Plasma Enzymes in Diagnosis (Clinical Enzymology)
This chapter discusses the principles of clinical enzymology, which have already been encountered in some of the preceding chapters. Enzymology can be defined as the assay of an enzyme(s) in body fluids, usually blood, that can be used diagnostically or to monitor a clinical condition.
An enzyme is a protein that catalyses one or more specific biochemical reactions. It is usually easier to measure enzyme activity in body fluids, by monitoring changes in either substrate or product concentrations, than to measure enzyme protein concentration directly, although this is sometimes done. However, measurement of the enzyme protein concentration is more specific and less prone to analytical variation.
Generally, enzymes are present in cells at much higher concentrations than in plasma. Some occur predominantly in cells of certain tissues, where they may be located in different cellular compartments such as the cytoplasm or the mitochondria. ‘Normal’ plasma enzyme concentrations reflect the balance between the rate of synthesis and release into plasma during cell turnover, and the rate of clearance from the circulation. The enzyme activity in plasma may be:
higher than normal, due to the proliferation of cells, an increase in the rate of cell turnover or damage or in enzyme synthesis (induction), or to reduced clearance from plasma,
lower than normal, due to reduced synthesis, congenital deficiency or the presence of inherited variants of relatively low biological activity – examples of the latter are the cholinesterase variants.
Sometimes macroenzymes are found, that is to say, a high-molecular-weight form of a native enzyme. Often these are enzymes [such as lactate dehydrogenase (LDH), creatine kinase (CK) and alkaline phosphatase (ALP)] complexed with immunoglobulins and are more common in individuals with autoimmune disease. It is important to recognize macroenzymes as they can sometimes cause diagnostic confusion. As we will now see, changes in plasma enzyme activities may be useful to detect and localize tissue cell damage or proliferation, or to monitor the treatment and progress of disease.
ASSESSMENT OF CELL DAMAGE AND PROLIFERATION
Plasma enzyme levels depend on the extent of cell damage and the rate of release from damaged cells, which, in turn, depends on the rate at which damage is occurring.
In the absence of cell damage, the rate of release depends on the degree of induction of enzyme synthesis and the rate of cell proliferation.
These factors are balanced by the rate of enzyme clearance from the circulation.
Acute cell damage, for example in viral hepatitis, may cause very high plasma aminotransferase activities that reduce as the condition resolves. By contrast, the liver may be much more extensively involved in advanced cirrhosis but the rate of cell damage is often low, and consequently plasma enzyme activities may be only slightly raised or within the reference range. In very severe liver disease, plasma enzyme activities may even fall terminally when the number of hepatocytes is grossly reduced (see Chapter 17).
Relatively small enzymes, such as amylase, can be cleared by the kidneys. Thus, plasma amylase activity may be high as a result of renal glomerular impairment rather than pancreatic damage. However, most enzymes are large proteins and may be catabolized by plasma proteases before being taken up by the reticuloendothelial system.
In healthy individuals, each enzyme has a fairly constant and characteristic biological half-life, a fact that may be used to assess the time since the onset of an acute illness. After a myocardial infarction, for example, plasma levels of CK and aspartate aminotransferase (AST) fall to normal before those of LDH, which has a longer half-life (see Chapter 22).
Localization of damage
Most of the enzymes commonly measured to assess tissue damage are present in nearly all body cells, although their relative concentrations in certain tissues may differ. Measurement of the plasma activity of an enzyme known to be in high concentration within cells of a particular tissue may indicate an abnormality of those cells, but the results will rarely enable a specific diagnosis to be made. For example, if there is circulatory failure after a cardiac arrest, very high plasma concentrations of enzymes originating from many tissues may occur because of hypoxic damage to cells and reduced rates of clearance.
The distribution of enzymes within cells may differ. Alanine aminotransferase (ALT) and LDH are predominantly located in cytoplasm, and glutamate dehydrogenase (although this is not usually measured clinically) in mitochondria, whereas AST occurs in both these cellular compartments. Different disease processes in the same tissue may affect the cell in different ways, causing alteration in the relative plasma enzyme activities.
