Chapter 13 Plasma proteins and enzymes
Proteins are present in all body fluids, but it is the proteins of the blood plasma that are examined most frequently for diagnostic purposes. Over 100 individual proteins have a physiological function in the plasma. Their principal functions, and some of the proteins, are indicated in Figure 13.1. Quantitatively, the single most important protein is albumin. With the exception of fibrinogen, the other proteins are known collectively as globulins. Changes in the concentrations of individual proteins occur in many conditions and their measurement can provide useful diagnostic information.
Some plasma proteins are enzymes (e.g. renin, coagulation factors). In addition to these, many primarily intracellular enzymes are detectable in plasma as a result of their loss from cells during normal cell turnover. The measurement of such enzymes provides a sensitive (although often relatively non-specific) indicator of tissue damage. Most of these enzymes are described in chapters of this book describing conditions in which their measurement is of particular value, but some general principles of the use of enzyme measurements are discussed in this chapter.
Measurement of plasma proteins
Total plasma protein
In very general terms, variations in plasma protein concentrations can be due to changes in any of three factors: the rate of protein synthesis, the rate of removal and the volume of distribution.
The concentration of proteins in plasma is affected by posture: an increase in concentration of 10–20% occurs within 30 min of becoming upright after a period of recumbency. Also, if a tourniquet is applied before venepuncture, a significant rise in protein concentration can occur within a few minutes. In both cases, the change in protein concentration is caused by increased diffusion of fluid from the vascular into the interstitial compartment. These effects must be borne in mind when blood is being drawn for the determination of protein concentration.
Only changes in the more abundant plasma proteins (i.e. albumin or immunoglobulins) will have a significant effect on the total protein concentration.
Except when patients have been given blood or proteins intravenously, a rapid increase in total plasma protein concentration is always due to a decrease in the volume of distribution (in effect, to dehydration). A rapid decrease is often the result of an increase in plasma volume. Thus, changes in plasma protein concentration can provide a valuable aid to the assessment of a patient’s state of hydration.
The total protein concentration of plasma can also fall rapidly if capillary permeability increases, because protein will diffuse out into the interstitial space. This can be seen, for example, in patients with septicaemia or generalized inflammatory conditions. Causes of increased and decreased total plasma protein concentration are summarized in Figure 13.2.
This technique was formerly widely used for the semi-quantitative assessment of plasma proteins, but now that specific assays are available for all the important proteins it only remains essential for the detection of paraproteins (monoclonal proteins produced by tumours of B-cell origin, particularly myeloma). Electrophoresis is usually performed on serum rather than plasma, because the fibrinogen present in plasma produces a band in the β region that might be mistaken for a paraprotein.
Electrophoresis, on cellulose acetate or agarose gel, separates the proteins into distinct bands: albumin, α1-and α2–globulins, β-globulins and γ-globulins. Plasma proteins are often still classified into groups according to their electrophoretic mobility (Fig. 13.3), although this classification has no relevance in relation to their function, with the exception that the normal immunoglobulins migrate (and are still often referred to) as γ-globulins.
Figure 13.3 Principal plasma proteins. Many other important proteins are present in only very low concentrations, for example thyroxine-binding globulin, transcortin and vitamin-D-binding globulin.
Figure 13.4 shows diagrammatically the appearance of normal serum (A) and serum containing a paraprotein (B) after electrophoresis on agarose gel and staining with a protein-sensitive stain. Note that in (B) there is a decrease in normal immunoglobulins, as characteristically occurs in patients with paraproteinaemia due to myeloma (see p. 231). Paraproteins typically migrate in the γ-region but (especially the IgA class) may migrate more anodally, towards the β region. Pattern (C) shows a polyclonal increase in immunoglobulins (as may occur in some autoimmune diseases and chronic infections).
Specific plasma proteins
Albumin, the most abundant plasma protein, makes the major contribution (about 80%) to the oncotic pressure of plasma. Oncotic pressure is the osmotic pressure due to the presence of proteins and is an important determinant of the distribution of extracellular fluid (ECF) between the intravascular and extravascular compartments.
