**7.5. BE, BB, and saturation of hemoglobin by O2**

Significant LD differences in total buffer bases (BB) and base excess (BE) were found in K/X and Z anesthesias (**Table 4**). BB moves from 40 to 60 mmol/l in all types of anesthesia and the BE from −8 to +12 mmol/l in both light parts of the rat regimen day under all types of anesthesia. Saturation of hemoglobin by oxygen was practically the same in all types of general anesthesia, and significant LD differences were not found except for K/X anesthesia, with higher saturation in the dark part of the rat regimen day. Significant differences of acid-base parameters between the single type of anesthesias are summarized in (**Table 5**).

**Figure 4.** pO2 in the light (yellow columns) and dark (blue columns) parts of rat regimen day in pentobarbital (P)-, ketamine/xylazine (K/X)- and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001 were considered to be a statistically significant difference between individual types of anesthesia. Red dashed lines represent ranges reported in **Table 1**.

**Figure 5.** HCO3 − in the light (yellow columns) and dark (blue columns) periods in pentobarbital (P)-, ketamine/xylazine (K/X) and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*\*p < 0.01 and \*\*\*p < 0.001 were considered to be a statistically significant difference between individual types of anesthesia. Red dotted lines represent the ranges reported **Table 1**.

Under this type of anesthesia, the mean value was significantly higher (p < 0.001) compared with both P and Z anesthesias in both light parts of the day (**Figure 7**). Moderate hyperkalemia

\*\*p < 0.01 statistically significant differences between the light and dark periods. P – pentobarbital P; K/X – ketamine/

**Ion P-light P-dark K/X-light K/X-dark Z-light Z-dark** *Na+* 145.08 ± 2.13 143.24 ± 1.7 140 ± 6.74 134.17 ± 5.56 133.97 ± 16.06 140.8 ± 7.67 *K+* 4.69 ± 0.31 4.91 ± 0.30 6.81 ± 1.42 8.85 ± 1.31\*\* 5.00 ± 0.71 4.68 ± 0.50 *Ca2+* 1.31 ± 0.05 1.33 ± 0.05 2.14 ± 0.07 — 0.99 ± 0.44 1.00 ± 0.38 *Cl<sup>−</sup>* 100.1 ± 1.21 100.51 ± 2.43 110.2 ± 2.39 — 104.8 ± 5.19 101.1 ± 5.1\*

(**Table 6**). Under P and Z anesthesias, plasma concentrations of Ca2+ were practically the same. In the light part of the day under K/X anesthesia, there was a significantly (p < 0.001) higher Ca2+ concentration versus P and Z anesthesias. In the dark part of the day under K/X anesthesia, the values were out of range of the ABL 800 Flex ion analyzer (**Figure 8**). Although significant differences were found between the different types of anesthesia in both light parts of the day, the animals were in relatively severe state of hypocalcemia,

Z anesthesia (**Table 6**). Hypochloremia occurred under P anesthesia in both light parts of the rat regimen day. Normochloremia to hyperchloremia occurred under both K/X and Z anesthesias in both light parts of the rat regimen day (**Figure 9**). In the dark part of the day under K/X anesthesia, the values were out of the detection range of the ABL 800 Flex ion analyzer. Significant differences in ion concentrations between the single type of anesthesias are summarized in (**Table 7**).

(P)-anesthetized, ketamine/xylazine (K/X)-anesthetized, and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*p < 0.05 and \*\*p < 0.01 were considered to be a statistically significant difference between individual types of anesthesia.

, no significant LD differences in plasma Ca2+ concentrations were found

chloride anions.

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in the light (yellow columns) and dark (blue columns) periods in pentobarbital

was found only under

was detected under P and Z anesthesias in both light parts of rat regimen day.

xylazine Z – zoletil; Na+ − sodium, K<sup>+</sup> − potassium, Ca2+ − calcium and Cl−

**Table 6.** Ion concentrations in arterial blood under individual types of anesthesia.

A significant (i.e., p < 0.05) LD difference in plasma concentrations of Cl−

Similar to Na+

Data presented as mean ± SD.\*p < 0.05,

especially when under P and Z anesthesias.

**Figure 6.** Plasma concentration of Na+

Red dashed lines represent ranges reported in **Table 2**.

#### **7.6. Ions**

LD differences for plasma Na<sup>+</sup> concentration were not detected under any of the selected general anesthesias (**Table 6**). The highest Na+ concentrations were under P anesthesia in the both light parts of the rat regimen day (light P vs. K/X, p < 0.01; P vs. Z, p < 0.01; dark P vs. K/X, p < 0.01; and nonsignificantly higher compared with Z anesthesia). In the light part of the day, the highest plasma concentration of Na+ was recorded under P anesthesia and the lowest concentration in Z anesthesia but with increasing dispersion of values. Based on our findings, it appears probable that the distribution of Na+ ions is significantly influenced by Z anesthesia (**Figure 6**). Under P anesthesia, regardless of the light or dark part of the day, hypernatremia was detected. In K/X and Z anesthesia, mean plasma Na+ concentrations moved from hyponatremic to hypernatremic.

