BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 7. ENZYMOLOGY

7.9. Application of enzymes in medicine

7.9.1. Enzyme diagnostics

Enzyme Diagnostics involves diagnosing a disease (or syndrome) based on the DETERMINATION OF ENZYME Activity in human biological fluids. The Introduction/14.html">Principles of Enzyme diagnostics are grounded in the following provisions:

✵ As a result of Cell damage, the concentration of intracellular Enzymes increases in the Blood or other biological fluids (e.g., urine);

✵ The amount of released enzyme is sufficient for its detection;

✵ The activity of enzymes in biological fluids detected upon cell damage remains stable for a sufficiently long time and differs from normal values;

✵ certain enzymes have a preferential or absolute localization in specific Organs (organ Specificity);

✵ there are differences in the intracellular Organization of A number of enzymes.

Causes leading to an increase in enzyme levels in the blood. Blood Plasma Enzymes can be divided into two groups. The first, relatively small group of enzymes is secreted into blood plasma by specific organs. For instance, the Liver synthesizes inactive precursors of the Blood Coagulation SYSTEM enzymes. The second group comprises a larger set of enzymes released from Cells during their normal functioning. Typically, these enzymes perform intracellular Functions and have no physiological significance in blood plasma. In a healthy individual, the activity of these enzymes in plasma is low and relatively constant, which is maintained by a steady balance between their release rate from cells and their degradation rate.

In many diseases, cell destruction occurs, and their contents, including enzymes, escape into the blood. Causes that provoke the release of intracellular contents into the bloodstream include impaired cell membrane permeability (during inflammatory processes) or compromised cell integrity (during necrosis). The determination of the activity of a number of enzymes in the blood in biochemical laboratories is used to diagnose Diseases of the Heart, liver, Skeletal Muscle, and other Tissues. The level of enzyme activity in plasma correlates with the degree of cell damage.

Knowledge of the subcellular localization of enzymes is of great importance for enzyme diagnostics. Thus, the appearance in blood plasma of enzymes with exclusively cytosolic localization indicates an inflammatory process; the presence of mitochondrial or nuclear enzymes points to deeper cellular damage, such as necrosis. However, an increase in Enzyme Concentration is not always associated with tissue damage. In cases of excessive cellular proliferation—for example, in oncoproliferative processes, increased synthesis rates of certain enzymes within cells, or impaired clearance (renal excretion capacity)—elevated concentrations of specific enzymes are observed in the blood. Normal values of enzyme activity in the blood of children and pregnant women differ from the reference ranges typical for healthy adults.

Isoenzymes. Enzymes that catalyze the same chemical reaction but differ in their Primary Protein Structure are called isoenzymes or isoenzymes. They catalyze the same type of reaction with fundamentally identical mechanisms, yet they differ from one another in kinetic parameters, activation conditions, and the CHARACTERISTICS OF THE apoenzyme-coenzyme bond.

The origins of isoenzymes are diverse, but most commonly they are caused by differences in The structure of the genes encoding these isoenzymes. Consequently, isoenzymes differ in the Primary Structure of their protein molecule and, accordingly, in their physicochemical properties. Methods for determining isoenzymes are based on these differences in physicochemical properties.

In terms of their structure, isoenzymes are primarily Oligomeric Proteins. Furthermore, a given tissue predominantly synthesizes specific types of protomers. As a result of specific combinations of these protomers, enzymes with diverse structures—isomeric forms—are formed. The detection of specific isomeric forms of enzymes allows their use in disease Diagnosis.

Lactate dehydrogenase isoforms. The enzyme lactate dehydrogenase (LDH) catalyzes the reversible oxidation of lactate to Pyruvate:

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Lactate dehydrogenase is an oligomeric protein with a Molecular Weight of 144,000 Da, consisting of four subunits of two types: M (from muscle) and H (from heart). The combination of these subunits forms the basis for the five isoforms of lactate dehydrogenase (Fig. 7.35, A). LDH₁ and LDH₂ are most active in The Heart muscle and Kidneys, while LDH₄ and LDH₅ predominate in skeletal Muscles and the liver. Other tissues contain Various Forms of this enzyme. LDH isoforms differ in electrophoretic mobility, which makes it possible to determine the tissue origin of LDH isoforms (Fig. 7.35, B).

