried out. Based on the stepwise discriminant analysis, mathematical models represented by a set of linear discriminant functions that determine the feasibility of prescribing velvet antler preparations have been developed. The highest diagnostic interest was obtained when using biochemical parameters.

Introduction. Against the background of intense training loads, signs of fatigue are registered in 40-60% elite athletes, which makes a substantial limiting effect on the effectiveness of training process and achieving records in sports [1-4]. Since currently there is no unified and generally accepted hypothesis on the development of the overtraining syndrome, the key moment in diagnosing this multi-factor pathological state is an assessment of laboratory markers, i.e. biochemical, neurohumoral and immunological indicators [5-6].

At the moment, there is a high need of developing new non-drug means of recovery, especially natural preparations that are able to increase physical performance of athletes, improve adaptation to extreme factors of sports activity and which at the same time do not belong to doping [7-8]. One of the high-potential means of the non-drug support of the organism against the background of intense physical loads are products of the velvet antler industry, which use in sports is justified and safe, and it was approved by the expert analysis from the All-Russian Scientific Research Center of Physical Culture and Sports № 12-5590-S (1996) [8-10]. Moreover, there is no information on a possibility to predict effectiveness of its use in athletes.

The purpose of this study is to develop models for predicting the effectiveness of velvet antler medication in athletes of winter cyclic sports during the preparatory period of the annual training cycle.

Methods and organization.  The study was carried out in the Federal Siberian Scientific and Clinical Center of the Federal Medical and Biological Agency (Krasnoyarsk) and the Tomsk Scientific and Clinical Center of Balneology and Physical Therapy, branch of the Siberian Federal Scientific and Clinical Center of the Federal Medical and Biological Agency (Tomsk) within the State Contract № 44.001.11.14 of the “Biomedical and Medicosanitary Support of Athletes of the Russian Federation National Teams” targeted program of the FMBA of Russia.

Selection of athletes of winter cyclic sports is made in the Regional Center of Sports Training “Academy of Winter Sports” (Krasnoyarsk) and the “Biathlon Academy (Krasnoyarsk). At the clinical stage of the study, we examined 86 athletes of winter cyclic sports (ski racing, biathlon), aged 18-30 years (average age is 21,90±4,19 years). Athletes of the main groups I (n=30) and II (n=30) received maral velvet antler powder in capsules at doses of 2-4 g/day, athletes of the control group III (n=26) received only placebo (powdered sugar in same capsules) and no other nutritional supplements. Administration of the powder and placebo took place at the preparatory stage; the intake course took 14 days. Clinical, laboratory and instrumental examinations of athletes were carried out before and after the intake course of the powder during the preparatory (September-October) period of the annual training cycle.

Hematological analysis was carried out with the МЕК 7222 hematological analyzer (Nihon Kohden, Japan). According to the leukogram results, we calculated the lymphocyte index (LI) and leukocyte index of intoxication (LII) according to the formula suggested by V.K. Ostrovskij et al. (2011). The biochemical indicators were identified with the AU-400 automated analyzer (Beckman Coulter, USA) and following methods: urea – uricase colorimetric method, glucose – hexokinase method, alanine transaminase (ALT) and aspartate transaminase (AST) –
UV-kinetic method, alkaline phosphatase – colorimetric method with p-nitrophenylphosphate, creatine kinase-MB – method of enzymatic inhibition with phosphocreatine, lactate – colorimetric lactone oxidase method, iron – colorimetric method with TPTZ, latent and total iron-binding capacity (TIBC) – colorimetric method with Nitroso-PSAP, total cholesterol (TC) and fractions – colorimetric method,  triglycerides – enzymatic colorimetric method, c-reactive protein – immunoturbidimetric method. Functioning of the lipid peroxidation system was studied with identification of following parameters: activity of extracellular catalase – colorimetric method, level of the malondialdehyde (MDA) – color reaction method with 2-thiobarbituric acid, level of ceruloplasmin – method with p-phenylenediamine. In order to conduct the aforementioned methods, we used the Photometer 5010 (Robert Riele GmbH, Germany). To identify the testosterone and cortisol level, we performed the immunochemiluminiscent test on the Immulite 1000 analyzer (USA).

