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Chronobiol Med > Volume 5(1); 2023 > Article
Babayan, Blagonravov, Hayrapetyan, Chibisov, Sarafyan, Gulyan, Danoyan, Danoyan, Tavaracyan, Gasparyan, Atoyan, Kostanyan, and Gabrielyan: Alterations of Water-Mineral Homeostatic System in Rabbits Under the Action of Stressor Factors


This study aimed to examine the alteration of circadian structures of electrolytes, trace elements, and white blood cell homeostasis under the action of stressors factors. Male rabbits were used in experimental work. Four-hour urine and blood specimens were collected over a span of 48–72 hours. Total Na (sodium), K (potassium), Ca (calcium), Mg (magnesium), Cu (copper), and Zn (zinc) were analyzed on atomic absorption spectrophotometer. Rhythm’s parameters have been estimated by the nonlinear least square method for sinusoidal rhythms and by dispersion analysis for nonsinusoidal rhythms. Intact rabbits’ chronoperiodical systems of water-mineral and white blood cell homeostasis were characterized by circadian structures. Acrophases of indices of water-mineral and white blood cell homeostasis in intact rabbits mostly had individual nature. Temporal structure of water-mineral and white blood cell homeostasis in rabbits under the action of stress were characterized by infradian rhythms or nonperiodical oscillations.


During the last years, interest in the role of electrolytes and trace elements in biological systems had increased [1-4]. The results of these investigations were contradictory. It was partly connected with the absence of unification methods, patient grouping, and normal values. Besides, these investigations were mostly carried out without taking into account the temporal structure of an organism. From the point of system approach, the problem of stress is closely connected with an individual reaction of every organism to factors of stress [5]. Chronobiological research indicated that many pathological functions are accompanied by disorders of chronoperiodical systems of an organism [5-17].
At the same time biorhythmological and the system approaches have not yet been applied to the stress complexly. The present study was undertaken for the purpose of examining the alteration of chronoperiodical system of water-mineral and white blood cell (WBC) homeostasis under the action of stressors factors.


Male rabbits (chinchilla breed) were used for the experimental work [18]. The first series of experiments was performed on intact animals (20 rabbits, the mass of 2608.0±96.0 g) and 25 rabbits were used in the second series of experiments with the mass of 2600.0±87.0 g. The rabbits (second series) were subjected to stress influence, daily 1.0–1.5-hour immobilization on the special mount, with their limbs exposed to the effects of electric stimulation (current frequency 100 Hz, current intensity 5–20 mA). The experiment lasted two weeks. Four-hour urine and blood specimens were collected for 48–72 hours over a span (in the second series these procedures started to be followed on the 12th day of the experimental work).
An assessment of the action of hypothalamo-hypophyseal-suprarenal system was made on the basis of the growth of the mesor and amplitude of the blood corticosterone rhythm. The quantitative determination of corticosterone contained in blood plasma was based on the radioimmunological analysis (RSLRAT Corticosterone, 3H) Kit (ICN Biomedical, RF). Total Na (sodium), K (potassium), Ca (calcium), Mg (magnesium), Cu (copper), and Zn (zinc) were analyzed on an atomic absorption spectrophotometer (Perkin-Elmer, USA). The distribution of WBCs was analyzed by examining the blood smear under the microscope. Rhythm’s parameters were estimated by dispersion analysis for nonsinusoidal rhythms and by nonlinear least squares method for sinusoidal rhythms [7,11,12,19].
The rhythms were grouped in accordance with the glossary of chronobiology which was subjected to some changes [12,19-21]. The rhythms with periods ranging from 3 to 20, 20 to 28, and 28 to 96 hours were considered to be ultradian, circadian, and infradian, respectively.
All procedures were performed in accordance with the ethical standards of the research committee and were implemented in accordance with the Declaration of Helsinki.


