CIRCADIAN CHANGES IN ELECTROLYTE CON CENTRA TIONS IN PLASMA AND ERYTHROCYTES IN TWO-WEEK-OLD CALVES

Skotnicka E., W. F. Skrzypczak, M. Ozgo: Circadian Changes in Electrolyte Concentrations in Plasma and Erythrocytes of Two-week-old Calves. Acta vet. Brno 1997, 66: 141-146. The aim of this study was to determine changes in the content of Na, K, CI, Ca, Mg and P in the plasma and erythrocytes of 14-day-old calves with simultaneously performed evaluation of the occurrence and analysis of the rhythms. The studies were carried out on a group of 10 Black and White, clinically healthy female calves aged 14 days. Seven days before and during the experiment, the animals were kept in uniform environmental conditions (LD = 16:8). Blood samples were collected seven times over a period of 24 hours, spaced by 4 hour intervals, i. e., at 9:00, 13:00, 17:00, 21 :00, 1:00,5:00 and 9:00. Wbole blood and plasma were used in the analysis. In the whole blood, hematocrit and concentrations of Na, K, CI, Ca, Mg and P were determined. The plasma osmolality was also determined. The concentrations of sodium, potassium and chlorides in the erythrocytes were estimated using an indirect method. In the present study, the circadian variations in plasma concentrations of sodium. potassium and chlorides and in its osmolality were found in two-week-old female calves. Changes in plasma content of sodium and potassium within twenty-four hours were significant (P<O.Ol). On the other hand, calcium, magnesium and phosphorus concentrations in plasma and concentrations of sodium, potassium and chlOlides in erythrocytes were stable over the twenty-four hour period under study. Female, calves, rhythms, plasma, erythrocytes, electrolytes, plasma osmolality Biological rhythms are a manifestation of adaptation of living creatures to the environmental conditions, and enable them to maintain their homeostasis, increasing also chances for survival (Btinning 1967;Jilge 1993;Mick etaI.I994). A stable level of mineral components in the blood testifies to efficiency of internal organs, above all to efficiency of kidneys which are the main regulator of isoinia and neurohormonal maturity of regulatory mechanisms (K 0 0 p man et aI. 1985, L u k e et aI. 1990; L u k e et al. 199 I; M c C a a 1992). Functional immaturity of kidneys was observed in calves in the first days of life (S pit z e r 1985; S k r z y pc z a k 1991). The response of this organ to hormonal stimulation is also different than in adult animals, e. g. to aldosterone (C u gin i et al. 1991; Sa fw e teet aI. 1992; Skrzypczak et aI. 1994; Steele et aI. 1994). It results from earlier studies that kidneys of newborn calves do not show functional changes within the circadian rhythm, thus indicating immaturity of the main mechanism enabling adaptation to varying environmental conditions over a twenty-four-hour period (HelIbrtigge 1960; Wolf et al. 1991; Safwate et aI. 1992; Weinert 1994). Circadian rhythms in the functions of calves' kidneys become visible about 10-14 day of life (K e t z 1960; S k r z y p c z a k et al. 1992). In connection with the above it is important, both from practical and applicable point of view, to find whether in calves which passed the second week of life there are changes in the blood level of main electrolytes over twenty-four-hour period and whether such changes influence internal stability of the blood cells (erythrocytes).

Biological rhythms are a manifestation of adaptation of living creatures to the environmental conditions, and enable them to maintain their homeostasis, increasing also chances for survival (Btinning 1967;Jilge 1993;Mick etaI.I994).
A stable level of mineral components in the blood testifies to efficiency of internal organs, above all to efficiency of kidneys which are the main regulator of isoinia and neurohormonal maturity of regulatory mechanisms (K 0 0 p man et aI. 1985, L u k e et aI. 1990; L u k e et al. 199 I; M c C a a 1992).
Functional immaturity of kidneys was observed in calves in the first days of life (S pit z e r 1985; S k r z y pc z a k 1991).The response of this organ to hormonal stimulation is also different than in adult animals, e. g. to aldosterone (C u gin i et al. 1991; Sa fw e teet aI.1992; Skrzypczak et aI. 1994;Steele et aI. 1994).It results from earlier studies that kidneys of newborn calves do not show functional changes within the circadian rhythm, thus indicating immaturity of the main mechanism enabling adaptation to varying environmental conditions over a twenty-four-hour period (HelIbrtigge 1960;Wolf et al. 1991;Safwate et aI. 1992;Weinert 1994).Circadian rhythms in the functions of calves' kidneys become visible about 10-14 day of life (K e t z 1960; S k r z y p c z a k et al. 1992).
In connection with the above it is important, both from practical and applicable point of view, to find whether in calves which passed the second week of life there are changes in the blood level of main electrolytes over twenty-four-hour period and whether such changes influence internal stability of the blood cells (erythrocytes).

