**8. Discussion**

**Figure 7.** Plasma concentration of K+

**Figure 9.** Plasma concentration of cl<sup>−</sup>

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

represent the ranges reported in **Table 2**.

122 Circadian Rhythm - Cellular and Molecular Mechanisms

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

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

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

**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

(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. 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 can significantly affect the functionality of the monitored system.

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 irrelevant.

#### **8.1. pH and blood gases**

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 mammalian heart. In this way, acidosis can dramatically prolong the delay of AV conduction. In combination with short cycle times, this may cause partial or complete AV block of conduction and, consequently, contribute to the development of bradyarrhythmias under conditions of local or systemic acidosis [99]. Hypoventilation in rats is associated with systemic acidosis, hypoxia and hypercapnia, decreased mesor, amplitude, as well as altered circadian rhythm of ventricular arrhythmia threshold from one peak to two peaks, with a smaller peak between 15:00 and 18:00 h and higher between 24:00 and 03:00 h [25].

Considering changes in blood gases from a chronobiological perspective, Ohshima et al. [109] and Iwase et al. [110] reported interesting results regarding the effects of histamine on ventilation and the balance of energy metabolism via H1 receptors in the brain. The hypothesis was tested on mice as to whether the ventilatory response to hypoxia fluctuated between the light and the dark period and whether histamine H1 receptors are necessary for circadian variation. The results demonstrated that during hypoxic conditions, minute ventilation in wild type

elimination were higher in the dark period. In H1 receptor knockout mice, changes

−

Chronobiology of Acid-Base Balance under General Anesthesia in Rat Model

http://dx.doi.org/10.5772/intechopen.75174

in minute ventilation were minimal because minute ventilation was relatively increased with

arterial blood, and serum levels of ketolate were increased, indicating metabolic acidosis. The results of that study assume that minute ventilation varies between the light and dark periods, and that H1 receptors play a role in the circadian variation of minute ventilation through acid-

Rectal temperature in rats measured before administration of anesthetic agent varies depending on the LD cycle, with significantly higher values in the dark (active) part of the day, indicating the preservation of the circadian rhythm of body temperature. After anesthetic administration, a significant drop in rectal temperature (rectal temperature before anesthetic administration versus rectal temperature 15 min after induction of anesthesia [p < 0.001]) has been observed under all types of anesthesia in both light parts of the rat regimen day [100]. Interestingly, LD differences in K/X and Z anesthesias were maintained, except for P anesthesia. These results confirm the well-known fact that thermoregulation is impaired under general anesthesia [111]. This basic process occurs when the body core temperature is redistributed to the surface of the skin by anesthetic-induced vasodilation and depression of hypothalamic thermoregulatory centers [112]. Thus, the loss of LD differences under P anesthesia confirms this fact, and that P likely also acts on the suprachiasmatic

Sustained anesthesia and hypothermia may be required under certain conditions of critical care. Data suggest that mild hypothermia (35–33°C), in combination with sustained anesthesia, may reduce the need for high levels of breathing volume and respiratory rate without significant changes in arterial oxygenation and acid-base balance. The risk for barotrauma in ventilated rats exposed to conditions similar to critical care could, therefore, be reduced by using lower volume/pressure ventilation in the presence of mild hypothermia and P anesthesia [113]. Moderate hypothermia in rats induced by sustained P anesthesia reduces ventilation but without a change in arterial oxygenation or acid-base balance, measured at normal body temperature. In theory, observations in spontaneously breathing rats indicate that a combination of moderate hypothermia and anesthesia can be safely used to maintain adequate ventilation with relatively low ventilation. It is assumed that such a maneuver, when used during mechanical ventilation, can prevent secondary pulmonary damage by allowing

Metabolism and pulmonary ventilation change over a 24 h period and exhibit circadian fluctuations. Because their changes are always synchronic, blood gases can remain stable in a narrow range. Piccione et al. [115] monitored arterial blood gases, pH, body temperature and respiratory rate in 5 cows and detected a circadian rhythm only for pCO2. In cows, blood

a lower adjustment of the volume and pressure of the ventilator [114].

consumption

125

and BE were elevated in

mice increased during the dark period. Hypoxia reduced metabolism, but O2

consumption in the light period. In this group, HCO3

base balance control and metabolism in mice [109, 110].

and CO2

respect to O2

nuclei of the hypothalamus.

Our results indicate that P, K/X, and Z anesthesias cause acidosis, hypoxia, and hypercapnia, especially in the light period of the rat regimen day. In the dark part of the day, values are closer to physiological ranges, except for P anesthesia [100]. It also appears that differences in pH, pO2 , and pCO2 differ among each type of general anesthesia, depending on the light period. The decrease in pH, observed in all types of anesthesia, is probably the result of a contemporaneous depression of pulmonary ventilation and decrease in body temperature in the light as well as in the dark part of the rat regime day.

