Introduction

The purposes of this chapter are to provide an overview of acid-base state in athletic horses at rest, and to introduce acid-base assessment in clinically normal horses at rest and performing exercise of different intensities and durations. The physicochemical approach to acid-base assessment will be explained and used to exemplify the origins of acid-base disturbances during exercise. Also, the impact of diet, alkalinizing agents, furosemide (frusemide, Lasix) and selected clinical conditions on acid-base status will be briefly explored. A brief discussion on the topic of acidosis and skeletal muscle fatigue is provided to address some of the myths and confusion surrounding lactate, H⁺ and muscle fatigue.

Why is it important to understand acid-base balance? Acid-base balance represents the integrated sum of many simultaneously acting physiological processes on physical and chemical state of body fluids. These processes include cellular respiration, ventilation at the lungs, renal function, hepatic function, and cellular (primarily skeletal muscle) ion transport processes. An understanding of acid-base state allows the practitioner to better understand the state of the horse at any point of time, how it got there, and to develop effective approaches for normalizing acid-base state when it had been pathophysiologically disturbed. This is important because ‘clinically normal’ acid-base state is tolerated within relatively wide physiological limits. We often read that plasma and intracellular pH are maintained within narrow limits; however, this contention is not supported by the research literature in any animal so far studied, including horses. In clinically normal humans and horses, plasma pH may vary from 7.0 to 7.6, although the norm lies close to 7.4. The large range of change in pH represents a 4-fold change in [H⁺], specifically 25 to 100 nEq/L, with normal plasma [H⁺] of 40 nmol/L. While this range of plasma [H⁺] can be tolerated, it is true that such changes are eventually accompanied by the activation of mechanisms that return [H⁺] back towards 40 nEq/L. Within skeletal muscle, pH at rest is about 7.0 ([H⁺] = 100 nEq/L) but with very high-intensity exercise muscle pH may approach 6.0 ([H⁺] = 1000 nEq/L). These are much wider ranges than generally appreciated. Also, they are tolerated, albeit briefly, and normal.

Let us start by considering what is meant by acid-base balance. In traditional terms acid-base balance refers to the balance between pH, PCO₂ and [HCO₃⁻] in physiological solution. While it is true that pH, representing the acidity or alkalinity of the fluid, is directly related to the PCO₂ and [HCO₃⁻], it is not true that changes in [HCO₃⁻] determine pH. The reason for this, as detailed below, is that it is not physically possible to change [HCO₃⁻] with simultaneously changing either the PCO₂, or the concentration of one or more of the ions in solution. Therefore, similar to pH, [HCO₃⁻] is dependent on the PCO₂ and the concentrations of all of the ions in solution. From this it follows that all of the ions in solution contribute to the acid-base state. Therefore acid-base state represents the physicochemical balance between PCO₂ and all ions in solution, including many large molecules such as albumin, lactate and intracellular proteins.

Acid-base balance in resting horses

The past 20 years has seen considerable interest in the acid-base state of racehorses at rest. This is in large part due to the practice of horsemen administering alkalinization agents to horses for the purpose of enhancing race performance, i.e. delaying onset of muscle fatigue.¹ The reader is referred to the following papers for discussion of ergogenic effects of alkalinizing agents.² Because of the potential of alkalinizing agents to enhance race performance, regulations have been put in place by all major racing jurisdictions. Most jurisdictions use a threshold for total CO₂ (TCO₂) of either 36 or 37 mmol/L for horses that have not been given furosemide; the corresponding [HCO₃⁻] thresholds are 34 and 35 mmol/L. Furosemide administration produces a dehydration with attendant changes in plasma ion concentrations, which typically raises TCO₂ by ~1.5 mmol/L. Therefore in racing jurisdictions that allow furosemide administration the TCO₂ testing threshold can be as high as 39 mmol/L. Racing jurisdictions also contend that all horses having a TCO₂ at or above the threshold have been given an alkalinizing agent for the purposes of enhancing exercise performance. Horsemen caring for horses that have been tested at or above the threshold are generally heavily penalized. There is scientific evidence both for and against the use of TCO₂ / [HCO₃⁻] testing thresholds, and this has led to considerable debate and legal wrangling. Two main points arise from objective review of the acid-base literature as it pertains to resting horses:

Overview of acid-base balance

Moderate to high intensity muscular exercise results in acidification of muscles and blood. The acidification primarily results from the generation of protons (H⁺) within contracting skeletal muscle. The protons are of the product of biochemical and physicochemical reactions associated with increased rates of anaerobic energy production that result in intracellular lactate accumulation, intracellular K⁺ depletion and increased CO₂ production.¹³ The resultant large and rapid efflux of acid equivalents from contracting muscle produces the systemic metabolic acidosis associated with moderate to high intensity exercise.

