The blood pH is tightly regulated

Most biomolecules contain ionizable groups that are subject to protonation and deprotonation. Consequently, all biological processes are pH dependent. The pH of plasma is approximately 7.40 in arterial blood and 7.35 in venous blood. The difference is caused by the higher concentration of carbonic acid in venous blood. Carbonic acid forms spontaneously from carbon dioxide and water, but the reaction is accelerated dramatically by the enzyme carbonic anhydrase in erythrocytes:

At 37°C and pH 7.4, there are approximately 800 molecules of dissolved CO₂ and 16,000 molecules of HCO₃⁻ for every molecule of H₂CO₃. The apparent pK for the overall reaction CO₂ + H₂O → HCO₃⁻ + H⁺ is 6.1. Thus the bicarbonate system acts as an effective buffer in the neutral to slightly acidic pH range.

Carbonic acid/bicarbonate is the most important physiological buffer system in the body. It is important because CO₂ and HCO₃⁻ are present in high concentrations in the interstitial and intracellular compartments as well as the plasma (Fig. 15.1). In addition, the CO₂ level can be regulated by the lungs and the HCO₃⁻ level by the kidneys.

Figure 15.1 Ionic compositions of blood plasma, interstitial fluid, and intracellular fluid.

Phosphate groups provide an additional buffer system:

The phosphate buffer is important only in the intracellular compartments, in which both inorganic phosphate and organically bound phosphate are plentiful.

Proteins provide additional buffering capacity through the ionizable groups in their side chains and their free amino and carboxyl termini.

Acidosis and alkalosis are common in clinical practice

Even small deviations from the normal blood pH lead to severe disturbances. An arterial pH lower than 7.35 is called acidemia, and an arterial pH exceeding 7.45 is called alkalemia. The pathological states leading to these outcomes are called acidosis and alkalosis, respectively.

Respiratory acidosis is caused by the abnormal retention of CO₂, and respiratory alkalosis is caused by hyperventilation. For example, a doubling in the rate of alveolar ventilation raises the blood pH from 7.40 to 7.62, and a 50% reduction in alveolar ventilation lowers the blood pH from 7.40 to 7.12 (Fig. 15.2).

Figure 15.2 pH change in plasma and extracellular fluids in response to changes in alveolar ventilation.

Metabolic acidosis is caused either by the overproduction of an organic acid or by failure of the kidneys to excrete excess acid. The normal urinary pH varies between 4.0 and 7.0, depending on the need to excrete excess protons. Conversely, metabolic alkalosis is caused by the abnormal loss of acids from the body (e.g., as a result of excessive vomiting).

Whenever the blood pH is abnormal, the body uses three lines of defense in an attempt to restore a normal blood pH:

Measurement of the plasma total carbon dioxide (CO₂ + H₂CO₃ + HCO₃⁻) distinguishes between metabolic and respiratory acidosis. In respiratory acidosis, the total carbon dioxide is elevated because CO₂ retention is, by definition, the cause of the acidosis. In metabolic acidosis, it is reduced because the patient hyperventilates in an attempt to eliminate excess carbonic acid. The converse applies to alkalosis.

Plasma proteins are both synthesized and destroyed in the liver

Immunoglobulins are synthesized by plasma cells, but most other plasma proteins are made by the liver. The liver synthesizes about 25 g of plasma proteins every day, which accounts for nearly 50% of the total protein synthesis in the liver.

With the important exception of albumin, most plasma proteins are glycoproteins. Their N-linked oligosaccharides end with sialic acid (N-acetylneuraminic acid) bound to galactose. A typical plasma protein remains in the circulation for several days, and during this time the terminal sialic acid residues are gradually chewed off by endothelial neuraminidases (Fig. 15.3). After the loss of the sialic acid, the exposed galactose at the end of the oligosaccharide binds to an asialoglycoprotein receptor on the surface of hepatocytes, followed by receptor-mediated endocytosis and lysosomal degradation.

Figure 15.3 Terminal sialic acid residues (Sia) of plasma glycoproteins are removed slowly by endothelial neuraminidases. The exposed terminal galactose residues (Gal) mediate binding to the hepatic asialoglycoprotein receptor.

Albumin prevents edema

Electrophoresis is the most important method for the separation of plasma proteins in the clinical laboratory (Fig. 15.4). It usually is performed at alkaline pH on a solid or semisolid support such as cellulose acetate foil or an agarose gel. This method separates the proteins by their charge/mass ratio. Five fractions can be identified by staining and densitometric scanning: albumin, and the α₁-, α₂-, β-, and γ-globulins.