We have confirmed the conclusions of other work investigating the effects of anesthesia on pulmonary ventilation. Induction of anesthesia in rats using P significantly increases pCO<sup>2</sup> and TCO2 , while pH is decreased [64, 101, 102]. P-induced anesthesia caused mild respiratory acidosis accompanied by an increase in arterial lactate levels. Urethane anesthesia leads to partially compensated metabolic acidosis. Hypothermia reduces metabolic acidosis and hypercapnia induced by P anesthesia. In urethane anesthesia, no difference was observed between hypothermic and normal values [103]. Alfaro and Palacios [104] compared acid-base balance in mildly hypothermic (30°C) and seriously hypothermic rats (20°C). The authors found that in the first group of hypothermic animals, respiratory alkalosis occurred with an increase in pH from 7.476 to 7.546 and a decrease in arterial bicarbonate from 22.9 to 16.8 mmol/L; in the second group, from 7.484 to 7.563 with a bicarbonate drop from 20.7 to 14.6 mmol/l. This pattern was clearly different in rats under P anesthesia (mild respiratory acidosis) and under urethane anesthesia (metabolic acidosis). Similar results were reported by Gaudy et al. [105]. Anesthesia may interfere with the development of processes that lead to the acid-base balance pattern observed in conscious animals. In 1997, Alfaro and Palacios [106] supplemented that their observations regarding the blood pH of normothermic anesthetized rats (body temperature Tb = 37°C) was also associated with an increase in plasma anions (lactate and Cl− ). More severe metabolic acidosis in rat blood were detected in urethane-induced hypothermia (Tb = 32°C).

Changes observed in rats anesthetized with the thiobarbiturate inactin were similar to urethane anesthesia, although they were generally less severe. Most subjects treated with barbiturates were significantly hypercapnic. Urethane anesthesia was characterized by a higher and more stable heart rate and greater pulse pressure. Arterial carbon dioxide and bicarbonate values in the urethane group were significantly lower at all sampling times than those obtained in the barbiturate groups [107]. In connection with hypercapnia, it is also interesting to note that mild hypercapnia increases peripheral tissue oxygenation in healthy individuals, which can improve resistance to infections after surgical intervention. Partial pressure of tissue oxygen, blood flow rate through the skin, cardiac index, and saturation of muscle oxygen increases linearly with partial CO2 pressure. The observed difference in peripheral oxygenation is clinically important because previous work has suggested that a comparable increase in tissue oxygenation reduces the risk of infection from 7–8%, to 2–3% [108].

Considering changes in blood gases from a chronobiological perspective, Ohshima et al. [109] and Iwase et al. [110] reported interesting results regarding the effects of histamine on ventilation and the balance of energy metabolism via H1 receptors in the brain. The hypothesis was tested on mice as to whether the ventilatory response to hypoxia fluctuated between the light and the dark period and whether histamine H1 receptors are necessary for circadian variation. The results demonstrated that during hypoxic conditions, minute ventilation in wild type mice increased during the dark period. Hypoxia reduced metabolism, but O2 consumption and CO2 elimination were higher in the dark period. In H1 receptor knockout mice, changes in minute ventilation were minimal because minute ventilation was relatively increased with respect to O2 consumption in the light period. In this group, HCO3 − and BE were elevated in arterial blood, and serum levels of ketolate were increased, indicating metabolic acidosis. The results of that study assume that minute ventilation varies between the light and dark periods, and that H1 receptors play a role in the circadian variation of minute ventilation through acidbase balance control and metabolism in mice [109, 110].

mammalian heart. In this way, acidosis can dramatically prolong the delay of AV conduction. In combination with short cycle times, this may cause partial or complete AV block of conduction and, consequently, contribute to the development of bradyarrhythmias under conditions of local or systemic acidosis [99]. Hypoventilation in rats is associated with systemic acidosis, hypoxia and hypercapnia, decreased mesor, amplitude, as well as altered circadian rhythm of ventricular arrhythmia threshold from one peak to two peaks, with a smaller peak between

Our results indicate that P, K/X, and Z anesthesias cause acidosis, hypoxia, and hypercapnia, especially in the light period of the rat regimen day. In the dark part of the day, values are closer to physiological ranges, except for P anesthesia [100]. It also appears that differences

period. The decrease in pH, observed in all types of anesthesia, is probably the result of a contemporaneous depression of pulmonary ventilation and decrease in body temperature in

We have confirmed the conclusions of other work investigating the effects of anesthesia on pulmonary ventilation. Induction of anesthesia in rats using P significantly increases pCO<sup>2</sup>

acidosis accompanied by an increase in arterial lactate levels. Urethane anesthesia leads to partially compensated metabolic acidosis. Hypothermia reduces metabolic acidosis and hypercapnia induced by P anesthesia. In urethane anesthesia, no difference was observed between hypothermic and normal values [103]. Alfaro and Palacios [104] compared acid-base balance in mildly hypothermic (30°C) and seriously hypothermic rats (20°C). The authors found that in the first group of hypothermic animals, respiratory alkalosis occurred with an increase in pH from 7.476 to 7.546 and a decrease in arterial bicarbonate from 22.9 to 16.8 mmol/L; in the second group, from 7.484 to 7.563 with a bicarbonate drop from 20.7 to 14.6 mmol/l. This pattern was clearly different in rats under P anesthesia (mild respiratory acidosis) and under urethane anesthesia (metabolic acidosis). Similar results were reported by Gaudy et al. [105]. Anesthesia may interfere with the development of processes that lead to the acid-base balance pattern observed in conscious animals. In 1997, Alfaro and Palacios [106] supplemented that their observations regarding the blood pH of normothermic anesthetized rats (body temperature

Tb = 37°C) was also associated with an increase in plasma anions (lactate and Cl−

metabolic acidosis in rat blood were detected in urethane-induced hypothermia (Tb = 32°C).