In the exercising horse, whole-body acid-base balance is dependent on the integrated responses of the muscular, respiratory, vascular, hepatic, cutaneous and renal systems. The muscular system, in addition to providing the locomotory force requirement for activity, generates considerable amounts of acid equivalents, resulting in acidification of the intracellular and extracellular fluid compartments. Non-contracting skeletal muscle also provides the largest tissue mass within the body for the removal of lactate and acid equivalents during high intensity exercise and the initial recovery period. The respiratory system plays a key role in eliminating acid equivalents as CO₂ at the lung in addition to extracting the oxygen needed to fuel aerobic cellular metabolism. The vascular system plays an integral role in the transport and distribution of acid and base equivalents throughout the body – this system provides for the ‘buffering’ of the acid-base disturbance by distributing acid equivalents from acid-generation sites (contracting skeletal muscle) to other sites (non-contracting skeletal muscle and other tissues).¹³ Within the vascular system itself, bicarbonate, plasma proteins and hemoglobin within red blood cells are also involved in the transport and temporary storage (buffering) of acid equivalents. The hepatic system is a major tissue mass involved in the removal of lactate from the vascular system, thereby removing acid equivalents from the circulation. The cutaneous system is heavily involved in the production and secretion of sweat to the surface of the skin during and immediately following moderate to high intensity exercise. Sweat contains large amounts of Na⁺, K⁺ and Cl⁻ and different rates of excretion of each ion affects acid-base state of blood leaving the skin.¹⁴ The kidneys are capable of excreting H⁺ and lactate at greatly elevated rates during recovery from high intensity exercise, aiding in the process of recovery from the acidosis of exercise.

Each of the systems described above is capable of modifying the water, electrolyte and acid-base composition of the extracellular (blood plasma, lymph, interstitial fluids) and intracellular fluid compartments. Therefore it must be appreciated that the acid-base status of the blood depends greatly on where and when the blood is sampled. Blood draining intensely contracting skeletal muscle has very high concentrations of H⁺, lactate, K⁺ and CO₂ while blood that drains relatively inactive tissues (jugular venous blood for example) has markedly lower concentrations of these metabolites and ions; arterial blood is intermediate in composition. Also, the magnitude of change is proportional to the intensity and duration of exercise, and the concentrations of these and other substances change with time of exercise and recovery.

Why is acid-base balance important? A detailed analysis of acid-base balance can provide a complete biochemical and physicochemical description of the state of the organism, or of individual organs and tissues within the body. Furthermore, severe acid-base disturbances are often associated with high intensity exercise, prolonged duration exercise and with many pathologies. Therefore an understanding of the origins of acid-base disturbance is of interest to both basic and clinical physiologists. Within the context of the present chapter, exercise physiologists remain keenly interested in acid-base balance because of a close association between acidification and muscle fatigue.¹³ The content of this chapter is primarily directed to moderate to high intensity exercise because exercise at these intensities produces a significant acid-base disturbance, while exercise at low intensities does not (unless markedly prolonged with underlying dehydration and metabolic abnormalities). For detailed reviews of skeletal muscle acid-base balance during exercise the reader is referred to Hultman and Sahlin¹⁵ and Lindinger and Heigenhauser.¹³ Treatments of plasma acid-base status have been provided by Constable,^16,17 Johnson et al.¹⁸ and Lindinger and Heigenhauser.¹³ There are also a number of brief reviews on the effects of exercise on acid-base status in horses.^16,17,19,20 There are also a number of clinical primers on assessing and treating acid-base disturbances.^17,21–23

