pH maintenance in animals

The maintenance of pH is a collection of processes that regulate the hydrogen ion concentration of body fluids. The total body fluids account for about 60 percent of body mass. The body fluids are divided into several compartments. The largest volume of body fluid is in the intracellular compartment, and this consists of about 40 percent of body mass. The extracellular fluid consists of interstitial fluid (about 15 percent of body mass) and several specialized compartments. The largest of the specialized compartments is the vascular space, or blood volume. About half of the blood volume is in blood cells that belong to the intracellular volume. The other half is plasma, which makes up about 5 percent of the body mass. There are several other small extracellular spaces, such as the cerebrospinal fluid and the aqueous and vitreous humors of the eyes, that comprise smaller percentages of the extracellular volume. Each of these compartments is regulated to maintain normal pH. The pH levels of the different compartments can be very different. In mammals, the extracellular pH, as measured in the plasma, is generally 7.4. The specialized fluids within the extracellular compartment can differ. For example, the cerebrospinal fluid has a pH of 7.32. Intracellular pH can vary from cell type to cell type but tends to be lower than extracellular pH. For example, skeletal muscle has an intracellular pH of 6.89, while red blood cells have a pH of 7.20.

pH Maintenance

The first line of defense in the maintenance of pH is using chemical buffers. Buffers are chemicals that resist pH change by absorbing hydrogen ions from acidic solutions or contributing hydrogen ions to alkaline solutions. They are made by producing a mixture of a weak acid and the salt of a weak acid. An example of a buffer is given by the bicarbonate buffer system. The components are H₂CO₃ (weak acid) and NaHCO₃ (salt of the weak acid). When a strong acid, such as HCl, is added to a solution containing this buffer pair, the H⁺ released from the highly dissociated HCl combines with HCO₃^–; that is completely dissociated from the NaHCO₃ to form weakly dissociated H₂CO₃. This prevents the large pH drop that would otherwise occur because the H₂CO₃ is a weak acid. This buffer system is primarily extracellular. The body fluids also contain intracellular buffer systems which can resist pH changes when acids are introduced.

Acids can be introduced in several ways. Exercise and oxygen deprivation cause a buildup of lactic acid as a result of anaerobic metabolism. Metabolism of sulfur-containing amino acids causes the production of sulfuric acid, another strong acid. Both aerobic and anaerobic metabolism cause the buildup of carbon dioxide, which combines with water to form carbonic acid; even though this is a weak acid, the large accumulation of carbon dioxide can lower the pH.

Acids produced by any of these means are buffered by one of the buffer systems in the body. In addition to the bicarbonate (NaHCO₃) buffer system described above, there are two intracellular buffers that participate in pH maintenance. In the phosphate buffer system, monosodium phosphate (H₂NaPO₄), a weak acid, dissociates to produce H⁺ and HNaPO^4–. The H⁺ is exchanged for Na⁺ by the kidneys or buffered on protein, leaving the salt disodium phosphate (HNa₂PO₄). This salt dissociates into Na⁺ and HNaPO₄^–. The HNaPO₄^– can then absorb H⁺ ions, which have dissociated from strong acids to form H₂NaPO₄, which is weakly dissociated. The phosphate buffer system is the least important of the buffer systems because there is so little phosphate in the body. The most important buffer system in the body is protein. Some of the individual amino acids in proteins can act as weak acids and accept H⁺ ions; the amino acid histidine is the most significant of these under the temperature and pH conditions of the body. Protein buffering is primarily an intracellular phenomenon, as the protein concentration in extracellular fluid is relatively low. The intracellular protein buffer makes up about three-quarters of the total body buffering capacity.