Changes observed in rats anesthetized with the thiobarbiturate inactin were similar to urethane anesthesia, although they were generally less severe. Most subjects treated with barbiturates were significantly hypercapnic. Urethane anesthesia was characterized by a higher and more stable heart rate and greater pulse pressure. Arterial carbon dioxide and bicarbonate values in the urethane group were significantly lower at all sampling times than those obtained in the barbiturate groups [107]. In connection with hypercapnia, it is also interesting to note that mild hypercapnia increases peripheral tissue oxygenation in healthy individuals, which can improve resistance to infections after surgical intervention. Partial pressure of tissue oxygen, blood flow rate through the skin, cardiac index, and saturation of muscle oxygen increases linearly with

pressure. The observed difference in peripheral oxygenation is clinically important

because previous work has suggested that a comparable increase in tissue oxygenation reduces

, while pH is decreased [64, 101, 102]. P-induced anesthesia caused mild respiratory

differ among each type of general anesthesia, depending on the light

). More severe

15:00 and 18:00 h and higher between 24:00 and 03:00 h [25].

the light as well as in the dark part of the rat regime day.

in pH, pO2

and TCO2

partial CO2

the risk of infection from 7–8%, to 2–3% [108].

, and pCO2

124 Circadian Rhythm - Cellular and Molecular Mechanisms

Rectal temperature in rats measured before administration of anesthetic agent varies depending on the LD cycle, with significantly higher values in the dark (active) part of the day, indicating the preservation of the circadian rhythm of body temperature. After anesthetic administration, a significant drop in rectal temperature (rectal temperature before anesthetic administration versus rectal temperature 15 min after induction of anesthesia [p < 0.001]) has been observed under all types of anesthesia in both light parts of the rat regimen day [100]. Interestingly, LD differences in K/X and Z anesthesias were maintained, except for P anesthesia. These results confirm the well-known fact that thermoregulation is impaired under general anesthesia [111]. This basic process occurs when the body core temperature is redistributed to the surface of the skin by anesthetic-induced vasodilation and depression of hypothalamic thermoregulatory centers [112]. Thus, the loss of LD differences under P anesthesia confirms this fact, and that P likely also acts on the suprachiasmatic nuclei of the hypothalamus.

Sustained anesthesia and hypothermia may be required under certain conditions of critical care. Data suggest that mild hypothermia (35–33°C), in combination with sustained anesthesia, may reduce the need for high levels of breathing volume and respiratory rate without significant changes in arterial oxygenation and acid-base balance. The risk for barotrauma in ventilated rats exposed to conditions similar to critical care could, therefore, be reduced by using lower volume/pressure ventilation in the presence of mild hypothermia and P anesthesia [113]. Moderate hypothermia in rats induced by sustained P anesthesia reduces ventilation but without a change in arterial oxygenation or acid-base balance, measured at normal body temperature. In theory, observations in spontaneously breathing rats indicate that a combination of moderate hypothermia and anesthesia can be safely used to maintain adequate ventilation with relatively low ventilation. It is assumed that such a maneuver, when used during mechanical ventilation, can prevent secondary pulmonary damage by allowing a lower adjustment of the volume and pressure of the ventilator [114].

Metabolism and pulmonary ventilation change over a 24 h period and exhibit circadian fluctuations. Because their changes are always synchronic, blood gases can remain stable in a narrow range. Piccione et al. [115] monitored arterial blood gases, pH, body temperature and respiratory rate in 5 cows and detected a circadian rhythm only for pCO2. In cows, blood gases remain highly stable for 24 h. Daily body temperature oscillations, respiratory rate, and probably many other factors affecting metabolism and pulmonary ventilation do not exclude excellent blood gas homeostasis.

activities and demonstrate circadian rhythmicity in these systems, acid/base balance parameters will also exhibit a parallel circadian rhythmicity. The functional efficiency of the respiratory and

therefore, remains: to what extent are changes in acid-base balance parameters still acceptable in in vivo rat models? Additionally, to what extent should the dependence on circadian rhythms

When considering parameters of acid-base balance, the most important is bicarbonate concentration. In general, given the impact of some processes on acid-base balance, it is advisable to especially consider changes in the concentrations of the major ions and their equilibrium to evaluate changes in the concentration of bicarbonate. The change in pH is secondary due to the change in the Henderson-Hasselbach equation. Eventual loss or addition of protons is immediately equalized by buffering mechanisms, and the capacity of which are significant with regard

Bicarbonate content in serum or plasma is a significant indicator of electrolyte dispersion and anion deficiency. Together with pH determination, bicarbonate measurements are used to diagnose and treat many potentially serious disorders associated with acid-base imbalance(s) in respiratory and metabolic systems. Concentration of bicarbonate reflects the acidity or alkalinity of the blood. In metabolic acidosis, the bicarbonate concentration is low, and in metabolic alkalosis, bicarbonate concentration is high. The actual concentration of bicarbonate reflects not only the metabolic component but also the respiratory component. For control of the respiratory component, standard bicarbonate is a better measure of the metabolic component than actual bicarbonate. Standard bicarbonate is inverse to the standard pH, which is pH under standard conditions (pCO2 = 40 mmHg, temperature 37°C, and 100% oxygen

The relationships between acid-base balance and ion management are closely connected. The

librium with other ions to preserve electroneutrality in the internal environment. For partial

According to the Henderson-Hasselbach equation, the pH of the internal environment depends

influence, resulting in serious functional consequences for the organism. In this case, if the concentration of a particular ion alters some pathological process, this change must be compensated by a change in the concentration of another ion to maintain electrical neutrality. Often, this compensation is afforded by changes in bicarbonate concentration. Biocarbonates, regard-

carbonate therapy in hyperkalemia, even in conditions of compensated blood pH [131].