Significant (i.e., p < 0.01) LD differences in plasma K<sup>+</sup> concentration were found only under K/X anesthesia, with higher values during the dark part of the rat regimen day (**Table 6**).


Bold values indicate statistically significant differences. P – pentobarbital P; K/X – ketamine/xylazine Z – zoletil; pCO<sup>2</sup> (kPa) – partial pressure of carbon dioxide; pO2 (kPa) – partial pressure of oxygen; HCO3 -(mmol/l) – bicarbonate; stHCO3 (mmol/l)-standard bicarbonate; ctCO2 – the sum of carbon dioxide bound to hemoglobin and carbon dioxide dissolved in plasma; BE (mmol/l) – base excess; BB (mmol/l) – total buffer bases; ctO<sup>2</sup> – the sum of oxygen bound to hemoglobin and oxygen dissolved in plasma, satO2 (%) – saturation of hemoglobin by oxygen.

**Table 5.** P values reflecting the statistical significance of differences in acid-base parameters among individual types of anesthesia in the light and dark parts of the rat regimen day.


Data presented as mean ± SD.\*p < 0.05,

**7.6. Ions**

**Figure 5.** HCO3

**Light**

**Dark**

LD differences for plasma Na<sup>+</sup>

−

plasma concentration of Na+

that the distribution of Na+

and Z anesthesia, mean plasma Na+

(mmol/l)-standard bicarbonate; ctCO2

and oxygen dissolved in plasma, satO2

Significant (i.e., p < 0.01) LD differences in plasma K<sup>+</sup>

**pH pO2 pCO2 HCO3**

anesthesias (**Table 6**). The highest Na+

120 Circadian Rhythm - Cellular and Molecular Mechanisms

concentration were not detected under any of the selected general

was recorded under P anesthesia and the lowest concentration in Z

ions is significantly influenced by Z anesthesia (**Figure 6**). Under P

concentrations moved from hyponatremic to hypernatremic.

– the sum of carbon dioxide bound to hemoglobin and carbon dioxide dissolved

concentration were found only under


– the sum of oxygen bound to hemoglobin

**<sup>−</sup> BE BB ctCO2 ctO2 satO2**

parts of the rat regimen day (light P vs. K/X, p < 0.01; P vs. Z, p < 0.01; dark P vs. K/X, p < 0.01; and nonsignificantly higher compared with Z anesthesia). In the light part of the day, the highest

and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*\*p < 0.01 and \*\*\*p < 0.001 were considered to be a statistically significant difference between individual types of anesthesia. Red dotted lines represent the ranges reported **Table 1**.

in the light (yellow columns) and dark (blue columns) periods in pentobarbital (P)-, ketamine/xylazine (K/X)-

anesthesia but with increasing dispersion of values. Based on our findings, it appears probable

anesthesia, regardless of the light or dark part of the day, hypernatremia was detected. In K/X

K/X anesthesia, with higher values during the dark part of the rat regimen day (**Table 6**).

**<sup>−</sup> stHCO3**

P-K/X 0.617 **0.001** 0.094 0.166 **0.01 0.01 0.01 0.05** 0.339 0.419 P-Z 0.869 **0.05 0.001 0.001 0.001 0.001 0.001 0.001 0.001** 0.401 K/X-Z 0.708 **0.001** 0.985 0.252 0.104 0.136 0.064 **0.01 0.05 0.01**

P-K/X **0.05** 0.137 **0.001 0.001 0.001 0.001 0.001 0.001 0.001** 0.251 P-Z **0.05** 0.707 **0.01 0.01** 0.559 **0.001 0.001 0.001 0.001** 0.730 K/X-Z 0.268 **0.01 0.001 0.001 0.001 0.001 0.01 0.001 0.01** 0.119 Bold values indicate statistically significant differences. P – pentobarbital P; K/X – ketamine/xylazine Z – zoletil; pCO<sup>2</sup>

(%) – saturation of hemoglobin by oxygen.

**Table 5.** P values reflecting the statistical significance of differences in acid-base parameters among individual types of

(kPa) – partial pressure of carbon dioxide; pO2 (kPa) – partial pressure of oxygen; HCO3

in plasma; BE (mmol/l) – base excess; BB (mmol/l) – total buffer bases; ctO<sup>2</sup>

anesthesia in the light and dark parts of the rat regimen day.

concentrations were under P anesthesia in the both light

\*\*p < 0.01 statistically significant differences between the light and dark periods. P – pentobarbital P; K/X – ketamine/ xylazine Z – zoletil; Na+ − sodium, K<sup>+</sup> − potassium, Ca2+ − calcium and Cl− chloride anions.

**Table 6.** Ion concentrations in arterial blood under individual types of anesthesia.

Under this type of anesthesia, the mean value was significantly higher (p < 0.001) compared with both P and Z anesthesias in both light parts of the day (**Figure 7**). Moderate hyperkalemia was detected under P and Z anesthesias in both light parts of rat regimen day.