The diagnostic precision of plasma enzyme analysis may be improved by the following:
Serial enzyme estimations The rate of change of plasma enzyme activity is related to a balance between the rate of entry and the rate of removal from the circulation. A persistently raised plasma enzyme activity is suggestive of a chronic disorder or, occasionally, impaired clearance.
Isoenzyme determination Some enzymes exist in more than one form; these isoenzymes may be separated by their different physical or chemical properties. If they originate in different tissues, such identification will give more information than the measurement of plasma total enzyme activity; for example, CK may be derived from skeletal or cardiac muscle, but one of its isoenzymes is found predominantly in the myocardium.
Non-specific causes of raised plasma enzyme activities
Before attributing a change in plasma enzyme activity to a specific disease process, it is important to exclude the presence of factitious or non-specific causes.
Slight rises in plasma ALT and AST activities are common, non-specific findings in many illnesses. Moderate exercise, or a large intramuscular injection, may lead to a rise in plasma CK activity; isoenzyme determination may identify skeletal muscle as the tissue of origin.
Some drugs, such as the anticonvulsants phenytoin and phenobarbital, may induce the synthesis of the microsomal enzyme γ-glutamyl transferase (GGT), and so increase its plasma activity in the absence of disease.
Plasma enzyme activities may be raised if the rate of clearance from the circulation is reduced. In the absence of hepatic or renal disease, this may occur if, for example, the plasma enzyme forms complexes with immunoglobulins, known as a macroenzyme.
FACTORS AFFECTING RESULTS OF PLASMA ENZYME ASSAYS
Analytical factors
The total concentration of all plasma enzyme proteins is less than 1 g/L. The results of enzyme assays are not usually expressed as concentrations, but as activities. Changes in concentration may give rise to proportional changes in catalytic activity, but the results of such measurements depend on many analytical factors, including:
substrate concentration,
product concentration,
enzyme concentration,
reaction temperature,
reaction pH,
presence of activators or inhibitors.
The definition of ‘international units’ does not take these factors into account, and the results from different laboratories, which are apparently expressed in the same units, may not be directly comparable. Therefore, plasma enzyme activities must be interpreted in relation to the reference ranges from the issuing laboratory. In some countries such as the UK there are plans to harmonize or converge laboratory reference ranges for certain analytes.
Non-disease factors
Examples of non-disease factors affecting enzyme activities include the following.
Age
Sex
Race/ethnicity
Plasma CK activity is higher in black people and Afro-Caribbeans than in white people.
Physiological conditions
Plasma ALP activity rises during the last trimester of pregnancy because of the presence of the placental isoenzyme. Several enzymes, such as AST and CK, rise moderately in plasma during and immediately after labour or strenuous exercise.
Plasma enzyme activities should therefore be interpreted in relation to the sex-, race-/ethnicity- and age-matched reference ranges of the issuing laboratory.
NORMAL PLASMA ENZYME ACTIVITIES
Individual enzymes of clinical importance are considered in the following section. Applications of their assays in defined clinical situations are discussed later in the chapter and in other chapters in this book.
Aminotransferases
The aminotransferases (ALT and AST) are enzymes involved in the transfer of an amino group from a 2-amino acid to a 2-oxoacid; they need the cofactor pyridoxal phosphate for optimal activity. They are widely distributed in the body. (See Chapter 17, which deals with hepatic disease, for further details.) The aminotransferases are used as part of the biochemical liver profile.
Aspartate aminotransferase
Aspartate aminotransferase (glutamate oxaloacetate aminotransferase, GOT) is present in high concentrations in cells of cardiac and skeletal muscle, liver, kidney and erythrocytes. Damage to any of these tissues may increase plasma AST levels.
Causes of raised plasma aspartate aminotransferase activities
Artefactual: due to in vitro release from erythrocytes if there is haemolysis or if separation of plasma from cells is delayed.
Physiological: during the neonatal period (about 1.5 times the upper adult reference limit).
Marked increase (may be greater than 5-10 times the upper reference limit or URL):
circulatory failure with ‘shock’ and hypoxia,
myocardial infarction,
acute viral or toxic hepatitis.