, this does not apply, and therefore, its regulation can be largely independent.

), these regulations are very sensitive but have only limited possibilities for rapid

CO3

. Regarding the regulation of most major ions

, reflecting the utility of hydrogen

). Therefore, the bicarbonate anion must be in equi-

[i.e., CO2

]), while

main reason is that one part of the bicarbonate buffer has no charge (H<sup>2</sup>

−

output will be increased. pH depends on changes of pCO2

Chronobiology of Acid-Base Balance under General Anesthesia in Rat Model

will also be higher at

http://dx.doi.org/10.5772/intechopen.75174

. The question,

127

cardiovascular systems is greater during periods of activity; therefore, pO2

be accounted for in the design of in vivo experiments involving general anesthesia?

these times, and CO2

**8.2. Acid-base balance and ion concentration**

to regulating proton concentration.

**8.3. Bicarbonate and acid-base balance**

the second component is charged (HCO3

on the ratio of bicarbonate concentration to pCO2

less of blood pH, alter the transcellular distribution of K+

saturation).

pressure of CO2

(Na+ , K+ , Cl<sup>−</sup>

If respiratory acidosis is induced after anesthesia, it is logical to adjust pulmonary ventilation so that the acid-base balance is adjusted to a physiological range. However, there is a problem with how to set up artificial ventilation to adjust acid-base balance parameters. The method of artificial ventilation for rats under general anesthesia has been in use since 1940 [116–119]. This can be a suitable procedure for creating experimental models observing the effect of pulmonary ventilation disorders on various functional systems. However, artificially controlled ventilation parameters using room air should be adequate to maintain acid-base balance. There are several types of normal artificial ventilation in rats that can be applied to maintain acid-base balance (**Table 8**).

The selection of anesthetic agent may be problematic with respect to the respiratory and cardiovascular systems [17, 18]. Changes in the functional performance of these systems lead to changes in acid-base balance. Conversely, changes in acid-base balance also reflect 24 h fluctuations in respiratory and cardiovascular function. Therefore, acid-base balance reference values may be problematic because acid-base balance only reflects the current state of the organism at a particular time of day. The results are then often compared with the average reference values, often regardless of dependence on the circadian rhythm of changes in acid-base balance. If both pH and partial pressures of the respiratory gases depend on respiratory and cardiovascular


**Table 8.** Previously published artificial lung ventilation parameters to maintain normal acid-base balance ranges in vivo in rats.

activities and demonstrate circadian rhythmicity in these systems, acid/base balance parameters will also exhibit a parallel circadian rhythmicity. The functional efficiency of the respiratory and cardiovascular systems is greater during periods of activity; therefore, pO2 will also be higher at these times, and CO2 output will be increased. pH depends on changes of pCO2 . The question, therefore, remains: to what extent are changes in acid-base balance parameters still acceptable in in vivo rat models? Additionally, to what extent should the dependence on circadian rhythms be accounted for in the design of in vivo experiments involving general anesthesia?

#### **8.2. Acid-base balance and ion concentration**

gases remain highly stable for 24 h. Daily body temperature oscillations, respiratory rate, and probably many other factors affecting metabolism and pulmonary ventilation do not exclude

If respiratory acidosis is induced after anesthesia, it is logical to adjust pulmonary ventilation so that the acid-base balance is adjusted to a physiological range. However, there is a problem with how to set up artificial ventilation to adjust acid-base balance parameters. The method of artificial ventilation for rats under general anesthesia has been in use since 1940 [116–119]. This can be a suitable procedure for creating experimental models observing the effect of pulmonary ventilation disorders on various functional systems. However, artificially controlled ventilation parameters using room air should be adequate to maintain acid-base balance. There are several types of normal artificial ventilation in rats that can be applied to

The selection of anesthetic agent may be problematic with respect to the respiratory and cardiovascular systems [17, 18]. Changes in the functional performance of these systems lead to changes in acid-base balance. Conversely, changes in acid-base balance also reflect 24 h fluctuations in respiratory and cardiovascular function. Therefore, acid-base balance reference values may be problematic because acid-base balance only reflects the current state of the organism at a particular time of day. The results are then often compared with the average reference values, often regardless of dependence on the circadian rhythm of changes in acid-base balance. If both pH and partial pressures of the respiratory gases depend on respiratory and cardiovascular

**Author (year) Respiratory rate, breaths/min Tidal volume, ml/100 g**

Schultz et al. [70] 65–70 Not determined

Häfner et al. [126] 30 Not determined

**Table 8.** Previously published artificial lung ventilation parameters to maintain normal acid-base balance ranges in vivo

Fagbeni et al. [120] 54 2 Richard et al. [121] 60 1 Guarini et al. [122] 55 2 Lott et al. [123] 70 1.5–2 Ohoi and Takeo [69] 40–60 1 Godin-Ribuot [124] 54 1.5 Oosting et al. [125] 60 3

Sun and Wainwright [71] 54 2

Tanno et al. [127] 44–55 1.5–2.5 Ravingerova et al. [128] 65–70 1.2 Wang et al. [89] 60–70 1.2 Neckař et al. [129] 65–70 1.2 Neckař et al. [130] 69 1.2

excellent blood gas homeostasis.