Acid-Base Disturbances

The most important extracellular buffer is the bicarbonate buffer system discussed above. This system is aided directly by a second line of defense against pH disturbances. This is called physiological buffering. Physiological buffering refers to the fact that the supply of the most important components of the bicarbonate buffer system, carbon dioxide and bicarbonate, can be controlled. The organs responsible for this control are the lungs (carbon dioxide) and kidneys (bicarbonate). For example, during exercise, the buildup of lactic acid consumes bicarbonate (HCO₃^–) and lowers pH. This is called metabolic acidosis. The low pH stimulates the rate of breathing, which in turn results in increased elimination of carbon dioxide by the lungs. This lowers the partial pressure (concentration) of carbon dioxide in the blood, reducing the amount of carbonic acid (H₂CO₃) that can dissociate to form H⁺. The reduction of carbon dioxide is called respiratory alkalosis. This respiratory alkalosis compensates for the metabolic acidosis caused by the depletion of bicarbonate. The kidneys provide the final correction for the initial metabolic acidosis. These organs accomplish this in two ways: reabsorption of bicarbonate and secretion of H⁺ into the urine in exchange for Na⁺. When bicarbonate concentration in the extracellular fluid is reduced by a metabolic acidosis, the hormone aldosterone, produced by the adrenal glands, stimulates the secretion of H⁺ and reabsorption of bicarbonate. This raises both the pH and bicarbonate concentration back to normal. The increased pH relaxes the stimulation of breathing, allowing the breathing rate to slow and the partial pressure of carbon dioxide to return to normal.

The above is an example of one of the four basic types of acid-base disturbance—metabolic acidosis (bicarbonate concentration reduced), metabolic alkalosis (bicarbonate concentration increased), respiratory acidosis (an increase in the partial pressure of carbon dioxide), and respiratory alkalosis (a decrease in the partial pressure of carbon dioxide). As before, metabolic acidosis is compensated for by respiratory alkalosis. This is part of a general pattern in which one condition is compensated for by the opposite condition. Another example would be a respiratory acidosis, lowering the pH and stimulating bicarbonate retention and H⁺ excretion in the kidneys to produce a compensatory metabolic alkalosis.

Body Temperature and Ion Exchange

Temperature also affects pH. This is usually of no consequence to warm-blooded animals, which have a constant body temperature. It is, however, of great consequence to cold-blooded animals, such as fish, amphibians, and reptiles, in which body temperatures vary with the environmental temperature. There is a decrease in pH by about 0.015 for each degree increase in temperature. This means that the pH at 5 degrees Celsius is 7.88, or 0.48 pH units higher than at mammalian body temperature (pH 7.40 at 37 degrees Celsius). This does not mean that cold-blooded animals do not regulate pH. While the regulation differs from that of warm-blooded animals in that pH varies, it is still regulated in the sense that it varies in a very predictable manner. By controlling partial pressures of carbon dioxide and bicarbonate ion concentrations in much the same way that mammals do, cold-blooded vertebrates maintain their body fluid pH at a constant 0.6 to 0.8 pH units higher than the pH of pure water at the same temperature. It is, thus, the relative alkalinity that is regulated rather than the pH.

While the kidneys of mammals are intimately involved in pH maintenance, the situation in lower vertebrates can be much different. Most fish primarily use ion exchange transport systems in their gills to regulate pH. Specialized cells in the exterior lining of the gill surfaces transport sodium ions (Na⁺) from the water into the blood in exchange for H⁺. Similarly, chloride ions (Cl^–) are transported into the animal in exchange for HCO₃^–. When fish become acidotic, they increase Na⁺/H⁺ exchange and decrease Cl^–/HCO₃^– exchange. This results in a net elimination of H⁺ and a net conservation of HCO₃^– to elevate the pH of the body fluids.