Acidosis and skeletal muscle fatigue

There is no question that high intensity muscle contraction results in intracellular acidification13,39,40 that generates an extracellular, systemic acidosis in the whole organism that can be very pronounced and long lasting.⁹ It is also clear that intracellular acidosis and fatigue are associative during high intensity exercise, with mounting evidence that increased [H⁺] reduces the calcium sensitivity of the contractile proteins.⁴¹ Furthermore, acidosis imposed prior to the period of high intensity exercise results in an earlier onset and more pronounced skeletal muscle fatigue.^42–44 Intracellular acidosis may, however, only exert these effects during high intensity muscle contraction. Recent evidence has shown that the contributions of intracellular acidosis to fatigue process have yet to be fully understood (Table 39.2). Westerblad and colleagues³³ have suggested that increased intracellular concentrations of inorganic phosphate may be a more important contributor to muscle fatigue than the increase in [H⁺]. Pedersen et al.^37,38 have shown that intracellular acidosis can contribute to sustaining muscle function during contractile activity by improving membrane excitability, an effect that appears to involve alterations of sarcolemmal chloride conductivity.^45,46

Table 39.2

Effects of increased intramuscular [H⁺] and functional consequence(s) in muscle

Decreased glycogenolytic (phosphorylase) activity24,25 → decreased anaerobic ATP production → ATP supply is limited → fatigue Decreased glycolytic (phosphofructokinase) activity24,26,27 → decreased anaerobic ATP production → ATP supply is limited → fatigue Decreased pyruvate dehydrogenase activity24 → decreased aerobic ATP production → ATP supply is limited → fatigue Decreased sarcolemmal and SR Ca2+-ATPase activity28–30 → elevated cytosolic [Ca2+] → decreased myosin ATPase activity29 → decreased rate of actin-myosin cross-bridge cycling → slowed rate of muscular contraction → fatigue Increased [H+] increases the [diprotonated inorganic phosphate] → Inhibition of actin-myosin cross-bridge interaction resulted31–33 Inhibition of Ca2+ binding to troponin C,29,34,35 resulting in decreased number of actin-myosin cross-bridges formed → decreased strength of force production → fatigue Increased acetyl-CoA, indicative of increased intramuscular triacylglycerol hydrolysis24 → increased citric acid cycle dehydrogenase activities → stimulate aerobic metabolism Decreased lactate efflux from muscle cells36 → prolongation of intracellular acidification by retaining a strong acid anion Increased intracellular [H+] → improved membrane excitability37,38

Skeletal muscle fatigue is also associated with an increased interstitial [K⁺] as a result of rapid rates of K⁺ loss through sarcolemmal K⁺ channels during the recovery phase of action potentials.47,48 This increase in interstitial [K⁺] results in a marked depolarization of the sarcolemma and decreased contractile force.^48,49 In contrast to the dogma that we have long been taught, the loss in both sarcolemmal excitability and tetanic force resulting from elevated interstitial [K⁺] (8–12 mEq/L) was actually reversed when intracellular acidosis (either 20 mmol/L lactic acid or increased CO₂) was imposed.^37,50,51 While these muscles were only stimulated to perform one contraction every 10 min, this allowed a separation between the fatigue associated with repetitive contraction versus that associated with sarcolemmal depolarization and intracellular acidification.

As summarized by Fitts⁵² and Allen,⁴⁰ increased [H⁺] does contribute to decreased force production during high intensity muscle contraction (see Fig. 39.1), and there is reasonably good evidence that these effects occur at the level of: (i) impaired Ca²⁺ binding to troponin C, which therefore impairs the ability of actin to form cross-bridges with myosin; (ii) slowing SR Ca²⁺-ATPase activity; (iii) increasing the leak of Ca²⁺ from the SR; (iv) and at key sites of biochemical control within glycogenolysis (decreased glycogen phosphorylase a activity) and glycolysis (decreased phosphofructokinase activity. The latter study also demonstrated an increased reliance on fat metabolism to meet the energy demands of contracting muscle during exercise in humans made acidotic by ingestion of 0.3 g/kg ammonium chloride.²⁴

It may be concluded that intracellular acidosis may have two main effects that, when taken together, are of long-term benefit for muscle function and survival (prevention of destruction resulting from overuse). First, acidification restores the sarcolemmal excitability and contractility resulting from elevated interstitial [K⁺], and the former is very important for cells maintaining the composition of their intracellular environment within physiological limits. Second, the muscle retains the ability to contract while the force and rate of contraction and rates of glycogenolysis / glycolysis are slowed as a result of the acidosis. This in turn slows the demand for energy and the production of acid equivalents, while allowing the animal to continue to move if need be.