Figure 15.4 Electrophoretic separation of plasma proteins on cellulose acetate foil at pH 8.6, densitometric scan. The electrophoretic pattern depends somewhat on the separation conditions, including support medium, pH, and ionic strength. ALB, Albumin.

Of these five fractions, only the albumin peak consists of a single major protein. Albumin is a single tightly packed polypeptide with 585 amino acids, without covalently bound carbohydrate. Its compact shape minimizes its effect on plasma viscosity. In general, compact proteins do not increase the plasma viscosity to the same extent as more elongated proteins of the same molecular weight (MW).

Albumin has a half-life of 17 days in the circulation. With its MW of 66,000 D and acidic isoelectric point (pI), it is able to avoid renal excretion, but it does cross the vascular endothelium of most tissues to some extent. Therefore it is present in interstitial fluid and lymph, but at lower concentrations than in the plasma.

Because the interstitial fluid volume is far larger than the plasma volume (12% and 4.5% of body volume, respectively), the total amount of albumin in the interstitial spaces slightly exceeds that in the vascular compartment. This albumin is returned to the blood by the lymph.

Although albumin accounts for only 60% of the total plasma protein, it provides 80% of the colloid osmotic pressure. This is because the colloid osmotic pressure depends on the amount of water and electrolytes that a protein attracts to its surface, and albumin is one of the most hydrophilic plasma proteins.

The colloid osmotic pressure is necessary to prevent edema. The hydrostatic pressure of the blood forces fluid from the capillaries into the interstitial spaces, and the colloid osmotic pressure of the plasma proteins is required to pull the fluid back into the capillaries (Fig. 15.5).

Figure 15.5 Importance of the colloid osmotic pressure for fluid exchange across the capillary wall. A net flow of water into the interstitium is observed at the arterial end of the capillary. This is balanced by a net flow into the capillary at its venous end.

Usually, edema develops when the albumin concentration drops below 2.0 g/dl. Other possible causes of edema include an increase in capillary permeability, venous obstruction, impaired lymph flow, and congestive heart failure with increased venous pressure.

Albumin binds many small molecules

Fatty acids, thyroxine, cortisol, heme, bilirubin, and many other metabolites bind noncovalently to albumin. Even approximately half the serum calcium and magnesium are albumin bound. Many drugs bind to serum albumin as well. In these cases, only the free, unbound fraction of a drug is pharmacologically active. Being noncovalent, albumin binding is reversible:

The dissociation constant K_D for the release of the drug from albumin is defined as

   (1)

This can be rearranged as

   (2)

As long as the molar concentration of albumin is far higher than that of the drug, the concentration of free, unbound albumin [Alb] approximates the total serum albumin concentration. Equation (2) indicates that in a patient whose albumin concentration is only half of normal, the ratio of free drug/bound drug is doubled. This is important because only the fraction of the drug that is not protein bound in the plasma equilibrates freely with the tissues. Therefore it is not the total drug concentration, but the concentration of the unbound drug, that determines the biological response.

If the patient has severe hypoalbuminemia, an otherwise desirable plasma level of a drug may actually be in the toxic range because an increased fraction of the drug is in the biologically active, unbound form. The commonly used laboratory tests for plasma drug levels do not distinguish between the free and bound fractions.

Some plasma proteins are specialized carriers of small molecules

Many of the proteins listed in Table 15.2 are specialized binding proteins that ferry endogenous substances through the blood.

Table 15.2 Characteristics of Some Plasma Proteins

Transthyretin, also called prealbumin because it moves slightly ahead of albumin during electrophoresis, participates in retinol transport. The liver releases stored retinol into the blood as a noncovalent complex with retinol-binding protein (RBP). RBP is a small protein of only 182 amino acids (MW 21,000), which has to bind to the larger transthyretin (MW 62,000) to avoid renal excretion.

Transthyretin also binds thyroid hormones. However, the major transport protein for these hormones is thyroxine-binding globulin (TBG), which binds thyroxine with 100 times higher affinity than does transthyretin. An increased level of TBG leads to an increased level of total circulating thyroid hormone, whereas its congenital absence leads to abnormally low levels. In both cases, however, the patient is healthy because the level of the free, unbound hormone is kept in the physiological range by homeostatic mechanisms.