We studied the humoral link of the systemic immunity with identification of a level of А, М, G class immunoglobulins (Ig) in blood serum, using the immunoturbidimetric method on the AU-400 biochemical analyzer (Beckman Coulter, USA). Content of circulating immune complexes (CIC) in blood serum was studied in the precipitation reaction with a polyethylene glycol solution. The phagocytic link of the immune system was evaluated with the method of V.M. Berman and E.M. Slavskaya (1958) with the S. Aureus (strain 209) testing culture. The metabolic activity of phagocytes was defined according to their ability to recover nitro blue tetrazolium (NBT) in the spontaneous and stimulated NBT test. The cytokine status was evaluated with the immunoenzymatic method with the Stat Fax 303 Plus® Microstrip Reader (Awareness Technology, USA) and “VektorBest” sets (Russia).

In order to assess physical fitness, we conducted a multi-stage loading test on the ERG-911BP stationary bicycle (Shiller, Switzerland). In the course of the bicycle ergometry (BEM), we assessed resistance to physical loads according to maximum (in W) and threshold capacity (W/kg) and physical performance according to the PWC170 test.

In order to statistically process the obtained data, we used the Statistica 8.0 software. Test for standard distribution was carried out using the Shapiro-Wilk test. In case of non-standard distribution, the data was presented in the form of “mean±standard derivation” (М±SD). For description, we used median (Me) and interquartile range in the form of Ме[LQ;UQ], where LQ – lower quartile, UQ – upper quartile. To define significance of differences between dependent samples, we used the Wilcoxon’s T-test, for independent samples – the Mann-Whitney U-test. We used the χ²-test and the Fisher’s exact test to define statistical significance of nominal signs’ prevalence. Correlation between variables was identified with the Spearman’s test (R). Building a model to predict effectiveness of velvet antler preparations was made using the stepwise discriminant analysis based on the Wilks' lambda and Mahalanobis distance. Statistical significance of the model was defined according to the Wilks' lambda. A critical significance level when evaluating statistical hypotheses was equal to 0,05.

Results and discussion. At the first stage of the study, we conducted the correlation analysis of relationships between examined indicators, which characterize a degree of endogenic intoxication and overstrain of adaptation mechanisms, and the indicators of physical performance. As the analysis’ results show, the level of physical performance that is identified with the PWC170 test had positive relationships of average force with content of hemoglobin, hematocrit and red blood cells, as well as a level of ferritin in blood as the main iron reserve needed when performing physical loads. The revealed negative correlation between levels of the TIBC of serum and the PWC170 test approves a presence of the aforementioned dependence. Content of uria in blood and malondialdehyde as indicators of endogenic intoxication affected physical performance (table 1).

Table 1

Correlations between indicators of physical performance and laboratory indicators characterizing the degree of endogenic intoxication and overstrain of adaptation mechanisms in athletes

Indicators	R	р
PWС170 & TIBC of serum	-0,376	0,009
PWС170 & TNF-α	-0,288	0,049
PWС170 & IL-1β	-0,441	0,001
PWC170 & cortisol	-0,465	0,001
PWC170 & NBTstim	0,390	0,006
PWC170 & PhIspont	0,298	0,042
PWC170 & LII	-0,445	0,002
PWC170 & hemoglobin	0,341	0,019
PWC170 & hematoctit	0,388	0,007
PWC170 & red blood cells	0,290	0,047
PWC170 & ferritin	0,292	0,046
PWC170 & malondialdehyde	-0,344	0,017
PWC170 & anabolism index	0,419	0,003
Threshold Capacity & testosterone	0,315	0,030
Threshold Capacity & urea	-0,401	0,005
Maximum Capacity & LI	-0,289	0,039
Double product (DP) & LII	-0,216	0,043

The revealed negative correlations of average force between indicators of physical performance and levels of inflammatory cytokines TNF-α and IL-1β, as well as a degree of functional activity of leukocytes (NBTstim and phagocytic index PhIspont), emphasize a significant level of immune disturbances in a development of the overtraining syndrome and a need to analyze immunological indicators when training athletes.