Assessment of acid-base balance and factors that affect acid-base regulation

In traditional terms, many of us remember being taught that acid-base balance is represented by the relationships among PCO₂, pH and the HCO₃⁻ in blood plasma. While this is true, using only these three variables provides for only a very limited understanding of the factors that contribute to acid-base imbalances. The approach taken within this chapter is to use a comprehensive, physicochemical approach to identify the causes or origins of acid-base disturbances during exercise, and to discuss how the disturbance is resolved during recovery from exercise.¹³ Several key papers have used this approach in horses.^9,12,53–58

While the traditional variables of acid-base – PCO₂, pH and the HCO₃⁻ – are useful in identifying whether an acid-base disturbance is metabolic or respiratory in nature, they are insufficient to identify the physicochemical origins of the acid-base disturbance. It is nonetheless important for the student of acid-base physiology to be familiar with the concepts presented using the traditional approach, and to be able to use these concepts as an important foundation on which to apply the physicochemical approach. This chapter will emphasize the use of the physicochemical approach as this method provides for a detailed physiological and clinical assessment of acid-base disturbances. It is worth pointing out that the traditional approaches to assessing acid-base status are not incorrect, but rather they were a simplification introduced in the 1960s to make use of readily available and relatively simple measurements of PCO₂ and pH. Technology developed from the 1970s onwards has simplified the measurements of the other important acid-base variables in blood plasma and skeletal muscle, allowing us to take a more comprehensive approach.

The physicochemical approach presented here was detailed by Peter Stewart.59–62 The foundation for this approach lies on the work of van Slyke and co-workers,^63,64 and builds on the work of many others including Hastings, Dill, Henderson and Siggaard-Anderson.¹³

Physicochemical characteristics refer to those properties and reactions that are physical and chemical in nature; they proceed in the absence of enzymes and life and occur as a result of the physical and chemical properties of the solvent and solute molecules. Also, biochemical reactions, those catalyzed by enzymes, may alter the physicochemical properties of a solution. However, for the purposes of discussing acid-base balance, biochemical reactions may be considered distinct from physicochemical reactions. The main physicochemical reactions are detailed below.

The advantages and disadvantages of the physicochemical approach are listed in Table 39.3. The development and widespread use of ion selective electrodes and combination blood gas–electrolyte analyzers has greatly simplified the process of obtaining the necessary measurements with the accuracy needed to perform detailed assessments of acid-base balance.¹³ The advantages of this approach lie in the ability to quantitatively determine the physical and chemical origins of acid-base disturbances. This is therefore a very powerful approach and an important step towards understanding acid-base physiology and pathophysiology. This approach provides an essential foundation for the effective treatment of pathological acid-base disorders.

Physicochemical determinants of acid-base balance

Prior to detailing the physicochemical reactions that increase [H⁺] within contracting skeletal muscle, it is necessary to provide an introduction to the physicochemical system of acid-base balance. This approach is founded on three underlying physical premises:

1. A dissociated proton molecule (H⁺) is only in physical existence for a fleeting instance of time, approximately 10⁻⁵ second. The proton is highly reactive, associating briefly with negative charges on proteins, ⁻OH molecules, HCO₃⁻ molecules and amino acids to name a few. The proton is therefore very unlike inorganic electrolytes such as Na⁺, K⁺ and Cl⁻ that are relatively unreactive.

2. Protons are a main constituent of water, the most prevalent molecule within the body. Water thus provides an almost limitless source of H⁺ for biochemical and physicochemical reactions. Protons are part of the solvent that comprises the milieu of the body. It is because of its ability to so rapidly dissociate and re-associate H⁺ and ⁻OH that water is the ‘universal’ solute.