Similar to Na+ , no significant LD differences in plasma Ca2+ concentrations were found (**Table 6**). Under P and Z anesthesias, plasma concentrations of Ca2+ were practically the same. In the light part of the day under K/X anesthesia, there was a significantly (p < 0.001) higher Ca2+ concentration versus P and Z anesthesias. In the dark part of the day under K/X anesthesia, the values were out of range of the ABL 800 Flex ion analyzer (**Figure 8**). Although significant differences were found between the different types of anesthesia in both light parts of the day, the animals were in relatively severe state of hypocalcemia, especially when under P and Z anesthesias.

A significant (i.e., p < 0.05) LD difference in plasma concentrations of Cl− was found only under Z anesthesia (**Table 6**). Hypochloremia occurred under P anesthesia in both light parts of the rat regimen day. Normochloremia to hyperchloremia occurred under both K/X and Z anesthesias in both light parts of the rat regimen day (**Figure 9**). In the dark part of the day under K/X anesthesia, the values were out of the detection range of the ABL 800 Flex ion analyzer. Significant differences in ion concentrations between the single type of anesthesias are summarized in (**Table 7**).

**Figure 6.** Plasma concentration of Na+ in the light (yellow columns) and dark (blue columns) periods in pentobarbital (P)-anesthetized, ketamine/xylazine (K/X)-anesthetized, and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*p < 0.05 and \*\*p < 0.01 were considered to be a statistically significant difference between individual types of anesthesia. Red dashed lines represent ranges reported in **Table 2**.

**Figure 7.** Plasma concentration of K+ ions in the light (yellow columns) and dark (blue columns) periods in pentobarbital (P)-anesthetized, ketamine/xylazine (K/X)-anesthetized, and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*\*\*p < 0.001 was considered to be a statistically significant difference between single types of anesthesia. Red dotted lines represent the ranges reported in **Table 2**.

**8. Discussion**

regimen day.

Bolded values indicate statistically significant differences.

**Light**

**Dark**

irrelevant.

**8.1. pH and blood gases**

The methodological character of this study was based on the chronobiological perspective of the initial state in acid-base balance and plasma ion concentration in arterial blood after application of commonly used anesthetics in experiments, as well as to differences in parameters of the internal environment between used the selected types of general anesthesia. The methodical characteristics of this study highlight the potential risks of experimental design. Each of the acid-base balance parameters reflects the current state of the internal environment, which

**Table 7.** Differences in plasma ion concentrations of individual types of anesthesia in the light and dark parts of the rat

**Na+ K+ Ca2+ Cl<sup>−</sup>**

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P-K/X **0.01 0.001 0.001 0.001** P-Z **0.01** 0.203 **0.05 0.01** K/X-Z 0.202 **0.001 0.001 0.01**

P-K/X **0.01 0.001 — —** P-Z 0.246 0.770 **0.01** 0.687 K/X-Z **0.05 0.001 — —**

If we only hypothetically assume that experiments are performed during working hours (i.e., in the light [inactive], part of the rat regimen day), the values presented in **Tables 1** and **2** are comparable with our results only from the light (inactive) part of the day. In the dark (i.e., active) part of the rat regimen day, the values―although significantly different among the individual types of general anesthesia―may be within the normal range but can also move out of range; this also applies to ion concentrations. In this case, therefore, comparisons are

The cardiovascular system is particularly sensitive to changes in the internal environment. For example, earlier work by Gerst et al. [95] did not detect an impact of respiratory acidosis and alkalosis on the threshold of heart vulnerability to ventricular fibrillation in dogs; however, together with hypoxia, they increased its threshold [96]. Conversely, metabolic acidosis reduces the ventricular fibrillation threshold, reduces the maximum diastolic potential, shortens the duration of action potentials, inhibits excitability, stimulates impulse conduction between Purkinje fibers and muscle tissue [97], worsens atrioventricular (AV) conduction, and inhibits AV node automation [98]. Acidosis affects the mechanical and electrical activity of the

can significantly affect the functionality of the monitored system.

**Figure 8.** Plasma concentration of Ca2+ ions in the light (yellow columns) and dark (blue columns) periods in pentobarbital (P)-anesthetized, ketamine/xylazine (K/X)-anesthetized, and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*p < 0.05, \*\*p < 0.01 and \*\*\*p < 0.001 were considered to be a statistically significant difference between individual types of anesthesia. Red dashed lines represent ranges reported in **Table 2**.

**Figure 9.** Plasma concentration of cl<sup>−</sup> ions in the light (yellow columns) and dark (blue columns) periods in pentobarbital (P)-anesthetized, ketamine/xylazine (K/X)-anesthetized, and zoletil (Z)-anesthetized rats. Data presented as mean ± SD. \*\*\*p < 0.001, \*\*p < 0.01 were considered to be a statistically significant difference between individual types of anesthesia. Red dashed lines represent the ranges reported in **Table 2**.


**Table 7.** Differences in plasma ion concentrations of individual types of anesthesia in the light and dark parts of the rat regimen day.