While the gills are the primary organ fish use to maintain acid-base balance, their kidneys also play an important role in pH maintenance, particularly in freshwater fish. When bodies of water become too acidic for aquatic and marine life, animals exhibit a variety of behaviors. Clownfish have decreased responses to threats, struggle to navigate with their diminished sense of smell, and swim farther from home than usual. The game fish named Cobia may grow small earbones that impede hunting and navigation. The kidneys of amphibians also play a role in pH maintenance. The major regulatory organs in these animals are the skin and urinary bladder. Aquatic salamander larvae increase Na⁺/H⁺ exchange and decrease Cl^–/HCO₃^– exchange across their skin to regulate internal pH. Terrestrial toads that are seldom in contact with water sometimes become dehydrated. They can use their urinary bladders for pH-regulatory ion exchanges by producing extremely acidic urine. Relatively little is known about the role of the reptilian kidney in pH maintenance. The alligator kidney plays a major role in acid elimination. They produce urine with a pH of 7.76. The urinary bladder of turtles also participates in pH regulation in much the same manner as the urinary bladder of the toad. The bird kidney plays a major role in pH maintenance, and its urine pH values are between 6.5 and 8, which is similar to the pH values of mammalian urine. Avian kidneys have cortical and medullary nephrons and they filter the bird’s total body water up to eleven times each day.

Studying pH Maintenance

The regulation of pH is usually studied by measuring the pH and the partial pressure of carbon dioxide of the blood. This measurement of the regulation of extracellular pH is a reflection of the overall acid-base status of the individual being studied. The first requirement is to be able to sample blood from an undisturbed, resting subject. This usually means an indwelling arterial cannula, through which blood from an artery can be sampled. The blood is then injected into a chamber in a blood-gas analyzer containing a glass pH electrode that measures the pH of the plasma. Additional blood is injected into another chamber that contains oxygen and carbon dioxide electrodes that measure the partial pressures of these two gases. Bicarbonate concentration can be calculated from the mathematical relationship among pH, the partial pressure of carbon dioxide, and the bicarbonate concentration when the former two are known. When making these kinds of measurements, it is important to measure the pH and partial pressure of the blood at the same temperature as the animal. Thermostated electrodes are used for this. The calculation used to estimate bicarbonate concentration requires the use of two constants which are also temperature-dependent. It is important to use the constants that are appropriate to the temperature of the animal and the measurement conditions.

Alternatively, bicarbonate concentration can be approximated by measuring the total amount of carbon dioxide (the sum of the carbon dioxide, carbonic acid, bicarbonate, and carbonate concentrations). Under physiological conditions, more than 99 percent of the carbon dioxide is in the form of HCO₃^–. When strong concentrated acid (HCl) is added to a measured volume of blood or plasma, all the carbonic acid, bicarbonate, and carbonate are released as carbon dioxide. This carbon dioxide can then be measured with a carbon dioxide electrode. The resulting total carbon dioxide, minus the concentration of carbon dioxide measured before adding the HCl, is very close to being equal to the concentration of bicarbonate.

While there are a number of indirect methods for measuring intracellular pH, the best results are achieved directly with micro pH electrodes which can be used to impale single cells. These techniques must be done on isolated tissues or anesthetized, restrained animals, whose acid-base status does not, in the least, resemble that of resting, undisturbed animals. The techniques are useful for studying pH maintenance at the cellular level and useful extrapolations can be made to the whole animal.

The usual approach to the study of pH maintenance is to induce a disturbance in the pH of an experimental animal and then to follow changes in the pH, partial pressure of CO₂, and the concentration of HCO₃^– in the blood. Breathing rates and volumes of air exchanged by the lungs can be measured to determine the contribution of the lungs to pH maintenance. For example, increasing the rate of breathing will increase the rate of elimination of carbon dioxide and lower the partial pressure of this gas in the blood. Urine collection can be done to assess the contribution of the kidneys to pH maintenance, and in aquatic animals that do not rely heavily on their kidneys for pH maintenance, changes in the composition of the water in which the animals are situated can be used to assess the contribution of the gills (fish) or skin (amphibians) to pH maintenance.