Fig. 7.35. Lactate dehydrogenase isoforms:

A - structure of various LDH isoforms; B - electrophoretic distribution and relative amounts of LDH isoforms in different organs;

C - content of LDH isoforms in blood plasma under normal and pathological conditions (electrophoretograms on the left, photometric scanning on the right)

The evolutionary emergence of different LDH forms is due to the Specific features of tissue oxidative METABOLISM. LDH₄ and LDH₅ isoenzymes (M-type LDH) function efficiently under anaerobic conditions, whereas LDH₁ and LDH₂ (H-type) operate under aerobic conditions when pyruvate is rapidly oxidized to CO₂ and H₂O rather than reduced to lactic acid.

In certain diseases, LDH activity is examined in blood plasma. Normally, LDH activity ranges from 170-520 U/L. Increased activity is observed in acute lesions of the heart, liver, and kidneys, as well as in megaloblastic and hemolytic anemias. However, this indicates damage to only one of the aforementioned tissues.

To establish a diagnosis, the analysis of blood plasma LDH isoforms by Electrophoresis is required. Fig. 7.35, B shows the plasma electrophoregrams of a healthy individual, a patient with myocardial infarction, and a patient with hepatitis. The detection of tissue-specific LDH isoforms in blood plasma is used as a diagnostic test for damage to the respective tissue.

Creatine kinase isoforms. Creatine kinase (CK) catalyzes the reaction of creatine phosphate formation:

The CK molecule is a dimer composed of Two Types of subunits: M (from muscle) and B (from Brain). These subunits form three isoenzymes: BB, MB, and MM. The BB isoenzyme is predominantly found in the brain, MM in skeletal muscles, and MB in the myocardium. CK isoforms exhibit different electrophoretic mobilities (Fig. 7.36).

Fig. 7.36. Structure and electrophoretic mobility of various creatine kinase isoforms

Normal CK activity should not exceed 90 IU/L. The determination of CK activity in blood plasma is of diagnostic importance in myocardial infarction (manifested by an elevated level of the MB isoform). The amount of the MM isoform can increase due to skeletal muscle trauma and injury. The BB isoform cannot cross the blood-brain barrier; therefore, it is practically undetectable in the blood even during a stroke and holds no diagnostic value.

Enzymology in myocardial infarction. Approximately 30% of patients with myocardial infarction present with an atypical clinical picture. Therefore, additional investigations are necessary to confirm myocardial injury.

During myocardial infarction, significant alterations in the blood activities of CK, LDH, and aspartate aminotransferase (AST) are observed, which depend on the time elapsed since the onset of the infarction and the extent of tissue damage. A typical curve illustrating Changes in the activity of these enzymes is shown in Fig. 7.37. Following coronary vessel occlusion, an initial increase in the activity of the MB isoform of CK is detected in the blood, but the enzyme is rapidly cleared from the bloodstream. The detection of elevated CK activity in blood plasma is the primary enzymodiagnostic criterion for myocardial infarction. If a patient presenting with retrosternal chest pain shows no change in CK activity, a diagnosis of myocardial infarction is unlikely.

Additional confirmation of the diagnosis of myocardial infarction is the detection of AST and LDH activities in the patients' blood. The dynamics of these activity changes are also illustrated in Fig. 7.37. Normal AST activity ranges from 5 to 40 IU/L. In myocardial infarction, AST activity increases within 4–6 hours, with peak activity observed over 2–3 days. The LDH level in blood plasma also rises a few hours after vascular occlusion, reaching peak activity on the 3rd–4th day, followed by a gradual return to normal levels. The magnitude of the increase in LDH activity correlates with the extent of myocardial damage.

Fig. 7.37. Changes in blood plasma enzyme activities during myocardial infarction