Moderate to slight increase (usually less than five times URL):
Hepatic steatosis [fatty liver or non-alcoholic fatty liver disease (NAFLD)],
cirrhosis (may be normal sometimes),
infectious mononucleosis (due to liver involvement),
cholestatic jaundice,
malignant infiltration of the liver (may be normal),
skeletal muscle disease,
after trauma or surgery (especially after cardiac surgery),
severe haemolytic episodes (of erythrocyte origin),
certain drugs.
Note that AST is not specific for hepatic disease.
Alanine aminotransferase
Alanine aminotransferase (glutamate pyruvate aminotransferase, GPT) is present in high concentrations in liver and, to a lesser extent, in skeletal muscle, kidney and heart.
Causes of raised plasma alanine aminotransferase activities
Marked increase (may be greater than 5-10 times URL):
circulatory failure with ‘shock’ and hypoxia,
acute viral or toxic hepatitis.
Moderate to slight increase (usually less than five times URL):
Hepatic steatosis (fatty liver or NAFLD),
cirrhosis (may be normal sometimes),
infectious mononucleosis (due to liver involvement),
liver congestion secondary to congestive cardiac failure,
cholestatic jaundice,
coeliac disease,
surgery or extensive trauma and skeletal muscle disease (much less affected than AST),
certain drugs.
Lactate dehydrogenase
Lactate dehydrogenase catalyses the reversible interconversion of lactate and pyruvate. The enzyme is widely distributed in the body, with high concentrations in cells of cardiac and skeletal muscle, liver, kidney, brain and erythrocytes; measurement of plasma total LDH activity is therefore a non-specific marker of cell damage.
Causes of raised plasma total lactate dehydrogenase activity
Artefactual: due to in vitro haemolysis or delayed separation of plasma from whole blood.
Marked increase (may be greater than 5-10 times URL):
circulatory failure with ‘shock’ and hypoxia,
myocardial infarction (see Chapter 22),
some haematological disorders: in blood diseases such as megaloblastic anaemia, acute leukaemias and lymphomas, very high levels (up to 20 times the URL in adults) may be found. In cases of lymphoma LDH can be used as a tumour marker. Smaller increases occur in other disorders of erythropoiesis, such as thalassaemia, myelofibrosis and haemolytic anaemias, renal infarction or, occasionally, during rejection of a renal transplant.
Moderate to slight increase (usually less than five times URL):
viral hepatitis,
malignancy of any tissue,
skeletal muscle disease,
pulmonary embolism,
infectious mononucleosis,
certain drugs.
Isoenzymes of lactate dehydrogenase
Five main isoenzymes can be detected by electrophoresis and are referred to as LDH1 to LDH5. LDH1, the fraction that migrates fastest towards the anode, predominates in cells of cardiac muscle, erythrocytes and kidney. The slowest moving isoenzyme, LDH5, is the most abundant form in the liver and in skeletal muscle.
Whereas in many conditions there is an increase in all fractions, the finding of certain patterns is of diagnostic value:
Predominant elevation of LDH1 and LDH2 (LDH1 more than LDH2) occurs after myocardial infarction, in megaloblastic anaemia and after renal infarction.
Predominant elevation of LDH2 and LDH3 occurs in acute leukaemia; LDH3 is the main isoenzyme elevated as a result of malignancy of many tissues.
Elevation of LDH5 occurs after damage to the liver or skeletal muscle.
It is rarely necessary to quantify LDH isoenzyme activity. A rise in LDH1 is most significant in the diagnosis of myocardial infarction. However, as LDH1 and, to a lesser extent LDH2 and LDH3, can use 2-hydroxybutyrate as well as lactate as substrate, some laboratories assay hydroxybutyrate dehydrogenase (HBD) as an index of LDH1 activity. Lactate dehydrogenase was used in the delayed diagnosis of a myocardial infarct (troponin has largely taken over this role from LDH) and also as a marker for certain tumours, for example lymphomas, and to help determine haemolysis (see Chapters 17 and 22).
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