A comprehensive review published 12 years ago (Huggins, C, and Tala-lay, P. Advances in Internal Medicine, 3:275, 1949) listed 8 enzymes in human blood serum that had been studied in relation to disease. Since that time more than 30 other serum enzymes have been investigated in various clinical situations. The rapid progress in clinical enzymology over the last decade has been greatly facilitated by the development of simple optical methods for the determination of enzyme activity, which are based on the absorption of visible or ultraviolet light by substrates or reaction products (Huggins and Talalay, loc. cit.). Direct spectrophotometric assay procedures were devised and applied extensively in more recent studies on serum enzymes in this laboratory.

In 1957 human serum was examined for the presence of some of the enzymes involved in lactose biosynthesis. The manufacture of lactose is a specific biochemical function of mammary tissue. It was considered that some of the enzymes responsible for this process might be retained in neoplasms of the mammary gland and that study of the serum levels of these enzymes might be of clinical value in carcinoma of the breast. Low levels of uridine diphospho glucose pyrophosphorylase were detected in human blood serum (203), but preliminary studies indicated that the activity of the enzyme was not changed in patients with breast cancer. The enzyme was measured by an optical method in which the pyrophosphorolysis of uridine diphosphoglucose was coupled, by the addition of appropriate enzymes, with the reduction of TPN. During the course of these investigations, it was discovered that normal human serum contained a number of TPN-linked dehydrogenases. The accidental finding that the serum levels of one of these enzymes, isocitric dehydrogenase, increased gready in certain liver disorders prompted a thorough study of serum TPN-specific dehydrogenases in disease.

TPN-Linked Enzymes In Blood Serum

TPN-specific dehydrogenases for glucose-6-phosphate, 6-phosphoglu-conate (6-PGD), and isocitrate (ICD) were found to be present in human blood serum, and optical methods were developed for their assay (204). The activity of 6-PGD and ICD in the sera of apparendy healthy individuals were of the same order of magnitude, namely, in the range of 50-250 millimicromoles of substrate oxidized per 1 ml. of serum per hour at 25°. Thus, on a molar basis, the levels of these TPN-linked dehydrogenases are about the same as those of aldolase and glutamic-pyruvic and glutamic-oxalacetic transaminases, but they are much lower than those of the DPN-linked malic and lactic dehydrogenases in serum. Although the activity of ICD and 6-PGD in red and white blood cells is much greater than that of serum, it appeared that these serum enzymes did not originate from blood cells. In normal individuals serum ICD and 6-PGD levels were independent of age, race, sex, and total serum protein concentration. In normal human serum it was not possible to detect TPN-linked dehydrogenases for glucose and glutamate, or the "malic enzyme" that catalyzes the reversible reductive carboxylation of pyruvic acid.

Serum ICD And 6-PGD In Disease

Studies on the serum ICD of 250 patients (162) showed that the activity of this enzyme was increased 4- to 30-fold in the early stages of acute viral hepatitis. The magnitude and sequence of changes in serum ICD in hepatitis resembled parallel alterations in serum glutamic-pyruvic transaminase. The serum levels of ICD were also elevated in some malignancies involving metastases to the liver, but they were usually in the normal range in cirrhosis of the liver and in extra-hepatic obstructive jaundice. A wide variety of other diseases, including myocardial infarction and many types of neoplasm, did not cause elevations of serum ICD. The alterations of serum ICD in liver disease were quite different from concomitant changes in serum lactic dehydrogenase and alkaline phosphatase and could not be correlated with flocculation phenomena or hyperbilirubinemia. It was concluded that measurement of serum ICD may be of value in following the course of viral hepatitis and in the differential diagnosis of intra- and extra-hepatic jaundice (although serum ICD levels are not infallible in the latter respect). Similar changes in serum ICD in liver disease were later observed by others (Okumura, M., and Spellberg, M. A. Gastroenterology, 39:305, i960; Franken, F. H.; Brauns, M. T.; Storck, G.; and Kazmeier, F. Klin. Wchnschr., 38:800, i960; Baron, D. N. J. Clin. Path., 5:740, 1960; Bodansky, O.; Schwartz, M. K.; Krugman, S.; Giles, J. P.; and Jacobs, A. M. Pediatrics, 25:807, i960).

Serum 6-PGD was found to be increased in the acute phase of viral hepatitis and occasionally in patients with malignant diseases with metastases to the liver. No striking alterations in serum 6-PGD were observed in other diseases (203).