126 Circadian Rhythm - Cellular and Molecular Mechanisms

maintain acid-base balance (**Table 8**).

in rats.

When considering parameters of acid-base balance, the most important is bicarbonate concentration. In general, given the impact of some processes on acid-base balance, it is advisable to especially consider changes in the concentrations of the major ions and their equilibrium to evaluate changes in the concentration of bicarbonate. The change in pH is secondary due to the change in the Henderson-Hasselbach equation. Eventual loss or addition of protons is immediately equalized by buffering mechanisms, and the capacity of which are significant with regard to regulating proton concentration.

Bicarbonate content in serum or plasma is a significant indicator of electrolyte dispersion and anion deficiency. Together with pH determination, bicarbonate measurements are used to diagnose and treat many potentially serious disorders associated with acid-base imbalance(s) in respiratory and metabolic systems. Concentration of bicarbonate reflects the acidity or alkalinity of the blood. In metabolic acidosis, the bicarbonate concentration is low, and in metabolic alkalosis, bicarbonate concentration is high. The actual concentration of bicarbonate reflects not only the metabolic component but also the respiratory component. For control of the respiratory component, standard bicarbonate is a better measure of the metabolic component than actual bicarbonate. Standard bicarbonate is inverse to the standard pH, which is pH under standard conditions (pCO2 = 40 mmHg, temperature 37°C, and 100% oxygen saturation).

#### **8.3. Bicarbonate and acid-base balance**

The relationships between acid-base balance and ion management are closely connected. The main reason is that one part of the bicarbonate buffer has no charge (H<sup>2</sup> CO3 [i.e., CO2 ]), while the second component is charged (HCO3 − ). Therefore, the bicarbonate anion must be in equilibrium with other ions to preserve electroneutrality in the internal environment. For partial pressure of CO2 , this does not apply, and therefore, its regulation can be largely independent. According to the Henderson-Hasselbach equation, the pH of the internal environment depends on the ratio of bicarbonate concentration to pCO2 . Regarding the regulation of most major ions (Na+ , K+ , Cl<sup>−</sup> ), these regulations are very sensitive but have only limited possibilities for rapid influence, resulting in serious functional consequences for the organism. In this case, if the concentration of a particular ion alters some pathological process, this change must be compensated by a change in the concentration of another ion to maintain electrical neutrality. Often, this compensation is afforded by changes in bicarbonate concentration. Biocarbonates, regardless of blood pH, alter the transcellular distribution of K+ , reflecting the utility of hydrogen carbonate therapy in hyperkalemia, even in conditions of compensated blood pH [131].

Our measurements indicated elevated levels of bicarbonate under P anesthesia, which, compared with the normal range (23.8–26.78 mmol/l in rats), would correspond to metabolic alkalosis, unless there were changes in other parameters of acid-base balance in both light parts of the day. However, under P anesthesia, we also found relatively severe acidosis, hypercapnia, hyperkalemia, and hypochloremia, which could signal the compensation of this state or the replacement of chlorides in the blood by bicarbonates. In this regard, P anesthesia induces more serious disruption of acid-base balance, independent of the cycle of alternating light and darkness. In K/X and Z anesthesias, these changes were more subtle, and when LD differences appear to be preserved, we assume that circadian rhythms are also preserved, and therefore, from a chronobiological point of view, these are appropriate types of general anesthesia.

and it only changes its distribution between compartments. From a whole-body perspective, potassium depletion will be a consequence of acidity, because its renal loss increases (so that heavier and longer-lasting acidosis will be accompanied by depletion of potassium at the current hyperkalemia). Similarly, alkalemia is accompanied by hypokalemia. However, the entire mechanism also works inversely: hyperkalemia causes acidosis and hypokalemia, on the other hand, leads to alkalosis. Simplified, we can imagine that potassium cations that

From the chronobiological point of view, however, this was not confirmed by our results. In each type of anesthesia, hyperkalemia was recorded, irrespective of whether the measurements were made in the light or dark part of the rat regimen day. Acidosis occurred only in the light part of the day under each type of anesthesia, while in the dark part of the day, the pH values also moved within normal ranges, but only under K/X and Z anesthesias These findings should, therefore, be taken into account to avoid application of particular anesthesias in the light part of the rat regimen day because positive correlations between pH and plasma

 concentration have been calculated for all types of anesthesia (P light r = 0.41, P dark = 0.16; K/X light r = 0.57, K/X dark r = 0.01, Z light r = 0.79, dark r = −0.22). What this means that the

was found under all types of anesthesia.

ing membrane processes, they touch primarily exciting tissues. In case of hyperkalemia, the concentration gradient decreases so that potassium escapes from the cell more slowly. However, the resting membrane potential becomes less negative and, therefore, in the initial phase of hyperkalemia, excitability increases (the resting potential is closer to the threshold). Increasing the potassium concentration in the extracellular environment by

Considering the electrophysiology of the heart, hyperkalemia affects the production and conduction of impulses, which can lead to ventricular fibrillation through several mechanisms:

space is the key factor determining the value of the resting membrane potential. Increases