Animals
The studies were carried out on a group of 10 Black and White, clinically healthy female calves at the age of 14 days.
The animals were kept in individual boxes in uniform environmental conditions (LD=16:8) seven days before and during the studies.They were fed dams'milk (from a bucket), 8 I per day, two times a day, at about 6:30 and 17:30.They were also given free access to hay and water.
. Before the experiment began, the external jugular vein of animals was catheterized to enable blood ,ampling without stress.
Whole blood and plasma were used in the analysis.To determine the content of studied components in erythrocytes.whole blood was hemolyzed adding 9 ml of deionized water to I ml of blood.To obtain the plasma, the remaining part of the blood was centrifuged.The plasma and the hemolyzate were stored at (-20) °C until analyses were done.

Biochemical analysis
In the whole blood hematocrit was determined, and concentrations of the following mineral components in the plasma (using the kits Analco, Poland): -calcium (complexon method using o-cresolophtaleins); -magnesium (complex on method using calmagit); -inorganic phosphmus (molybdate method).
In the plasma and the hemolyzate, concentrations of following elements were measured: -sodium and potassium (name photometry method using a FJapho-40 photometer); -chlorides (potentiometric titration method using a Spexon-I 00 chlorimeter).
The plasma osmolality was also established (cryoscopic method using a Knauer osmometer).Concentration of sodium, potassium and chlorides in the erythrocytes were estimated using the indirect method based on the solution strength law, using hematocrit and the plasma and whole blood concentration of ions according to the following fmmula (J ani a k 1989): where: Cer = ion concentration in erythrocytes; Cpt = ion concentration in plasma; Cwo = ion concentration in whole blood; Ht = hematoclit.

Statistical analysis
The results were statistically evoluated using analysis of variance and Duncan's D-test (Statgraphics v. 5.0 software).The Chronos package was used to establish the occurrence of the rhythm cycles and acrophases.