Efflux Of Alcohol And Polyol Dehydrogenases Into Blood Serum

The liver of man and other mammals is a rich source of two DPN-linked dehydrogenases that oxidize alcohols. One of these enzymes, alcohol dehydrogenase, catalyzes inter alia the oxidation of ethanol to acetaldehyde. The other enzyme, polyol dehydrogenase (195, 197), catalyzes the oxidation of some 5-, 6-, and 7-carbon polyols to the corresponding ketose sugars, sorbitol being a particularly active substrate. Alcohol and polyol dehydrogenases could not be detected in the serum of normal individuals. But high levels of both these enzymes were found in the sera of patients in the acute phase of viral hepatitis (203). The presence of these enzymes in blood serum is thus a qualitative systemic index of viral disease of the liver. Independent studies by U. Gerlach (Klin. Wchnschr., 37:93, 1959) confirmed the entry of polyol dehydrogenase into the blood serum in certain liver disorders.

Serum Aldolase

A method was developed for the rapid colorimetric estimation of aldolase in blood and tissues of animals (Sibley, J. A., and Lehninger, A. L. J. Biol. Chem., 177:859,1949). With this procedure it was found that a "normal" amount of the enzyme existed in rat tumors. The occurrence of aldolase in normal blood serum and its elevation in the serum of tumor-bearing animals was also demonstrated. These experiments thus fully confirmed the wartime experiments of Warburg and Christian (Biochem. Zeitschr., 314: 399, 1943) and have made it possible by somewhat simpler procedure to follow the course of experimental therapy of tumors in rats. It was found, for example, that treatment with urethane or certain other tumor growth inhibitors also caused a reduction of the serum aldolase level (Sibley, J. A., and Lehninger, A. L. J. Nat. Cancer Inst., 9:303, 1949). In human patients certain other pathological states caused elevations of serum aldolase levels, for example, hepatitis and other liver disorders. An incidental finding that progressive muscular dystrophy causes significant elevations in serum aldolase has since been developed into a valuable diagnostic and prognostic aid in these diseases (Dreyfus, J.-C; Schapira, G.; and Schapira, F. Ann. N.Y. Acad. Sc., 75:235-49, 1958).

Origin And Fate Of Serum Enzymes

Early experiments by O. Warburg (Biochem. Zeitschr., 314:399, 1943) demonstrated that injected aldolase rapidly disappears from the blood stream. Simdar findings were made with respect to seminal acid phosphatase (Huggins C, The Harvey Lectures, 42:148, 1947). ICD was purified from rat heart and injected intravenously into rats; serial estimations of serum ICD levels revealed that the half-life of the injected enzyme was in the neighborhood of 1 hour (203). The rate of inactivation of ICD by blood serum in vitro was very slow. Serial studies were also made of serum ICD, 6-PGD and lactic, alcohol, and polyol dehydrogenases following various types of experimental liver injury in the rat (carbon tetrachloride poisoning, partial hepatectomy, ligation of the bde duct). There was no uniformity in the elevation of these serum enzymes. The changes in serum ICD and alcohol and polyol dehydrogenases tended to occur earlier in female as compared to male rats. It could be concluded from these experiments that the normal serum and tissue levels of these enzymes, and their intracellular localizations, are by no means the only factors which determine the extent of their outflow into the circulation from dead or damaged tissues (203). In accord with this conclusion were the findings (162, 203) that the ICD activities of normal human liver and cardiac muscle were about the same and that serum ICD increases gready following acute injury to the liver parenchyma but not in myocardial infarction.

Enzymes In Human Seminal Plasma

The phosphomonoesterases and proteolytic enzymes of human seminal plasma have been studied extensively (154), but relatively little is known about the levels and properties of other enzymes in this fluid. A survey of respiratory and glycolytic enzymes in the seminal plasma of man (158) showed that 3 oxidizing enzymes were present in particularly high concentration, namely, lactic, malic, and isocitric dehydrogenases. Low levels of a TPN-specific glutathione reductase and of a DPN-linked polyol dehydrogenase were found in seminal plasma, but 10 other enzymes could not be detected. The ICD of seminal plasma was shown to originate mainly from the prostate gland and to be completely specific for TPN (pyridine nucleotide transhydrogenase was absent from this fluid). The activity of ICD in human seminal plasma is very much greater than that of other body fluids, being about 200 times higher than that of blood serum and more than 1,000-fold greater than the level in cerebrospinal fluid. It was observed in these studies that human seminal plasma catalyzed a rapid and extensive degradation of pyridine nucleotides, principally by the action of nucleotide pyrophosphatase. Also, it was found that seminal plasma contains relatively large amounts of pyruvate as well as fructose. The levels of seminal malic, lactic, and isocitric dehydrogenases could not be correlated with any clinical conditions.