Steroid hormones have two binding proteins: transcortin for glucocorticoids, and sex hormone–binding globulin for androgens and estrogens. Only the unbound fraction of the hormone determines the biological response. Therefore variable levels of the binding proteins can complicate the interpretation of hormone levels measured in the clinical laboratory. What the laboratory measures routinely is the concentration of the total (free + protein-bound) hormone, but the strength of the biological response depends only on the unbound fraction.

The hormone-binding proteins buffer the plasma concentration of the free, unbound hormone, in the same way that a pH buffer buffers the concentration of free protons.

Haptoglobin and hemopexin are binding proteins with a very different function. After intravascular hemolysis, the hemoglobin that is released from the ruptured erythrocytes dissociates into α-β dimers that are too small (MW 33,000D) to escape renal excretion. To prevent the loss of hemoglobin with its valuable iron, the hemoglobin binds to haptoglobin, and any free heme binds to hemopexin. These complexes are cleared by phagocytic cells and hepatocytes, respectively (Fig. 15.6).

Figure 15.6 Fate of hemoglobin (Hb) after intravascular hemolysis. Hpt, Haptoglobin; Hpx, hemopexin; RBC, red blood cell.

Because haptoglobin is degraded along with its bound hemoglobin, the serum haptoglobin level is depressed in all hemolytic conditions, sometimes to near zero. Haptoglobin does not bind the myoglobin that is released from damaged muscles. Therefore the haptoglobin level is normal in muscle diseases. Because the common laboratory tests for “blood” in the urine do not distinguish between hemoglobin and myoglobin, measurement of serum haptoglobin can distinguish between hemoglobinuria and myoglobinuria.

Deficiency of α₁-antiprotease causes lung emphysema

The proteolytic cascades that normally occur during blood clotting and immune responses are modulated by circulating protease inhibitors. Some of these inhibitors are very selective, but others inhibit a large number of proteases.

α₂–Macroglobulin binds a great variety of proteases and even forms covalent bonds with them. These protease-inhibitor complexes are ingested by phagocytes, followed by lysosomal degradation. α₂-Macroglobulin is considered a backup protease inhibitor that comes into play when more selective inhibitors fail.

α₁-Antiprotease is also known as α₁-protease inhibitor or α₁-antitrypsin. It inhibits many serine proteases, with highest affinity for elastase from white blood cells. In the laboratory, its activity is measured as the trypsin inhibitory capacity (TIC).

More than 75 genetic variants of α₁-antiprotease are known. One of them, the Z allele, codes for a protein that cannot be secreted from the hepatocytes in which it is synthesized. Some ZZ homozygotes succumb to neonatal hepatitis or infantile cirrhosis, presumably because the accumulating protein damages the hepatocytes. Those who escape serious liver damage are prone to lung emphysema.

Ordinarily, emphysema is caused by the smoldering inflammation of chronic bronchitis and is seen mainly in long-term smokers. Chronic inflammation destroys the septa of the lung alveoli, reducing the surface area available for gas exchange.

The observation of early-onset lung emphysema in individuals with α₁-antiprotease deficiency suggests that excessive proteolytic activity contributes to tissue damage in chronic bronchitis. Macrophages and neutrophils spill lysosomal proteases during phagocytosis. These proteases must be kept in check by α₁-antiprotease, which is present not only in the blood but also in bronchial secretions and interstitial fluid. The lungs are especially vulnerable to out-of-control proteases because they are exposed to inhaled bacteria and other foreign particles that must be scavenged continuously by neutrophils and alveolar macrophages.

α₁-Antiprotease can be inactivated by smoking. An essential methionine residue in the protein becomes oxidized to methionine sulfoxide by components of cigarette smoke. This contributes to the development of chronic bronchitis and emphysema in smokers, even those without a genetic defect in the protease inhibitor system.

The prevalence of α₁-antiprotease deficiency in the white population of the United States is about 1 in 7000. Eighty percent of these patients eventually will develop emphysema, many at an early age. Treatment is strict avoidance of smoking. α₁-Antiprotease can be administered by intravenous injection or by inhaler to the lungs.

Levels of plasma proteins are affected by many diseases

Plasma protein electrophoresis is a valuable aid in the diagnosis of many diseases. Figure 15.7 summarizes some typical patterns.

Figure 15.7 Plasma protein electrophoresis in various disease states. A, Normal. ALB, Albumin. B, Immediate response pattern. C, Delayed response pattern. D, Liver cirrhosis. E, Protein-losing conditions (nephrotic syndrome, protein-losing enteropathy). F, Monoclonal gammopathy (“paraprotein”).