Results and Discussion
The results obtained in the experiment indicate the occurrence of circadian changes in concentrations of sodium and potassium in the plasma of two-week-old calves (Table 1).
The concentration of sodium and potassium in the plasma of calves showed circadian variability with an acrophase for sodium at 20:08 and an acrophase for potassium at 8:35, The plasma osmolality and plasma chloride concentrations changed over a 24-hour period as well, but these changes were not significant.Similar results were obtained by Kanabrocki et al. (1973), They observed, while studying the circadian changes in the mineral composition of human blood (at LD 15:9) that the acrophase for sodium ions occurred between 19:00 and 20:00, whereas for potassium ions between 23:00 and 24:00, The highest concentration of chlorides was found by them at about 22:00, that is at the beginning of the dark phase.The observed changes in the Na, K and CI concentrations in the plasma of the studied calves were probably a result of the circadian periodicity in the function of kidneys (A i z man et al. 1983; Koopman et al. 1985;Wolf et al. 1991;Katinas et al. 1992;Safwate et al. 1992;Ai z m an et al. 1994).Distinct circadian changes in the activity of kidneys in young calves were observed by K e t z (1960), and by S k r z y p c z a k et al. (1992).It is wellknown that the activity of kidneys in the neonatal period shows great differences in comparison with the period offull somatic maturity.The blood flow through the kidneys is lower, as well as the level of glomerular filtration rate; the tubular resorption and secretion processes in tubules, particularly of water and electrolytes.are less efficient, and the excretability of excessive amounts of sodium and water is smaller (Dalton 1968; S krz y p c zak 1989; S krzy pc z ak 1991; Sa fw a te et al. 1992).The circadian rhythms in the activity of kidneys in calves begin to occur during the second week of the postnatal life in response to regular changes in the blood level of hormones controlling the function of kidneys and the activity of their receptors (I t 0 h et at.1985; Rob bill a rd et al. 1988;Ballauf etal.1991;Nielsen etal.1991;Wolf etal.1991:Skrzypczak etal.1992;Ruddy et al. 1993).
The findings of B aranow-B arano wski et al. (1980) indicate that a gradual increase in the activity of adrenal cortex occurs in the postnatal period.Similar results were obtained by Hart man n et al. (1987) and S pit z e r (1982).These authors are of the opinion that a rapid increase in the activity of the adrenal cortex after birth is due to a low supply of electrolytes (mainly Na) and to their increased need during the organisms development process.Skrzypczak (1991) showed that the mineralocorticoid regulation of Na and K excretion is different in calves when compared to adult animals.Other authors also point out lower efficiency of the RAA system in calves (H art m an n et al. 1987; S krzyp c z ak 1991 ).
S z c z epa Ii s k a et al. (1967) believe that the circadian rhythm of diuresis is due to blood fluctuations of vasopressine during a 24-hour period.These authors found that the circadian rhythm of the A VP level does not depend on the motoric activity phase as it occurs in the case of aldosterone secretion.Attention is called to consistence of diuresis fluctuations and the level of ADH in the blood with the course of changes in the excretion of electrolytes controlled by separate mechanisms (M c C a a 1992).
The increase in the excretion of sodium, potassium and chlorides is observed during the organism's active phase (Luke et al. 1991;Kemp et al. 1922;Ruddy et al. 1993; B 0 e m k e et al. 1994).It was shown, among other things, that the rhythm of potassium excretion in humans is consistent with the rhythm of glomerular filtration, the plasma flow through kidneys and the diuresis rhythm ( K 0 0 p man et al. 1989).
S k r z y p c z a k et al. (1992) in studies performed on two-week-old calves observed a higher resorption of sodium and chlorides in the activity period (day-light time), lower resorption during the night hours, and increased urine excretion of these electrolytes at night (despite the lowered glomerular filtration load).These authors state that high tubular resorption of potassium in the night hours contributes to a decrease in the urine excretion of this ion during that time of day.
The twelve-hours shift observed during the experiment in the phase of potassium rhythm in relation to the rhythm of sodium is confirmative for dependence between opposed tubular resorption of sodium and potassium.
In the studied calves, no circadian changes in the concentration of calcium, magnesium and phosphorus were observed, their plasma levels being stable (table 1).Mar k 0 wit z et al. (1984), however, when studying changes in the Ca, Mg and Pconcentrations in the blood of children, observed their circadian periodicity.S z y s z k a et al. (1992) in studies on circadian variability in the plasma concentration of magnesium in humans found sinusoidal changes of concentration with an acrophase in the evening hours and its decrease in the morning hours.They explain the lowest concentration of magnesium at that time of day by the peak in activity of catecholamines, aldosterone and ACTH.In the present experiment, the concentrations of sodium, potassium and chlorides were stable in the erytrocytes, despite the circadian changes in their plasma (Table 2).
These results confirm the ability of red cells to preserve homeostasis of the cell interior and indicate that cellular membranes of the erythrocytes are in possession of a mechanism securing the integrity of intracellular environment with physiological fluctuations in the plasma levels of electrolytes.
In conclusion: 1.The circadian variations in the plasma concentration of sodium and potassium were found in two-week-old calves.These changes were significant (P<O,OI).2. No significant changes were found in the chloride, calcium, magnesium and phosphorus concentration in the plasma, plasma osmolality and erythrocytes sodium, potassium and chloride concentration.3. Physiological changes in the plasma concentration of sodium and potassium do not affect the electrolyte homeostasis of erythrocytes.

Table I
Circadian changes of plasma Na, K, CI, Ca, Mg and P concentrations and plasma osmolality in examined calves (n=lO, x ± SD)

Table 2
Circadian changes of erythrocytes Na, K, Cl concentrations in examined calves