In hypoalbuminaemic states, the decreased plasma oncotic pressure disturbs the equilibrium between plasma and interstitial fluid, with the result that there is a decrease in the movement of the interstitial fluid back into the blood at the venular end of the capillaries (Fig. 13.5). The accumulation of interstitial fluid is seen clinically as oedema. The relative decrease in plasma volume results in a fall in renal blood flow. This stimulates the secretion of renin, and hence of aldosterone, through the formation of angiotensin (secondary aldosteronism, see p. 24). This results in sodium retention and thus an increase in ECF volume, which potentiates the oedema.
Figure 13.5 Pathogenesis of oedema in hypoalbuminaemia. The normal balance of hydrostatic and oncotic pressures is such that there is net movement of fluid out of the capillaries at their arteriolar ends and net movement in at their venular ends (indicated here by arrows). Oedema can thus be due to an increase in capillary hydrostatic pressure, a decrease in plasma oncotic pressure or an increase in capillary permeability.
There are many possible causes of hypoalbuminaemia (Fig. 13.6), a combination of which may be implicated in individual cases. For example, in a patient with malabsorption due to Crohn’s disease, a low albumin may reflect both decreased synthesis (decreased supply of amino acids due to malabsorption) and increased loss (directly into the gut from ulcerated mucosa).
Hyperalbuminaemia can be either an artefact, for instance as a result of venous stasis during blood collection or overinfusion of albumin, or be a result of dehydration. Albumin synthesis is increased in some pathological states as a response to protein loss, but this never causes hyperalbuminaemia.
Measurements of albumin concentration are frequently used in relation to the provision of nutritional support. This topic is discussed in detail in Chapter 20, but it should be noted that, because of its relatively long plasma half-life (approximately 20 days), plasma albumin concentration is not a useful marker of the response to nutritional support in the short term (<10 days).
Plasma albumin concentration is also used as a test of liver function. Because of its relatively long half-life in the plasma, albumin concentration is usually normal in acute hepatitis. Low concentrations are characteristic of chronic liver disease, being due to both decreased synthesis and an increase in the volume of distribution as a result of fluid retention and the formation of ascites.
Albumin is a high-capacity, low-affinity transport protein for many substances, such as thyroid hormones, calcium and fatty acids. The influence of low plasma albumin concentration on measurements of thyroid hormones and calcium is considered on pp. 154 and 209, respectively. Albumin binds unconjugated bilirubin, and hypoalbuminaemia increases the risk of kernicterus in infants with unconjugated hyperbilirubinaemia. Salicylates, which displace bilirubin from albumin, can have a similar effect.
Many drugs are bound to albumin in the blood, and a decrease in albumin concentration can have important pharmacokinetic consequences. Phenytoin, for example, is highly protein bound, so a decrease in plasma albumin will increase the concentration of free drug and thus the risk of toxicity if the dose of phenytoin is not reduced.
A number of molecular variants of albumin exist. In bisalbuminaemia, the variant protein has a slightly different electrophoretic mobility from normal albumin and a pair of albumin bands is seen on electrophoresis: there are no clinical consequences. Analbuminaemia is a rare, inherited condition in which the plasma albumin concentration is less than 1 g/L. People with this condition tend to suffer episodic mild oedema, but are otherwise well.
This α1-globulin is a naturally occurring inhibitor of proteases. Its significance is related to the clinical consequences of inherited disorders of α1-antitrypsin synthesis. These can cause emphysema, occurring at a younger age (third and fourth decades) than is usual for this condition, and neonatal hepatitis, which can progress to cirrhosis.
Homozygotes for the normal protein are termed Pi (protease inhibitor) MM. Over 70 alleles of the gene have been described. α1-Antitrypsin deficiency is most frequently due to homozygosity for the Z allele (PiZZ), this genotype having a frequency of about 1 in 3000 in the UK. In affected individuals, plasma α1-antitrypsin concentration is reduced to between 10% and 15% of normal. The defect is due to a single amino acid substitution, which causes the protein to form aggregates that cannot be secreted from the liver and, as a result, cause liver damage. The abnormal protein shows decreased glycation, but this is probably a consequence, not the cause, of its retention in hepatocytes.