• at decreased negativity of the resting membrane potential, the difference between resting and threshold potential is lower and depolarization is more easily induced. If the negativity of the resting membrane potential continues to fall, the negativity of the threshold

• the value of the resting membrane potential also determines the number of sodium chan-

myocardium, the resting membrane potential is reduced from −90 to −80 mV.

current and thus to a decrease in the negativity of the membrane potential. In the

If we generally consider the consequences of changes in plasma K+

increasing the potential leads to blockage of voltage-gated Na+

leakage if the rat is in general anesthesia. In the dark part of the rat regimen day,

shifts the pH to the alkalinity, respectively. Alkalosis

Chronobiology of Acid-Base Balance under General Anesthesia in Rat Model

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129

in the direction from the intracellular into extracellular

input into the cell. The lower the nega-

channels are activated and depolarization

concentration leads to a decrease in the gradient to a decreased out-

concentration affect-

channels, and consequently,

move through the plasma membrane are exchanged for protons.

K+

increases K+

no pH dependence on K+

excitability decreases.

• the concentration gradient of K+

potential also begins to decrease.

nels that open during depolarization to allow Na+

tive resting membrane potential, the less the Na+

in the extracellular K+

ward K+

occurs slower.

increase in plasma concentration K+

#### **8.4. BE and BB**

BE relates to a true excess of base in the range (above or below) of the total BB. Normally, BB is 48–49 mmol/l. If BB is 40 mmol/l, it means that the buffer base was decreased by almost 8 mmol/l or BE is −8 mmol/l (also known as base deficiency). If BB is 60 mmol/l, it indicates that the base of the buffer is increased by approximately 12 mmol/l, or BE is +12 mmol/l. Fifty percent of BB is produced by bicarbonate and 25% by other buffers (proteins, phosphates, sulfates). In our experiments, BE and the total BB moved within the normal ranges, which would mean that buffering capacity was sufficient not only in the dark but also in the light period of the rat regimen day and under all types of anesthesia.

#### **8.5. Ions**

#### *8.5.1. Potassium and acid-base balance*

As early as the 1950s and 1960s, the relationship between extracellular potassium, bicarbonates, and blood pH was recognized. Relatively small changes in potassium concentration in the cell compartment can result in large changes in plasma potassium concentration. As a result, plasma potassium concentration may be reduced, normal, or elevated, despite normal stores of potassium in the body. The main regulator of transcellular potassium distribution is the pH of the extracellular fluid, which is reflected in blood pH. It was demonstrated that lowering the pH of blood increases serum potassium levels and vice versa [132–135]. It has recently been found that the concentration of extracellular bicarbonate―apart from its effect on extracellular pH―affects a wide range of metabolic reactions [136–139]. During this time, there was contradictory evidence that changes in blood hydrogen carbonate concentration in isohydric conditions alter plasma potassium concentration [140–143] in normokalemia, and no information regarding the role of bicarbonates in hypokalemia or hyperkalemia was available. At the increase of pH about 0.1, kalemia is increased about 0.5–0.6 mmol/l.

In acidemia, a number of "redundant" protons will enter the cells in which they will buffer. Consequently, a cation is transferred through the plasma membrane, which would in itself lead to a change in membrane potential. Instead of the proton, another cation is transferred from the intracellular to the extracellular space. Because the conductivity of the plasma membrane is highest for K+ ions, primarily potassium ions will be transferred. Acidemia in this scenario leads to hyperkalemia. The total amount of potassium in the body does not increase, and it only changes its distribution between compartments. From a whole-body perspective, potassium depletion will be a consequence of acidity, because its renal loss increases (so that heavier and longer-lasting acidosis will be accompanied by depletion of potassium at the current hyperkalemia). Similarly, alkalemia is accompanied by hypokalemia. However, the entire mechanism also works inversely: hyperkalemia causes acidosis and hypokalemia, on the other hand, leads to alkalosis. Simplified, we can imagine that potassium cations that move through the plasma membrane are exchanged for protons.

Our measurements indicated elevated levels of bicarbonate under P anesthesia, which, compared with the normal range (23.8–26.78 mmol/l in rats), would correspond to metabolic alkalosis, unless there were changes in other parameters of acid-base balance in both light parts of the day. However, under P anesthesia, we also found relatively severe acidosis, hypercapnia, hyperkalemia, and hypochloremia, which could signal the compensation of this state or the replacement of chlorides in the blood by bicarbonates. In this regard, P anesthesia induces more serious disruption of acid-base balance, independent of the cycle of alternating light and darkness. In K/X and Z anesthesias, these changes were more subtle, and when LD differences appear to be preserved, we assume that circadian rhythms are also preserved, and therefore, from a chronobiological point of view, these are appropriate types of general anesthesia.