Acute-phase reactants are plasma proteins whose levels change within 1 or 2 days after acute trauma or surgery, and especially during infections and inflammation (Table 15.3). Their synthesis is controlled by stress hormones and cytokines that are released in these conditions. The albumin peak is reduced, whereas the α₂ peak is often increased because one of its major components, haptoglobin, is a positive acute-phase reactant.

Table 15.3 Acute-Phase Reactants*

* The levels of these plasma proteins are either elevated or reduced in many acute illnesses.

The most sensitive acute-phase reactant is C-reactive protein. Its plasma level rises up to 100-fold in bacterial infections and to a lesser degree in some other stressful conditions. C-reactive protein binds avidly to some bacterial polysaccharides and seems to participate in the innate immune response.

γ-Globulins are increased in many chronic diseases, including infections, malignancies, and liver cirrhosis. The nonselective stimulation of immunoglobulin synthesis in these conditions is called polyclonal gammopathy.

Nephrotic syndrome is caused by damage to the glomerular basement membrane in the kidneys. As a result, plasma proteins, especially those of low MW, are lost in the urine. The albumin peak and most of the globulin peaks are depressed, but the α₂ peak is increased. The α₂-macroglobulin in this fraction is so large (MW 725,000 D) that it is retained while the smaller plasma proteins are lost.

Similar patterns of decreased albumin and increased α₂-globulin are seen in protein-losing enteropathy, when plasma proteins are lost through a large inflamed area in the intestine, and in extensive burns, when plasma proteins seep through the denuded body surface.

Abnormalities in the concentrations of minor plasma proteins cannot be divined from the electrophoretic pattern and have to be determined by sensitive immunological methods. For example, α-fetoprotein is synthesized in the fetal liver but occurs in only trace amounts in normal adult blood. Its levels are increased in most patients with hepatocellular carcinoma. Effectively, the cancer cells revert to a fetal phenotype that entails α-fetoprotein production in addition to rapid proliferation. More importantly, α-fetoprotein is used for the prenatal diagnosis of open neural tube defects. In these severe malformations, α-fetoprotein leaks from the fetal blood into amniotic fluid and even into the maternal blood (Fig. 15.8).

Figure 15.8 Use of α-fetoprotein in amniotic fluid for the diagnosis of neural tube defects in the fetus. Maternal serum can be screened for α-fetoprotein (A), and suspect results are followed up by amniocentesis (B).

Blood components are used for transfusions

Blood transfusions necessitate blood group matching, and there is a risk of transmitting acquired immunodeficiency syndrome (AIDS), hepatitis, and other diseases. Also, not every patient requires the same blood component. An anemic patient needs red blood cells, a patient with nephrotic syndrome needs albumin, a patient with a clotting disorder needs clotting factors or platelets, and a patient with an immunodeficiency can be treated with immunoglobulins. Table 15.4 lists some of the most important blood products and their uses.

Table 15.4 Plasma Components Available for Therapeutic Use

Immunoglobulins bind antigens very selectively

The immunoglobulins make up approximately 20% of the total plasma protein. Most move in the γ-globulin region during electrophoresis, but there are immunoglobulins under the β and α₂ peaks as well.

The immunoglobulins function as antibodies that bind tightly to antigens. An antigen is, by definition, any molecule that induces the formation of a matching antibody. An antigen must fulfill two requirements:

Antigen-antibody binding is noncovalent and therefore reversible. Formation of the antigen-antibody complex initiates the elimination of the antigen. Phagocytic cells can both endocytose soluble antigen-antibody complexes and phagocytize antibody-coated bacteria and viruses.

The most astounding property of antibodies is their diversity. Every person has more than one million structurally different antibodies, each with its own unique antigen-binding specificity. This diversity enables the body to recognize and eliminate almost any imaginable antigen.

Antibodies consist of two light chains and two heavy chains

All antibodies have the same general structure, as shown for immunoglobulins of the G1 class (IgG1) in Figure 15.9. The molecule consists of four disulfide-bonded polypeptides: two identical heavy chains with MW of 53,000 D each and two identical light chains with MW of 23,000 D each. The molecule has the shape of the letter Y. Each of the two arms of the Y is formed by the amino-terminal half of a heavy chain and a complete light chain. The stem consists of the carboxyl-terminal halves of the two heavy chains.

Figure 15.9 Structure of human immunoglobulin G1 (IgG1). Each domain (V_L and C_L in the light [L] chains; V_H, C_H1, C_H2, and C_H3 in the heavy [H] chains) is a globular portion of the molecule, stabilized by an intrachain disulfide bond. Fab,

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