The most informative were the revealed correlations between indicators of physical performance and hormonal parameters that characterize the activity of catabolic and anabolic processes when loads are applied (cortison, testosterone and anabolism index), which corresponds with earlier studies [11].

The revealed negative correlations between leukocyte indices of endogenic intoxication (LII, LI) and a level of physical performance are of interest. They allow considering these simple estimate indicators defined in case of routine clinical blood test as informative for early diagnostics of the overtraining syndrome in athletes (table 2).

Table 2

Correlations between leukocyte indices of intoxication and parameters of the endocrine and metabolic state and physical performance in athletes

Indicators	R	р
LII & PWС 170	-0,445	0,002
LI & Maximum Capacity	-0,289	0,039
LII & DP	-0,216	0,043
LII & eosinophils	-0,401	<0,0001
LII & ceruloplasmin	0,346	0,001
LII & malondialdehyde	0,384	0,004
LII & testosterone	-0,217	0,048
LII & erythropoietin	-0,209	0,046
LII & PhIstim	0,284	0,038
LII & TNF-α	0,366	0,032
LII & (ALT)	0,224	0,041

In our study, both LI and LII appeared to be closely correlated with separate biochemical indicators of endogenic intoxication (MDA, ceruloplasmin, ALT) and indicators of immune reactivity (TNF-α, PhIstim). The revealed negative correlation between a degree of endogenic intoxication according to the LII index and a level of erythropoietin is of special interest, which indicates a reduced oxygen transfer activity of blood in case of developing endogenic intoxication against the background of loads.

The obtained results demonstrated that using leukocyte indices showing interaction between different subclasses of leukocytes allows giving an additional information on a degree of endogenic intoxication and a state of the adaptation system and immune response in athletes.

To predict the expected effectiveness of using the velvet antler powder in athletes, we carried out the stepwise discriminant analysis, the result of which was a development of mathematical models presented by the set of linear discriminant functions that determine the feasibility of prescribing velvet antler preparations. Decision rules (discriminant functions) were linear classificatory functions of the following type:

d_j(x₁,x₂,…,x_n) = b_0,j + b_1,jx₁ + b_2,jx₂  + … + b_n,jx_n,

where d_j is a linear discriminant function for the j-subgroup; b_0,j – a constant for the j linear discriminant function; b_1,j, b_2,j, …, b_n,j – coefficients for x₁, x₂, …, x_n in j linear discriminant function; x₁, x₂, …, x_n – signs’ values of a classified patient.

As a learning sample, we defined two groups of athletes, in which an increase of physical performance occurred as a response to intake of the velvet antler powder in the first case, there was no effect in the second case. As a criterion of the influence’s effectiveness, we chose an increase of the PWC170 and threshold capacity indicators by 15% regarding initial values: if it is higher than 15%, then the effect of using the powder is present, if it is less than 15%, then there is no effect.  

The classification was performed in a following way: the U1 coefficient characterized a group of athletes with ineffective use of the powder, the U2 coefficient – athletes with an effective use of the powder. A possibility of achieving the result was evaluated as follows: if U1>U2, then using the powder would be ineffective, if U1<U2, then it would be effective.

We conducted the stepwise discriminant analysis for each group of indicators:

1. Hematological: hemoglobin, hematocrit, red blood cells, leukocytes, neutrophils, lymphocytes, eosinophils, estimate indices of endogenic intoxication, i.e. LII and LI.
2. Biochemical: urea, glucose, creatine kinase (CPK), creatine kinase-MB (CPK-MB), lactate, iron, cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), atherogenic index (AI), catalase, MDA, testosterone, cortison, anabolism index, erythropoietin, ferritin.
3. Immunological: PhIspont, PhIstim, NBTspont, NBTstim, IgA, IgМ, IgG, TNF-α, IL-1β, IL-4, IL-6, C-reactive protein.