BE relates to a true excess of base in the range (above or below) of the total BB. Normally, BB is 48–49 mmol/l. If BB is 40 mmol/l, it means that the buffer base was decreased by almost 8 mmol/l or BE is −8 mmol/l (also known as base deficiency). If BB is 60 mmol/l, it indicates that the base of the buffer is increased by approximately 12 mmol/l, or BE is +12 mmol/l. Fifty percent of BB is produced by bicarbonate and 25% by other buffers (proteins, phosphates, sulfates). In our experiments, BE and the total BB moved within the normal ranges, which would mean that buffering capacity was sufficient not only in the dark but also in the light period of

As early as the 1950s and 1960s, the relationship between extracellular potassium, bicarbonates, and blood pH was recognized. Relatively small changes in potassium concentration in the cell compartment can result in large changes in plasma potassium concentration. As a result, plasma potassium concentration may be reduced, normal, or elevated, despite normal stores of potassium in the body. The main regulator of transcellular potassium distribution is the pH of the extracellular fluid, which is reflected in blood pH. It was demonstrated that lowering the pH of blood increases serum potassium levels and vice versa [132–135]. It has recently been found that the concentration of extracellular bicarbonate―apart from its effect on extracellular pH―affects a wide range of metabolic reactions [136–139]. During this time, there was contradictory evidence that changes in blood hydrogen carbonate concentration in isohydric conditions alter plasma potassium concentration [140–143] in normokalemia, and no information regarding the role of bicarbonates in hypokalemia or hyperkalemia was avail-

able. At the increase of pH about 0.1, kalemia is increased about 0.5–0.6 mmol/l.

In acidemia, a number of "redundant" protons will enter the cells in which they will buffer. Consequently, a cation is transferred through the plasma membrane, which would in itself lead to a change in membrane potential. Instead of the proton, another cation is transferred from the intracellular to the extracellular space. Because the conductivity of the plasma mem-

scenario leads to hyperkalemia. The total amount of potassium in the body does not increase,

ions, primarily potassium ions will be transferred. Acidemia in this

the rat regimen day and under all types of anesthesia.

*8.5.1. Potassium and acid-base balance*

128 Circadian Rhythm - Cellular and Molecular Mechanisms

brane is highest for K+

**8.4. BE and BB**

**8.5. Ions**

From the chronobiological point of view, however, this was not confirmed by our results. In each type of anesthesia, hyperkalemia was recorded, irrespective of whether the measurements were made in the light or dark part of the rat regimen day. Acidosis occurred only in the light part of the day under each type of anesthesia, while in the dark part of the day, the pH values also moved within normal ranges, but only under K/X and Z anesthesias These findings should, therefore, be taken into account to avoid application of particular anesthesias in the light part of the rat regimen day because positive correlations between pH and plasma K+ concentration have been calculated for all types of anesthesia (P light r = 0.41, P dark = 0.16; K/X light r = 0.57, K/X dark r = 0.01, Z light r = 0.79, dark r = −0.22). What this means that the increase in plasma concentration K+ shifts the pH to the alkalinity, respectively. Alkalosis increases K+ leakage if the rat is in general anesthesia. In the dark part of the rat regimen day, no pH dependence on K+ was found under all types of anesthesia.

If we generally consider the consequences of changes in plasma K+ concentration affecting membrane processes, they touch primarily exciting tissues. In case of hyperkalemia, the concentration gradient decreases so that potassium escapes from the cell more slowly. However, the resting membrane potential becomes less negative and, therefore, in the initial phase of hyperkalemia, excitability increases (the resting potential is closer to the threshold). Increasing the potassium concentration in the extracellular environment by increasing the potential leads to blockage of voltage-gated Na+ channels, and consequently, excitability decreases.

Considering the electrophysiology of the heart, hyperkalemia affects the production and conduction of impulses, which can lead to ventricular fibrillation through several mechanisms:


• repolarization is the result of opening K+ channels and the subsequent outward K+ current. For unclear reasons, the amount of K+ from the cell paradoxically increases with increasing extracellular K+ concentration. In hyperkalemia, therefore, acceleration of repolarization occurs.

Hypercalcemia is a state when the serum Ca2+ level is greater than 2.8 mmol/l and ionized Ca2+ is greater than 1.4 mmol/l. At values above 4 mmol/l, "chemical death" may occur when cardiac arrest may occur. Hypertension and arrhythmias occur at the hypercalcemia. On the ECG, QT interval is shortened. Hypocalcemia is accompanied by an increase in neuromuscu-

maintenance of the electrical neutrality, the anion deficiency is supplemented by an increase

and hypochloremic alkalosis develops. In summary, substitution of chlorides in the blood

After summarizing the results from the analysis of acid-base balance parameters (**Table 6**), we concluded that there are differences in the final status of the rat internal environment that

In the light part of the day, under P anesthesia, the rats are in a state of acidosis, hypercapnia, and hypoxia, and elevated levels of bicarbonate have been reported. Similarly, it is also in the dark, but with mild acidosis, hypercapnia and hypoxia with a moderate decrease to normal

Under K/X anesthesia, we found a dependence on LD cycle in all monitored parameters. In the

pO in the hypoxic range, relatively large range of bicarbonate (from reduced to increased levels) levels and lower saturation (around 85%) were observed. In the dark part of the day, from

level of bicarbonate. Different values were in saturation of hemoglobin by oxygen, where higher saturation was during the dark (active) part (around 90%). The efficiency of the buffer

Under Z anesthesia, the status was as follows: acidosis, hypercapnia, hypoxia to normoxia, and normal levels of bicarbonate in the light part of the day. In the dark part of the day, the state of

hypoxia to normoxia at a normal to moderately elevated level of bicarbonate. The saturation of hemoglobin by oxygen fluctuated around 89% in both light parts of the rat regimen day, and

