Abstract
Recently, placental alkaline phosphatase (PLAP) has been suggested as a tumour marker in patients with seminomas (S), since elevated serum levels of PLAP were found with high frequency in these patients. The present immunoperoxidase study of 33 testicular germ cell tumours was undertaken to localize PLAP in the various types of these tumours as well as in the carcinoma-in-situ (CIS) pattern. Eighteen out of 19 (95%) S were PLAP positive compared to nine out of 14 (64%) non-seminomas (NS). In the NS the positive staining reaction was localized to tumour components of embryonal carcinoma (EC) in six cases, of choriocarcinoma (CC) in one and of S in two, while components of yolk sac tumour and teratoma were PLAP negative. The number of positively stained cells in S was much higher than in EC. The staining reaction was pronounced in the syncytiotrophoblast of CC and in some syncytiotrophoblast-like cells present in S.
The staining reaction product was mainly confined to the cell membrane in the positive tumour types. In 20 out of 24 cases with CIS various numbers of CIS cells were PLAP positive, while PLAP was not found in normal germinal epithelium. Sixty three per cent of S patients had serum values above 1.0 micrograms/l, while such values occurred in 21% of NS patients. The tissue staining pattern for PLAP was found to correspond to the preoperative serum value. On the basis of these findings it is concluded that PLAP may be a useful marker in patients with S. Serum levels of PLAP may be used diagnostically in patients with testicular tumours and for monitoring therapy and detection of recurrences in patients with S. For optimal utility of this marker, determinations of serum profiles of PLAP are recommended. Finally, demonstration of PLAP in CIS indicates a functional relationship between CIS and S supporting the hypothesis that CIS is the precursor state of these tumours.
Enzymes in serum
The enzymes measured in serum, plasma and extravascular fluids for diagnostic purposes are biocatalysts. Even minor quantities are sufficient to attain the equilibrium of a chemical reaction by lowering the activation energy. Enzymes have a reaction specificity meaning that a chemical reaction can only be catalyzed by an enzyme specifically required for this reaction. Furthermore, enzymes have a substrate specificity meaning that only a specific substance or substance group functions as a reactant and is converted to the product.
Factors influencing enzyme activity
The enzymes in serum come from tissue cells or result from secretory enzymes entering the blood. The tissue enzymes mainly originate from the cells’ main metabolic chains. In the cells, they are either dissolved in the cytoplasm or bound to cell structures such as the mitochondria. Secretory enzymes such as peptidases and hydrolases are usually secreted in an inactive form, while only a few enzymes such as cholinesterases pass into the plasma in an active form. Enzyme activity in the plasma depends on factors governing the extent of release. The release of cell-specific enzymes is regulated by the extent of cell damage. Increased enzyme production of individual cells or the proliferation of enzyme-producing tissue are decisive for the release of secretory enzymes.
Enzyme release: Low activity of cell-specific enzymes in the blood of healthy individuals is based on the impermeability of the metabolically active cell membrane. Any pathological process affecting the cell membrane’s energy supply, for example inadequate supply with ATP or other high-energy substrates due to ischemia or anoxia, can cause disintegration of the cell membrane and consecutive release of enzymes. First, the membrane potential is upset. K+ leaves the cell and Na+ and water enter the cell causing swelling. Subsequent Ca2+ entry activates hydrolases and peptidases which results in the destruction of intracellular structures and leakage of the cell membrane. Cytoplasmic enzymes are the first to appear in the blood, followed by mitochondrial and membrane-bound enzymes. The extent and rate of enzyme release depend on the size and the concentration gradient of a given enzyme between cytoplasm and extracellular space. In healthy individuals, intracellular enzyme concentration is very much higher than the concentration in plasma. For example, the AST and ALT activities in the liver, kidney, heart and skeletal muscle are higher than in the plasma of a healthy individual as follows:
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- AST 7000 fold, 4500 fold, 8000 fold and 5000 fold
- ALT 2800 fold, 1200 fold, 400 fold and 300 fold.
Enzymes pass from the interstitium to the blood either via direct transfer across the capillary wall or indirectly via the lymphatic pathways. Direct transfer is the case in well-vascularized tissue such as the liver parenchyma, and indirect transfer takes places in tissue with a less permeable capillary membrane such as the muscles. The extent of enzyme release depends on the enzyme’s intracellular localization. Enzymes dissolved in cytoplasm appear in the blood relatively soon after cell damage with an easily measurable activity. Enzymes bound to subcellular structures such as the mitochondria take longer. As a rule, an enzyme pattern corresponding to the intracellular enzyme distribution appears in the blood within 24 hours after cell damage including necrosis.
Changes in enzyme production: Changes in enzyme production can manifest as reduced or elevated activity in the blood compared to the reference interval without the presence of cell damage. Reduced activity is less in the focus of diagnostic attention as it is mainly based on genetic dysfunction of the tissue enzymes such as ALP. In contrast, reduced activity in secretory enzymes is often based on a reduction in the relevant enzyme-producing tissue, for example reduced CHE in liver cirrhosis. Increased enzyme release into the blood without the presence of tissue damage can have the following causes:
- An increase in the number and/or biochemical activity of tissue cells. The proliferation and increased activity of osteoblasts in adolescents, for example, causes elevated ALP due to the increased production of the bone-specific isoenzyme.
- Enzyme induction. Tissue cells increasingly produce enzymes, for example, chemical stimulation of the hepatocytes by alcohol, barbiturates or phenytoin enhances the production of GGT by these cells. Moreover, obstruction of the bile ducts, for example, stimulates the synthesis of the liver isoenzyme ALP.
- Development of new tissue. During the last trimenon of pregnancy, for example, the placenta synthesizes ALP determined as placental isoenzyme in the blood of the pregnant woman. A similar isoenzyme can also be produced by malignant germ cell tumors of the testes.
Tissue damage and enzyme elevation
The most common enzyme elevations are caused by damage of the liver, myocardium, skeletal muscle and erythrocytes. Liver-induced enzyme elevations result from direct damage of the cell membrane by viruses, the toxic effect of drugs and poisons or tissue hypoxia. The latter usually causes centrilobular necrosis of hepatocytes and can result from acute right heart failure, portal hypertension or arterial hypoxia, for example, in cases of shock. In contrast, hepatic infarctions are rare because the liver has a dual blood supply from the hepatic arteries and the portal vein system.
The situation is different for the heart. As a rule, occlusion of the end arteries results in hypoxemic necrosis of myocytes due to the segmental blood supply of the myocardium. An enzyme pattern corresponding to that of the myocardial cell appears in the blood within 24 hours.
Damage to the skeletal muscles associated with enzyme release is manifold, the most important being injuries, hypoxic necroses, inflammations, infections, degenerative diseases, toxic damage (alcohol), uremia and neurogenic myopathies.
The lysis of erythrocytes causes the release of LD, the enzyme with the highest activity in these cells. A distinction is made between in-vivo and in-vitro hemolysis. In-vivo hemolysis occurs within the blood vessel (intravascular) and is immune-mediated in most cases, while in-vitro hemolysis is based on the destruction of the erythrocytes during blood sampling or several days of storing of whole blood prior to analysis.
