The analysis of enzymes in blood plasma has played a central role in the diagnosis of several disease processes. Many enzymes are functional constituents of blood. Examples include pseudocholinesterase, lipoprotein lipase, and components of the cascades that trigger blood clotting and clot dissolution. Other enzymes are released into plasma following cell death or injury. While these latter enzymes perform no physiologic function in plasma, they can serve as biomarkers, molecules whose appearance or levels can assist in the diagnosis and prognosis of diseases and injuries affecting specific tissues. Following injury, the plasma concentration of a released enzyme may rise early or late, and may decline rapidly or slowly. Proteins resident to the cytoplasm tend to appear more rapidly than those from subcellular organelles. Factors that determine the speed with which enzymes and other proteins are removed from plasma include their susceptibility to proteolysis and their permeability to renal glomeruli.
Quantitative analysis of the activity of released enzymes or other proteins, typically in plasma or serum but also in urine or various cells, provides information concerning diagnosis, prognosis, and response to treatment. Assays of enzyme activity typically employ standard kinetic assays of initial reaction rates. Table 7–2 lists several enzymes of value in clinical diagnosis. These enzymes are, however, not absolutely specific for the indicated disease. For example, elevated blood levels of prostatic acid phosphatase are associated typically with prostate cancer, but also may occur with certain other cancers and noncancerous conditions. Consequently, enzyme assay data must be considered together with other factors elicited through a comprehensive clinical examination. Factors to be considered in interpreting enzyme data include patient age, sex, prior history, possible drug use, and the sensitivity and the diagnostic specificity of the enzyme test.
TABLE 7–2Principal Serum Enzymes Used in Clinical Diagnosis ||Download (.pdf) TABLE 7–2 Principal Serum Enzymes Used in Clinical Diagnosis
|Serum Enzyme ||Major Diagnostic Use |
Aspartate aminotransferase (AST, or SGOT)
Alanine aminotransferase (ALT, or SGPT)
|Amylase ||Acute pancreatitis |
|Ceruloplasmin ||Hepatolenticular degeneration (Wilson disease) |
|Creatine kinase ||Muscle disorders and myocardial infarction |
|γ-Glutamyl transferase ||Various liver diseases |
|Lactate dehydrogenase isozyme 5 ||Liver diseases |
|Lipase ||Acute pancreatitis |
|β-Glucoscerebrosidase ||Gaucher disease |
|Phosphatase, alkaline (isozymes) ||Various bone disorders, obstructive liver diseases |
Enzymes Assist Diagnosis of Myocardial Infarction
An enzyme useful for diagnostic enzymology should be relatively specific for the tissue or organ under study, should appear in the plasma or other fluid at a time useful for diagnosis (the “diagnostic window”), and should be amenable to automated assay. The enzymes used to confirm a myocardial infarction (MI) illustrate the concept of a “diagnostic window,” and provide a historical perspective on the use of different enzymes for this purpose.
Detection of an enzyme must be possible within a few hours of an MI to confirm a preliminary diagnosis and permit initiation of appropriate therapy. Enzymes that only appear in the plasma for 12 hours or more following injury are thus of limited utility. The first enzymes used to diagnose MI were aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase. AST and ALT proved less than ideal, however, as they appear in plasma relatively slowly and are not specific to heart muscle. While LDH also is released relatively slowly into plasma, it offered the advantage of tissue specificity as a consequence of its quaternary structure.
Lactate dehydrogenase (LDH) is a tetrameric enzyme consisting of two monomer types: H (for heart) and M (for muscle) that combine to yield five LDH isozymes: HHHH (I1), HHHM (I2), HHMM (I3), HMMM (I4), and MMMM (I5). The relative proportions of each subunit in the cells of a particular organ is determined by tissue-specific patterns in the expression of the H and M genes. Isozyme I1 predominates in heart tissue, and isozyme I5 in the liver. Thus, when LDH levels rise in blood plasma, the identity of the injured tissue can be inferred from its characteristic pattern of LDH isozymes. In the clinical laboratory, individual isozymes can be separated by electrophoresis and detected using a coupled assay (Figure 7–12). While historically of importance, the assay of LDH has been superseded as a marker for MI by proteins that appear in plasma more rapidly than LDH.
Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reaction scheme shown on the left. (NBT, nitroblue tetrazolium; PMS, phenazine methosulfate.) At right is shown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct; B is normal serum; and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.
Creatine kinase (CK) has three isozymes: CK-MM (skeletal muscle), CK-BB (brain), and CK-MB (heart and skeletal muscle). CK-MB has a useful diagnostic window. It appears within 4 to 6 hours of an MI, peaks at 24 hours, and returns to a baseline level by 48 to 72 hours. As for LDH, individual CK isozymes are separable by electrophoresis, thus facilitating detection. Assay of plasma CK levels continues in use to assess skeletal muscle disorders such as Duchene muscular dystrophy. Today, however, in most clinical laboratories the measurement of plasma troponin levels has replaced CK as the preferred diagnostic marker for MI.
Troponin is a complex of three proteins involved in muscle contraction in skeletal and cardiac muscle but not in smooth muscle (see Chapter 51). Immunological measurement of plasma levels of cardiac troponins I and T provide sensitive and specific indicators of damage to heart muscle. Troponin levels rise for 2 to 6 hours after an MI and remain elevated for 4 to 10 days. In addition to MI, other heart muscle damage also elevates serum troponin levels. Cardiac troponins thus serve as a marker of all heart muscle damage. The search for additional markers for heart disease, such as ischemia-modified albumin, and the simultaneous assessment of a spectrum of diagnostic markers via proteomics, continues to be an active area of clinical research.
Additional Clinical Uses of Enzymes
Enzymes also can be employed in the clinical laboratory as tools for determining the concentration of critical metabolites. For example, glucose oxidase is frequently utilized to measure plasma glucose concentration. Enzymes are employed with increasing frequency as tools for the treatment of injury and disease. Tissue plasminogen activator (tPA) or streptokinase is used in the treatment of acute MI, while trypsin has been used in the treatment of cystic fibrosis. Intravenous infusion of recombinantly produced glycosylases has been approved for the treatment of several lysosomal storage diseases including the Gaucher disease (β-glucosidase), Pompe disease (α-glucosidase), Fabry disease (α-galactosidase A), and Sly disease (β-glucuronidase).