The results showed that the majority of the intact animals were characterized by circadian rhythms of water-mineral homeostasis and WBCs. The rhythms had a certain value of mesors and amplitudes.
Acrophases of the rhythms had an individual character and, at the same time, we can notice that there were 6–12 hour differences between acrophases of the same indices of water-mineral homeostasis in the plasma of blood and urine. Under the influence of stressors factors, the water-mineral and WBC systems reorganized a temporal structure of the functions of its indices which were characterized in a transformation of circadian rhythms into nonperiodical oscillations or in a formation of mainly infradian fluctuations (for blood and urine indices 39%–34% and 43%– 31%, respectively, and for WBCs 60%–20%). Circa- and ultradian rhythms respectively made 13% and 14%, 15% and 11% (Figure 1).
The rhythms of corticosterone had infradian period in all cases and had been statistically significant (Table 1). The rhythms of blood eosinophils had ultradian period. The corticosterone mesor and amplitude were higher in comparison to intact rabbits (Table 1). Mesor and amplitude of blood lymphocytes were statistically significantly higher (p<0.001) in comparison to intact rabbits.
Mesors of blood neutrophils and eosinophils were statistically significantly smaller (p<0.01) in comparison with the results of the intact rabbits. The results showed that mesors of Na, Cu, and Zn excretion rhythms were statistically significantly higher (p< 0.01) in comparison with the data of intact rabbits.
Mesor and amplitude of Ca excretion were statistically significantly smaller in comparison with the result of intact rabbits (Table 1).


Our data indicated that in intact rabbits, temporal organization of the electrolytes, trace elements, and WBS homeostasis were sinusoidal and circadian (Figure 1). This data witnessed about an internal synchronization by periods of rhythms which assured the relative stationarity of chronoperiodical system. That synchronization was inherent to the ordinary functions of the organism [10-14,17,22]. There was a relatively constant concentration of electrolytes and trace elements in extra- and intracellular space of an organism. Comparing the data of literature with our results, we can suppose that new neuroendocrine status of organism reorganizes the circadian chronostructure of water-mineral excretion for preservation of relative stationary of the macro- and microelements composition in the internal space at the stress.
The research results gave reason to consider that the complex of reactions of water-mineral chronoperiodical system forms in organism which is a defense reaction to effects of pathological factors. Their essence was a reorganization of circadian rhythms of the chronoperiodical system. In different components of water-mineral chronoperiodical system the reorganization had different character. The chronostructure of water-mineral homeostasis of blood was characterized by only a periods’ and amplitudes’ changes. Efferent components (urinary excretion of indices of water-mineral homeostasis) were characterized by changes of periods, mesors, and amplitudes. Logically, one could suppose that due to the excessive liability of parameters of the temporal structure of an efferent component of the water-mineral system, the constancy was preserved for mesors of electrolytes and trace elements homeostasis of blood. That was the constancy of rhythms of the indices of blood water mineral homeostasis and the high degree of lability of parameters of rhythms of the executive apparatus. That makes a water-mineral chronoperiodical system a precise mechanism providing stability of water-mineral homeostasis of an organism under the influence of pathological factors on the basis of a self-regulation principle.
We were unable to compare our data with the results of other authors since similar investigations in intact animals at the stress have not been found in available literature.
Chronic stress results in elevated levels of glucocorticoids via activation of the hypothalamic-pituitary-adrenal axis and high levels of glucocorticoids [23]. The hormones adrenalin, noradrenalin, and glucocorticoids, typically associated with a stress response, exert diverse effects on leukocytes [23,24].
The academic examination stress induces changes in the distribution of peripheral blood mononuclear cells [25]. Endogenous cortisol was an important factor in the regulation of lymphocyte numbers. Lymphocyte levels and plasma cortisol concentrations of healthy subjects had circadian variation that were inversely related [26]. Several authors had reviewed the effects of stress on lymphocyte activity [27]. Our results showed that at the stress, mesors of corticosterone and lymphocytes were statistically significantly higher than in the intact animals.
In rabbits (intact and at stress) acrophases of indices of watermineral homeostasis and WBCs mostly had individual nature. However, our data witnessed that in every intact rabbit there was a 1–6 hour difference between acrophases of corticosterone and lymphocyte. In every rabbit under the action of stressor factors, acrophase of lymphocyte was in antiphase of the acrophase of corticosterone. Probably, our data indicated the direct influence on chronoperiodical systems of organism by the stressors factors, and internal synchronization between parameters of the rhythms were transformed to desynchronization.
The infra-, circa-, and ultradian rhythms which we have determined and also statistically nonsignificant oscillations give us a reason to consider individual reactions of water-mineral and WBC chronoperiodical systems of the animals under the action of stressors factors.