It was concluded that P anesthesia is not the most appropriate type of general anesthesia to use in chronobiological rat models. It is likely to produce a more acidic environment than K/X and Z anesthesias, and although an LD difference in P anesthesia was not recorded, the pH values

normal to alkaline pH, hypocapnia, moderate decreased to normal pO2

system moved within the normal range in both light parts of the day.

the internal environment was from acidic to normal, hypercapnia, and pO2

BB and BE were also in the normal range; thus, buffering capacity remained intact.

 values but with elevated levels of bicarbonate. Saturation of hemoglobin by oxygen was at the same level in both light parts of the rat regimen day, and at approximately 87%, the efficiency of the buffer system was not impaired because the values were within the normal range.

loss (e.g., vomiting), the concentration of the other major ions is not altered, and for

does not change; therefore, ventilation is maintained

Chronobiology of Acid-Base Balance under General Anesthesia in Rat Model

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131

ranging from normocapnia to hypercapnia,

but with a reduced

moved from mild

lar excitability, but myocardial contractility decreases.

*8.5.3. Chlorides and acid-base balance*

in the bicarbonate concentration. pCO2

occurs at the expense of hydrogen carbonates.

depend on the LD cycle and on the type of anesthesia.

light part of the day, unambiguous acidosis, pCO2

During Cl<sup>−</sup>

**9. Conclusions**

pO2

Electrolyte abnormalities are becoming an increasingly important cause of arrhythmias. In humans monitored using electrocardiography, spiky and narrow T-waves (acceleration of repolarization) are the most common manifestations, QRS complex enlargement and prolongation of the PQ interval (slow depolarization). If hyperkalemia deepens, atrial activity may disappear, and ventricles are stimulated from AV node with resulting bradycardia. In severe hyperkalemia, the QRS complex expands, with consequent risk for ventricular fibrillation and cardiac arrest.

Although electrocardiographic (ECG) changes in hyperkalemic rats are poorly understood, it is clear that excess plasma potassium may also alter cardiac excitation. In addition, the effects of hyperkalemia on ECG in rats may differ from other species that do not have ST segments and longer QT intervals. At testing, the effects of two local anesthetics (bupivacaine and lidocaine) at normocalcemia and hyperkalemia were found that hyperkalemia with concentration 9.0 mmol/l had little effect on heart rate or AV conduction in the absence of bupivacaine or lidocaine. Nevertheless, the effect of local anesthetics on slowing the ventricular rate was significantly enhanced. For bupivacaine, ventricular deceleration to 50% vs. control, during hyperkalemia, was performed almost completely through inhibition of AV conduction whereas for lidocaine through not only inhibition of AV conduction but also atrial rate. Regardless of the mechanism, hyperkalemia of this grade increased the ventricular slowing effect of bupivacaine and lidocaine [144]. Kuwahara et al. [145] described changes in rat ECG in dependence on K+ levels. In moderate hyperkalemia, an increased amplitude of T wave occurred. The duration of the PR interval and the QRS complex was slightly reduced, and the P wave disappeared in most rats at potassium levels above 8.0 mmol/l. In advanced hyperkalemia (plasma potassium concentration higher than 7.5 mmol/l), conduction was suppressed in all parts of the heart.

As for hypokalemia, except impacts on other functions and systems, heart failure and cardiac rhythm are typical of cardiac symptoms. On the ECG, low, flat, or inverted T-waves and prolongation of the QT interval can be seen. Supraventricular and ventricular extrasystoles occur episodically.

#### *8.5.2. Calcium and acid-base balance*

Similarly as the proton is exchanged for the potassium cation, a calcium cation is also exchanged for protons. Plasma proteins play a key role in this mechanism. Blood plasma proteins behave as buffers, primarily due to carboxyl groups and amino groups. As regard the carboxyl groups, these groups are in protonic, nondissociated state (-COOH) in the acidic environment. In the alkaline environment, they begin to buffer and their dissociation into the carboxylate -COO− occurs, which is able to bind very effective especially Ca2+. It means that in the case of acidosis, the -COOH does not change to -COO<sup>−</sup> , and in the case of alkalosis, it dissociates to -COO<sup>−</sup> and H+ and the calcium binds to -COO<sup>−</sup> .

It can also be said that the pH depends on what part of the calcium will be ionized and what part will be nonionized. The practical consequence is that alkalosis leads to ionized hypocalcemia, acidosis, on the contrary, to ionized hypercalcemia. Although total calcium does not change, we have to realize that ionized calcium is metabolically active, especially when it comes to membrane processes.

Hypercalcemia is a state when the serum Ca2+ level is greater than 2.8 mmol/l and ionized Ca2+ is greater than 1.4 mmol/l. At values above 4 mmol/l, "chemical death" may occur when cardiac arrest may occur. Hypertension and arrhythmias occur at the hypercalcemia. On the ECG, QT interval is shortened. Hypocalcemia is accompanied by an increase in neuromuscular excitability, but myocardial contractility decreases.

#### *8.5.3. Chlorides and acid-base balance*

During Cl<sup>−</sup> loss (e.g., vomiting), the concentration of the other major ions is not altered, and for maintenance of the electrical neutrality, the anion deficiency is supplemented by an increase in the bicarbonate concentration. pCO2 does not change; therefore, ventilation is maintained and hypochloremic alkalosis develops. In summary, substitution of chlorides in the blood occurs at the expense of hydrogen carbonates.