As a result of using the stepwise discriminant analysis, we created rules for classification from 9 initial hematological indicators, which include 3 most informative ones. The use of them allows predicting the powder’s effect with a probability of 93,8% (F (2,13) = 14,958; p= 0,004).  

Taking into account the obtained data, discriminant functions for predicting the effectiveness of using the powder according to hematological indicators had the following form:  

U1 (no effect) = -153,35 - 1,09*NE + 70, 41*RBC + 68,64* LII

U2 (present effect) = -125,51 - 0,54*NE + 58,54* RBC + 64,01* LII,

where U – linear discriminant function; NE – a percentage of neutrophils according to the data from leukocyte formula; RBC – a number of red blood cells; LII – leukocyte index of intoxication.

The prediction rule (discriminant functions) according to biochemical indicators took the following form (F(7,8)=5,671; p=0,013, percentage of correct prediction 98,3%):

U1 (no effect) = -76,25+0,24*cor + 2,00*AI + 6,05*LDL + 4,32*CPC-MB - 6,56*urea -1,12*lact

U2 (present effect) = -76,31 + 0,224*cor - 5,06*AI + 14,69*LDL + 7,02* CPC-MB -12,94*urea - 5,54* lact,

where U – linear discriminant function; cor – level of cortisol in blood; AI – atherogenic index; LDL – level of low-density lipoproteins; CPC-MB – level of creatine kinase-MB; urea – level of urea; lact – level of lactate.

From 12 initial immunological indicators, we chose 4 most informative ones, using which allows predicting the powder’s effect with a possibility of 87,5% (F (4,11)=6,103; p=0,042). 

Considering the data obtained, the discriminant functions for predicting the effectiveness took the following form:

U1 (no effect) = -59,97 + 0,14*NBTspont - 1,45* IgM + 4,83* IL6 + 7,49* IgG

U2 (present effect) = -54,83 - 0,03* NBTspont + 1,80* IgM + 8,79* IL-6 + 6,71* IgG,

where U – linear discriminant function; NBTspont – values of the NBT spontaneous test; IgM – level of immunoglobulin M; IL-6 – level of interleukin-6; IgG – level of immunoglobulin G.

Conclusion. The conducted analysis justifies the feasibility of identifying laboratory markers in early diagnostics of fatigue and reduced physical performance in athletes. It was established that leukocyte indices of endogenic intoxication are closely correlated with indicators of physical performance, lipid peroxidation, level of testosterone and erythropoietin and can be used as markers of developing fatigue in athletes.

The effectiveness of using the velvet antler powder as a mean to correct fatigue and overtraining in athletes of winter cyclic sports during the preparatory period can be predicted using the developed models presented by the set of linear discriminant functions defining the feasibility of prescribing velvet antler preparations. The highest diagnostic interest of predicting the powder’s effect was obtained when using biochemical indicators (cortisol, atherogenic index, low-density lipoproteins, creatine kinase-MB, urea and lactate).

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Information about the authors: Svetlana Viktorovna Vereshchagina – Head of the Laboratory Diagnostics Department of the FSBI FNCC FMBA of Russia, Krasnoyarsk, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.; Vasilij Aleksandrovich Fokin – Doctor of Medical Sciences, Professor of the Department of Medical and Biological Cybernetics of the FSBEI of HE SibSMU MOH Russia, Tomsk, е-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.; Irina Nikolaevna Smirnova –  Doctor of Medical Sciences, Head of the Therapeutic Division of the Department of Prevention and Restorative Treatment of Occupational Diseases of the Branch of the TSRIBiP FSBI SibFSCC of FMBA of Russia, Seversk, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.; Natal’ya Gennad’evna Abdulkina – Doctor of Medical Sciences, Deputy General Director for Scientific and Clinical Work of the FSBI SibFSCC of FMBA of Russia, Seversk, e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it..