Funding Statement


Conflicts of Interest

The authors have no potential conflicts of interest to disclose.

Availability of Data and Material

The datasets generated or analyzed during the study are available from the corresponding author on reasonable request.

Author Contributions

Conceptualization: Lyusya A. Babayan, Hamlet G. Hayrapetyan. Data curation: Lyusya A. Babayan. Formal analysis: Arman B. Danoyan, Haykaz E. Danoyan. Investigation: Lyusya A. Babayan, Michail L. Blagonravov, Hamlet G. Hayrapetyan, Sergey M. Chibisov. Methodology: Haykaz E. Danoyan. Project administration: Lyusya A. Babayan. Resources: Lyusya A. Babayan. Supervision: Lyusya A. Babayan. Validation: Lyusya A. Babayan. Visualization: Ani R. Tavaracyan, Narine A. Gasparyan, Naira Kh. Atoyan, Hasmik L. Kostanyan, Margarita T. Gabrielyan. Writing—originaldraft: Lyusya A. Babayan. Writing—review & editing: Pargev K. Sarafyan, Ara K. Gulyan, Arman B. Danoyan, Haykaz E. Danoyan, Ani R. Tavaracyan, Narine A. Gasparyan, Naira Kh. Atoyan, Hasmik L. Kostanyan, Margarita T. Gabrielyan.

Figure 1.
Summary data of distribution (%) of ultradian rhythm (UR), circadian rhythm (CR), and infradian (IR) rhythm of statistically significant temporal organizations of water-mineral homeostasis. IR1B, blood index in intact rabbits; IR1U, urine index in intact rabbits; SRB, blood index at stress; SRU, urine index at stress; SNR, statistically nonsignificant rhythms.
Table 1.
Mesors (M) and amplitudes (A) of plasms (1), erythrocytes (2), and urinary (3) excretion of the electrolytes and trace elements and ultra- (U), circa- (C), and infradian (I) distribution (%) of the statistically significant rhythms (S) at stress
Indices Biological materials S (%) U (%) C (%) I (%) M±SE A±SE
Volume of urine (3) 50 0 25 75 226.9±7.7 201.0±3.1
Na (1) 50 40 0 60 137.9±1.9 6.3±1.25
(2) 40 100 0 0 25.9±1.0 3.6±0.7
(3) 50 25 25 50 4.22±0.51*** 2.51±0.5
K (1) 70 29 57 14 4.4±0.3 0.52±0.05
(2) 40 50 0 50 82.3±3.4 4.7±1.0
(3) 75 33 50 17 16.53±2.69 10.38±2.69*
Coefficient Na/K (1) 70 14 43 43 32.8±1.98 5.3±0.59
(2) 40 50 50 0 0.3±0.01 0. 5±0.001
(3) 63 0 40 60 0.27±0.05 0.19±0.05
Ca (1) 80 0 0 100 2.2±0.22 0.4±0.03
(3) 63 20 20 60 34.2±1.53*** 24.6±4.61***
Mg (1) 60 33 0 67 1.3±0.09 0.2±0.02**
(3) 50 0 25 75 65.39±11.5 53.8±15.3
Cu (1) 70 14 43 43 11.3±1.3 2.8±0.2*
(3) 50 0 25 75 0.17±0.02** 0.12±0.03
Zn (1) 60 17 0 83 24.5±0.7 5.6±0.6
(3) 62 60 0 40 2.12±0.22** 1.49±0.32
Corticosterone (1) 70 0 0 100 33.2±2.2** 31.9±2.3**

* p<0.05;

** p<0.01;

*** p<0.001; they were calculated comparably with data of the intact animals.

Mesors and amplitudes were calculated by 100 mg mass of the body the following units: volume of urine, mL/h; Na (sodium)—(1, 2) mmol/L, (3) μmol/h; K (potassium)—(1, 2) mmol/L, (3) μmol/h; Ca (calcium)—(1) mmol/L, (3) nmol/h; Mg (magnesium)—(1) mmol/L, (3) nmol/h; Cu (copper)—(1) μmol/L, (3) nmol/h; Zn (zinc)— (1) μmol/L, (3) nmol/h; corticosterone, nmol/L.


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