3. Because of these physical attributes of protons and water, it is physically impossible to add protons to a physiological solution without adding water. Take hydrochloric acid (HCl) as an example. HCl exists only in aqueous form and is characterized by a very high concentration of Cl⁻ and H⁺ in solution. The H⁺ is an integral part of the aqueous system. It will be described below that it is the strong acid anion Cl⁻ that makes this solution so acidic. The strong acid anion Cl⁻ can be neutralized by the addition of an equivalent amount of the strong base cation Na⁺ to the solution, but without an accompanying acid anion such as Cl⁻, HCO₃⁻, of H₂PO₄⁻. Thus NaOH would be added – the strong anions Cl⁻ and Na⁺ remain fully dissociated in solution while there occurs a rapid reaction between H⁺ and ⁻OH that decreases [H⁺]. The resultant solution is saline at neutral pH.

The physicochemical approach to acid-base balance recognizes that there are three groups of independent variables that determine the concentrations of the traditional acid-base variables pH and [HCO₃⁻]. The three groups of independent variables are: (1) the strong ion difference (SID), which represents the sum (charge considered) if the strong acid anions and strong base cations; (2) the total weak acid concentration (A_tot), which represents the sum (charge considered) of the weak acids and bases; and (3) the carbon dioxide (CO₂) concentration, which is usually measured and used as the partial pressure of CO₂ (PCO₂).

Strong ions and strong ion difference

The terms strong acid anion and strong base cation were introduced in the preceding section and they will be defined here. The term ‘strong’ refers to the fact that the ion will be fully, or nearly so, dissociated in aqueous solutions (Fig. 39.2). Most of the inorganic ions are ‘strong’ and hence nearly fully dissociated within the body fluids (Table 39.4). Some organic ions are also strong, such as lactate⁻ (acid dissociation constant of 3.9) and phosphocreatine²⁻ (PCr²⁻; acid dissociation constant of 4.5). Anions possess negative charge while cations possess positive charge. An anion is an acid by definition because the addition of that strong anion, in the absence of an accompanying strong base, will result in acidification of the solution. Using the example of HCl above, the addition of HCl to plasma will result in acidification. Similarly, the addition of HLactate will also result in acidification. In contrast, the addition of the strong base Na⁺ in the absence of accompanying strong anion (as NaHCO₃⁻) will result in alkalinization. The values for the key variables used in the physicochemical assessment of acid-base balance, for resting horses, are provided in Table 39.5.

Table 39.4

A summary of acid-base terminology

The following definitions are placed in order of functional similarities, as opposed to alphabetical order. Base: any cation in biological fluids65 Buffer base: base equivalent to the sum of buffer anion concentrations (including [HCO3−]) in mEq/L65 base excess / deficit: represents the accumulation of non-volatile base / acid in the blood (excludes plasma [HCO3−] and blood hemoglobin concentration)66 Alkali (alkaline) reserve: the proton buffering ability of plasma bicarbonate when bases or non-volatile acids are added to or taken from the body fluid65 [Atot]: a physicochemical term that defines the total concentration of weak anions in solution60–62 [SID]: a physicochemical assessment term that refers to the sum of all strong base cations minus the sum of all strong acid anions60–62 [SID] = Σ[strong base cations] – Σ[strong acid anions] Strong ion: that ions that are fully, or nearly so, dissociated in physiological solutions. In general, if the dissociation constant is ≤ 4.5, then the molecule is considered to be a strong anion; if the dissociation constant is greater than 9, then the molecule is considered to be a strong cation. Anion gap: a traditional term that is defined as: anion gap = ([Na+] + [K+]) – ([Cl−] + [HCO3−]) Strong ion gap: a term coined by Constable et al.23 as an alternate way of determining the concentration of unmeasured strong ions in plasma: strong ion gap = 2.24 × total [protein] (g/dl) / (1 + 10(6.65−pH)) – AG     (where AG is anion gap). Unmeasured anions: unmeasured anions contribute to the anion gap, strong ion difference and strong ion gap. The unmeasured anions include both strong (SO42−, some amino acids, pyruvate) and weak (inorganic phosphate, carbonate, carbamates, some amino acids) anions. The negative charges on plasma protein contribute to the anion gap, strong ion difference and strong ion gap, but this is usually a ‘measured’ anion.