The development of emphysema is believed to be due to a lack of natural inhibition of the enzyme neutrophil elastase, which results in destructive changes in the lung. Not all PiZZ homozygotes develop liver or lung disease. The risk of developing emphysema is greatly increased by smoking: cigarette smoke oxidizes a thiol group at the active site of α1-antitrypsin, decreasing the inhibitory activity of what small amounts of the protein are present.
PiMZ heterozygotes have plasma α1-antitrypsin concentrations that are about 60% of normal; there is probably only a very slightly increased tendency for these individuals to develop lung disease when compared with normal PiMM homozygotes. Although homozygotes for the relatively common S allele have markedly reduced α1-antitrypsin activity, they rarely present clinically. PiSZ heterozygotes, however, have increased susceptibility to lung disease, albeit not to the same extent as those with PiZZ.
Accurate phenotyping is required for the screening of an affected individual’s family members. This involves the use of special techniques, such as isoelectric focusing, to allow identification of individual proteins. Genotypic antenatal screening is possible, using the polymerase chain reaction (PCR) to amplify fetal DNA obtained by chorionic villus sampling.
α1-Antitrypsin is an acute phase protein. Its concentration increases in acute inflammatory states and this may be sufficient to bring a genetically determined low concentration of the protein, for example in a PiMZ heterozygote, into the normal range. However, even with an acute phase response, the α1-antitrypsin concentration in PiZZ homozygotes never rises above 50% of the lower limit of the normal range.
Haptoglobin is an α2-globulin. Its function is to bind free haemoglobin released into the plasma during intravascular haemolysis. The haemoglobin–haptoglobin complexes formed are removed by the reticuloendothelial system, thereby conserving iron, and the concentration of haptoglobin falls correspondingly. Thus, a low plasma haptoglobin concentration can be indicative of intravascular haemolysis. However, low concentrations due to decreased synthesis are seen in chronic liver disease, metastatic disease and severe sepsis.
Haptoglobin is an acute phase protein and its concentration also increases in hypoalbuminaemic states such as the nephrotic syndrome. It demonstrates considerable genetic polymorphism: the molecule consists of pairs of two types of subunit, α and β, and while the β-chain is constant, there are three alleles for the α-chain. However, as far as is known, these different proteins are functionally similar and their existence is not known to be of clinical significance.
α2-Macroglobulin is a high molecular weight protein (820 kDa) that constitutes approximately one-third of the α2-globulins. Hepatic synthesis of the protein increases in the nephrotic syndrome and, because it is too large to be filtered even through a damaged glomerular basement membrane, plasma concentrations rise. Like α1-antitrypsin, α2-macroglobulin is an inhibitor of proteases, although it has a broader spectrum of activity.
This is a copper-carrying protein, which functions as a ferroxidase and superoxide scavenger. Its synthesis and plasma concentration are greatly reduced in Wilson’s disease (p. 100). Its concentration is increased in pregnancy (an oestrogen-related effect). It is an acute phase protein.
This β-globulin is the major iron-transporting protein in the plasma; normally about 30% saturated with iron, it is characteristically more than 60% saturated in haemochromatosis. Its measurement as an index of nutritional status is discussed in Chapter 20. Transferrin and ferritin are discussed in more detail in Chapter 17. Ferritin is also an iron-carrying protein, and measurement of its plasma concentration is used as a test for assessing body iron stores. The plasma concentration of carbohydrate-deficient transferrin can be used to assess exposure to alcohol (see p. 95), although it is neither a specific nor a sensitive test.
Acute phase proteins and the acute phase response
The term ‘acute phase response’ encompasses a complex range of physiological changes that occur following trauma and in burns, infection, inflammation and other related conditions. It comprises haemodynamic changes, increases in the activity of the coagulation and fibrinolytic systems, leukocytosis, changes in the concentration of many plasma proteins and systemic effects, particularly pyrexia. It is mediated by a host of cytokines, tumour necrosis factor and vasoactive substances.