Examples of experimental manipulations that can produce pH disturbances in animals include infusion of acids and bases into the blood to produce metabolic acidosis and alkalosis, respectively. Alternatively, animals can be exercised to elevate lactic acid in their blood to produce a metabolic acidosis. Respiratory acidosis and alkalosis can be induced by artificially altering breathing rates. Alternatively, elevation of the partial pressure of carbon dioxide in the air or water of an experimental animal will produce respiratory acidosis.

pH Maintenance and Homeostasis

Maintenance of pH is part of the overall phenomenon of homeostasis or constancy of the internal environment in a changing external environment. The primary reason for maintaining a consistent acid-base balance is the need to keep constant the conformation (shape) of proteins. Proteins are very sensitive to changes in pH, and the usual consequence of such changes is a change in the shapes of proteins. Protein conformation is critically important to proper protein function.

All enzymes (proteins that cause necessary biochemical reactions to occur) have very specific shapes that must be maintained for them to function properly. Changes in pH cause alterations in those shapes and compromise the effectiveness of the enzymes. Many other proteins require certain conformational states. Cell membranes contain proteins that determine which chemicals can enter and leave the cell’s interior. These proteins are called carriers if they actively transport specific substances into or out of the cell interior. Alternatively, membrane proteins can act as specific channels that allow passive movement of only specific molecules or ions across the membrane. Both carriers and channels are sensitive to pH-induced conformational changes. There are other membrane-bound proteins called receptors that respond best to hormones and other chemical messengers when the pH is maintained at the norm. Proteins in the immune system, such as antibodies, also require proper pH to function optimally.

Cold-blooded animals do not regulate their pH to a specific point, but instead regulate the difference between their pH and the pH of pure water at the same temperature. This relative alkalinity ensures constant protein conformation in the same way that constant pH at constant temperature ensures constant protein conformation in warm-blooded animals because the weak acid nature of proteins causes them to be partially dissociated from H⁺ in the pH and temperature range experienced by animals. The conformation of protein is determined, to a large extent, by the net electrical charge (distribution of + and – charges over the protein). The net charge is influenced by the degree of dissociation of the protein and H⁺. As discussed above, the dissociation in protein that is important to pH maintenance is the dissociation of H⁺ from histidine. As long as the dissociated fraction of histidine remains constant, the protein conformation will remain constant. As long as the relative alkalinity is maintained, the fractional dissociation of histidine remains constant and, thus, protein conformation does not change. In reality, the regulation of mammalian pH at 7.4 (at 37 degrees Celsius) is one specific example of this general rule. The relative alkalinity of mammalian blood at 37 degrees is the same as the relative alkalinity of fish blood at 5 degrees or of reptile blood at 42 degrees.

In a study that spanned 105 laboratories in seven countries, 131 scientists uncovered a link between pH and lactate levels and brain energy metabolism dysfunction that led to neuropsychiatric and neurodegenerative disorders in animal studies. These disorders included autism spectrum disorders, schizophrenia, bipolar disorder, and Alzheimer's disease, among others. Additionally, in several studies using mice, individuals with schizophrenia and bipolar disorder have shown decreased levels of brain pH. This link provides further insight into the importance of pH management in all animals, even humans.

Principal Terms

Acidosis: A body fluid pH of less than 7.4 at 37 degrees Celsius

Alkalosis: A body fluid pH greater than 7.4 at 37 degrees Celsius, the opposite of acidosis

Anaerobic Metabolism: Metabolism in the absence of oxygen that leads to the production of lactic acid, a strong acid

Partial Pressure: The pressure exerted by a specific gas in a mixture of gases, such as the atmosphere; it is analogous to concentration

pH: The negative logarithm of the hydrogen ion concentration, with higher hydrogen ion concentrations indicating lower pH; the pH scale goes from 0 to 14, with a pH of 7 being neutral, values below 7 indicating acidity, and values above 7 indicating alkalinity

Strong Acid: An acid that dissociates almost completely into its component ions; hydrochloric acid, for example, dissociates almost completely into hydrogen ions and chloride ions

Weak Acid: An acid that does not dissociate to a great extent; carbonic acid, for example, dissociates to produce some ions, but most of the molecules remain in their original forms

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