Table 39.5

Physiologically important acid-base variables, and their concentrations, in arterial plasma and skeletal muscle of horses at rest

VARIABLES	PLASMA		SKELETAL MUSCLE
Dependent variables	‘normal’ value	normal range	‘normal’ value	normal range
[H+] nEq/L	40	33–45	100	71–126
pH	7.40	7.35–7.48	7.0a,b	6.90–7.15a,b
[HCO3−] mEq/L	28	22–34	10a	8–12a
Independent variables
PCO2 mmHg	40	35–45
[total CO2] mmol/L	30	23–36	10a	8–12a
[SID] mEq/L	40	37–43	110c	60–130c
Strong ions
[Na+] mEq/L	140	132–146	15	8–30
[K+] mEq/L	3.7	2.7–4.7	140	110–165
[Ca2+] mEq/L	2.5	2–3	0.4	0.2–1.0
[Mg2+] mEq/L	1.0	0.5–2.0	5	3–7
[Cl−] mEq/L	105	99–109	10	5–25
[lactate−] mEq/L	1.0	0.5–1.5	1.5	1.0–2.0
[PCr2−] mEq/L	na	na	15	12–18
[SO42−] mEq/L	0.5	0.3–0.7	–	–
Weak ions
[Atot] mEq/L	12	11–13	140	120–170
[plasma protein] g/dl	5.5	5.0–6.0	na	na
[albumin] g/dl			na	na
[globulins] g/dl			na	na
[HPO42−] + [H2PO4−] mmol/L	2.7	2.0–3.5	8	7–9
carnosine mmol/L	na	na	6b	3–9b
Protein histidine concentration mmol/L	–	–	46b	30–60b

na = not applicable.

^aSahlin et al.⁶⁷ – human muscle.

^bHultman and Sahlin¹⁵ – human muscle.

^cLindinger.13,68

The concentrations of strong acid anions and strong base cations within a fluid compartment are summed, with consideration of the charge, to yield the strong ion difference, or [SID].

Within plasma and the extracellular fluid compartment, the [SID] may be calculated as:

[SID] (mEq/L)=([Na+]+[K+]+[Mg2+]+[Ca2+])− ([Cl−]+[lactate−]+[SO42−])

Note that it is the free or ionized concentrations of the divalent ions that must be used, and not the total concentration; considerable amounts of the divalent ions are bound to plasma proteins or to each other. The concentrations measured using ion selective electrodes are that of the free or ionized or dissociated ion in the aqueous portion of the solution (i.e. mEq/L of plasma water), so long as the instrument does not use a calculation to modify the ‘concentration’ to total (not free) concentration or mEq/L of plasma. Thus, while these divalent ions are ‘strong’, the interactions with charged moieties on protein molecules removes some of the ion from solution. In practice, the free concentrations of the divalent cations and anions are approximately equivalent and can be ignored, leaving:

[SID]plasma (mEq/L)=([Na+]+[K+])−([Cl−]+[lactate−])

In some treatments of acid-base balance using the physicochemical approach, [lactate⁻] is also ignored. However [lactate⁻] cannot be ignored in the exercising and recovering animal.

Within skeletal muscle, PCr²⁻ and Mg²⁺ must be used within the equation because their free concentrations are large and change substantially during exercise:

[SID]muscle (mEq/L)=([Na+]+[K+]+[Mg2+])− ([Cl−]+[lactate−]+[PCr2−])

The strong ions are important determinants of the concentrations of [H⁺] and [HCO₃⁻] because they directly affect the associated state of H₂O, and thereby determine the concentrations of H⁺ and ⁻OH.

A decrease in the SID (without concurrent change in PCO₂ or A_tot), due to either a decrease in strong cation concentration OR an increase in strong anion concentration will increase [H⁺] and decrease [HCO₃⁻] – an acidification occurs. Conversely, an increase in SID has an alkalinizing effect and decreases [H⁺] and increases [HCO₃⁻].

Weak acids and bases, and [A_tot]

The term ‘weak’ refers to those anion acids and cation bases that are not fully dissociated in solution. Thus when sodium phosphate (Na₂HPO₄) is added to an aqueous solution, two Na⁺ are added and fully dissociates and a weak anion HPO₄²⁻ is added. In contrast to Na⁺, the HPO₄²⁻ cannot achieve full dissociation due to reactions of the molecule with H⁺ within the solution. Thus the HPO₄²⁻ is also partially and instantaneously transformed into H₃PO₄, H₂PO₄⁻ and PO₄³⁻ (Fig. 39.2). This physical attribute of phosphate is what makes phosphates, and many other weak acid anions such as bicarbonate and albumin, good proton ‘buffers’. The predominant weak acid anion in plasma and ECF is albumin, while the predominant weak acid anions within skeletal muscle cells are the histidine residues on proteins.