Increases occur in the plasma concentrations of C-reactive protein and procalcitonin (see below), protease inhibitors, caeruloplasmin, α1-acid glycoprotein, fibrinogen and haptoglobins: these are a result of increased synthesis, mediated primarily by interleukin-6 (IL-6) and other cytokines. At the same time, there are decreases in the concentration of albumin, prealbumin and transferrin: these are mainly a result of increased vascular permeability, mediated by prostaglandins, histamine and other vasoactive substances.
C-reactive protein (CRP) is so called because of its property of binding to a polysaccharide (fraction C) from the cell walls of pneumococci. It may have a general function in defence against bacteria and foreign substances. Its concentration can increase 30-fold from a normal value of less than 5 mg/L during the acute phase response, for which it is a valuable marker, particularly in the context of monitoring patients with inflammatory conditions such as rheumatoid arthritis and Crohn’s disease. Its measurement appears to be both more sensitive and more specific than measurements of the erythrocyte sedimentation rate (ESR) and plasma viscosity in this respect. The CRP concentration begins to rise at about 6 h after the initiation of an acute phase response and reaches a peak after about 48 h before beginning to fall. The concentrations of α1-acid glycoprotein and fibrinogen rise and fall more slowly: peak concentrations occur at about 70 and 90 h, respectively. A raised CRP concentration is unequivocal evidence of an inflammatory response, but viral infections do not usually cause a raised CRP, and neither do some autoimmune diseases, for example systemic lupus erythematosus and scleroderma. Measurements of lower concentrations of CRP (in the range 0.1–5 mg/L, so-called ‘high sensitivity CRP’) may be useful as a marker of cardiovascular risk.
Procalcitonin is a 116 amino acid protein that undergoes cleavage in the C-cells of the thyroid to produce calcitonin. Procalcitonin is another acute phase protein; its plasma concentration increases to particularly high levels in the acute phase response to infection. Neither the function of procalcitonin in the acute phase response, nor its origin (it is not secreted by C-cells), is certain. It is not a substitute for CRP as a marker of an acute phase response, but its measurement may provide additional information, because it appears to have greater sensitivity and specificity for infection than CRP, and to be a better prognostic indicator.
Other plasma proteins
Measurements of other plasma proteins may provide useful information in particular circumstances. Measurement of coagulation factors (fibrinogen, factor VIII and others) is usually carried out in haematology laboratories and is essential in the investigation of some bleeding disorders. Measurement of the proteins of the complement system is of considerable value in the investigation of some diseases with an immunological basis. The apolipoproteins are considered in detail in Chapter 14. The importance of hormone-binding proteins, such as cortisol-binding globulin and sex hormone-binding globulin, is considered in Chapters 8 and 10, respectively. Plasma proteins used in the assessment of nutritional status are discussed in Chapter 20. Measurement of the plasma concentration of β2–microglobulin is of value in monitoring patients with myeloma (see p. 232). The measurement of plasma proteins derived from tumours (tumour markers) is discussed in Chapter 18.
Transudates and exudates
The protein concentration of pleural fluid or abdominal ascites is occasionally measured to determine whether the sample is a transudate (fluid with a low protein content derived by filtration across capillary endothelium) or an exudate (fluid with a high protein content actively secreted in response to inflammation). A value of 25 or 30 g/L is often taken as the dividing line between the two types of fluid, but this is not a reliable criterion as the protein content of both is very variable. Measurement of lactate dehydrogenase (see later) may be helpful in equivocal cases, as its activity is higher in exudates than in transudates, although values are also influenced by the plasma concentration of the enzyme. A chylous leak from the thoracic duct may be confirmed by the detection of a high triglyceride concentration in the pleural fluid.
The critical question is more often whether the ascites or pleural fluid is infected or if it is related to the presence of a tumour. This can only be determined by microbiological and cytological examination, so protein measurement is of limited value.