The main weak acids and bases within the extracellular fluid compartment are albumin, globulin, phosphate and bicarbonate. Bicarbonate, however, is part of the CO₂ system and thus is not used in the calculation, or estimation, of [A_tot]. As for the strong ions, the weak ions also directly affect the concentrations of H⁺ and HCO₃⁻ in solution. Within skeletal muscle it is primarily the histidine moieties on proteins that contribute to [A_tot], with creatine, Pi, ATP and other molecules also contributing.⁶⁸ While it is theoretically possible to measure the concentration of weak acids and bases in both extracellular and intracellular fluid compartments, this tends to be prohibitive and appears not to be necessary to be able to effectively estimate acid-base state. Rather, an effective [A_tot] and apparent dissociation constant K’a have been determined in equine plasma and rat skeletal muscle (Table 39.6). A value for [A_tot] has not been determined in equine or human skeletal muscle. Muscle [A_tot] is equivalent to the non-bicarbonate proton buffering capacity of adult rat plantaris muscle,^68–70 and is similar to that of human vastus lateralis.^15,71,72 When rat plantaris values for [A_tot] and K’a were applied to human muscle, reasonably good data were generated.⁶⁸ Equine muscle, compared to human muscle, has a much greater non-bicarbonate proton buffering capacity: 43 mEq.kg dry muscle⁻¹.pH⁻¹ in trained humans, vs 58 and 93 mEq.kg⁻¹.pH⁻¹ in untrained and trained equine skeletal muscle.⁷³ Assuming proportionality with rat hindlimb skeletal muscle (buffer capacity of ~40 mEq.kg⁻¹.pH⁻¹ = [A_tot] of ~140 mmol/L⁶⁸), this translates to an [A_tot] of ~315 mmol/L in trained equine muscle.

Table 39.6

Values of the constants used within the acid-base equations

PARAMETER	CONSTANT	REFERENCE
KA – plasma	2.11 or 2.12 × 10−7 Eq/L	Constable 1997;17 Staempfli et al. 199974
	1.74 × 10−7 Eq/L	Waller et al. 201012
KA – resting muscle	1.64 × 10−7 Eq/L	Lindinger 199568
KA – exercised muscle	1.98 × 10−7 Eq/L	Lindinger 199568
K3	6.0 × 10−11 Eq/L	Stewart 198362
KC	2.46 × 10−11 (Eq/L)2/mmHg	Stewart 198362
K’w	4.4 × 10−14 (Eq/L)2	Stewart 198362

The carbon dioxide system

The concentration of CO₂ is the third independent variable of acid-base balance. Carbon dioxide is effectively a strong acid, and because it is a major end-product of cellular respiration is often referred to as a respiratory acid. Also, the primary means for eliminating excess CO₂ from the body is through the respiratory system.13,18

Carbon dioxide is a strong acid by virtue of its ability to combine with water to increase the concentration of H⁺ while at the same time increasing the weak acid [HCO₃⁻]. This reaction effectively acidifies the solution to which CO₂ has been added. The majority (about 95%) of the total CO₂ within the body is in the form of HCO₃⁻, with much smaller amounts of H₂CO₃, CO₃²⁻, dissolved CO₂ (CO_2(d)) and some that is bound to amino groups on protein to form carbamino compounds. The chemical reactions involved in the hydration and dehydration of CO₂ are:

CO2+H2O⇌H2CO3⇌H++HCO3−⇌2H++CO32−

Solving equations to determine acid-base balance

With this background, the following five mass action equations and one equation expressing electrical neutrality of solutions describe the physicochemical characteristics of any aqueous, physiological solution:60–62

Water dissociation:

K′ w = [H+]⋅[−OH]

Weak electrolyte system:

KA⋅[HA]=[H+]⋅[A−]

[Atot]=[HA]+[A−]

Carbon dioxide system:

Kc⋅PCO2=[H+]⋅[HCO3−]

K3⋅[HCO3−]=[H+]⋅[CO32−]

Electrical neutrality:

[SID]+[H+]−[HCO3−]−[A−]−[CO32−]−[−OH]=O

It is noteworthy that [H⁺] appears in each of these equations and its dependence on the concentrations of strong and weak acids/base and CO₂ is evident. These six equations can be combined into a single equation that may then be solved for [H⁺] when the three independent variables and the constants are known:

[H+]4+{KA=[SID]} [H+]3={KA([SID]−[Atot])−(KCpCO2+K′ w)}   [H+]2–{KA(KCpCO2+K′ w)+K3KCpCO2}   [H+]–KAK3KCpCO2=0

Contracting skeletal muscle: proton generating and removing reactions

When considering the acid-base changes that occur in blood during exercise, it is important to have an understanding of the changes that occur within skeletal muscle because that tissue forms 40–60% of the mass of the horse.⁷⁵ Contracting skeletal muscle generates the disturbance^76,77 and non-contracting cells are capable of ameliorating the disturbance.^78,79 The role of non-contracting muscle may be small to negligible in horses performing moderate to high intensity exercise because most skeletal muscles are used for locomotion and maintenance of posture. That is in contrast to bipedal humans where many activities require leg muscles and leave many other muscles relatively inactive. This section will thus focus on the time course and magnitude of changes that occur within contracting skeletal muscle, primarily gluteus medius, during moderate to high intensity exercise.

Exercise is a consequence of muscular contraction, and muscular contraction results in an increase in cellular energy demand compared to the resting state. The increased energy demand is due to activation of myosin ATPase needed for release of actin-myosin cross-bridge interaction, increased activity of sarcoplasmic reticulum Ca²⁺-ATPase activity resulting from increased cytosolic [Ca²⁺] and increased rates of Na,K-ATPase activity needed to maintain transmembrane Na⁺ and K⁺ gradients and repolarization of the muscle membrane potential.

The acid-base changes that occur within contracting skeletal muscle and in blood during exercise are the results of the biochemical (metabolic) and physicochemical reactions that occur within contracting muscle cells.¹³ The onset of muscular contraction sets into motion a series of biochemical events that result in stimulation and inhibition of numerous metabolic pathways. Those pathways within the aerobic energy systems are relatively slow to increase, while those of the anaerobic pathways (ATP utilization, phosphocreatine degradation, glycolysis) increase rapidly. Thus the onset of exercise (rest to work transition) may be associated with muscular acidification for reasons described below. Similarly, transitions from low to high work rates, as well as exercise at moderate to high intensities, result in increased rates of anaerobic metabolism. Full activation of aerobic pathways may be achieved within minutes of the onset of exercise, but until this is achieved anaerobic pathways continue to supply ATP. Activation of aerobic metabolism results in increased mitochondrial respiration with CO₂ production – while this CO₂ is acidic, its rate of production and removal from the cell can easily be matched by CO₂ elimination rates at the lung. Therefore aerobic CO₂ production can be ignored in most discussions of the acid-base changes of exercise.

Muscle characteristics and acid-base

Skeletal muscle is composed of different fiber types, some of which produce acid equivalents at high rates (the anerobic, fast twitch, glycolytic fibers) while others do not (the aerobic, slow twitch, oxidative fibers). Fiber types continue to be classified on the basis of their twitch characteristics, oxidative / glycolytic capacities and on their myosin heavy chain composition.⁸⁰ The acid-base changes that occur reflect the fiber type composition of the contracting muscles, and may reflect breed differences and type of activity performed. This is tempered by the high degree of variability in muscle fiber type proportions in most horse breeds.^81,82

Within individual muscle groups, such as the well-studied gluteus medius of equids, skeletal muscle fibers of different composition are in close proximity and form integrated functional units that are selectively recruited by appropriate motor units depending on the locomotory requirements of the animal.⁸³ Muscle fibers with high oxidative capacity that have low glycoytic capacity, slow contractile properties with low myosin ATPase activity, are fatigue resistant and primarily function in the maintenance of posture (Table 39.7). These slow twitch oxidative fibers have the ability to oxidize all the pyruvate generated from glycolysis and, during exercise, they have the ability to take up and oxidize lactate released into the interstitium from nearby glycolytic muscle fibers – the intramuscular lactate shuttle.⁸⁴

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