The Clinical Significance of Alterations in Transaminase Activities of Serum and Other Body Fluids

THE CLINICAL SIGNIFICANCE OF ALTERATIONS IN TRANSAMINASE ACTIVITIES OF SERUM AND OTHER BODY FLUIDS Felix Wr6blewski Division of Clinical Investigation. Sloan-Kettering Institute and Department of Medicine. Memorial Center for Cancer and Allied Diseases. New York. New York

Page 1 Scope of Chapter ...................................................... 314 314 2 Transamination ....................................................... 314 2.1. Nonenrymatic ..................................................... .................... 315 2.2. Enzymatic .............................. .................... 319 3. Methods of Measuring Transaminase Activity of .................... 319 3.1. Chromatographic Technique 320 3.2. Spectrophotometric Techniques ...................................... 322 3.3. Colorimetric Techniques ............................................ 323 4. Tissue Distribution of Transaminase Activity ............................. 323 4.1. Animal Tissues .................................................... 323 4.2. Human Tissues.................................................... 324 5 Alterations of Serum Transaminase in Cardiac Disease ..................... 5.1. Experimentally Produced Pathological Cardiac States .................. 324 324 5.1.1. Transmural Myocardial Infarction ............................. 5.1.2. Subendocardial or Focal Myocardial Infarction . . . . . . . . . . . . . . . . . .325 326 5.1.3. Other Types of Cardiac Tissue Injury .......................... 5.2. Clinical Cardiac Disease States ...................................... 326 5.2.1. Myocardial Infarction ...... ..................... 326 , and Others . . . . . . . . . . 328 5.2.2. Carditis, Pericarditis, Acute 329 6 Alterations of Serum Transaminase in Hepatic Disease..................... 6.1. Experimentally Produced Pathological Hepatic States.................. 329 330 6.2. Clinical Hepatic Disease States ...................................... ......................... 331 6.2.1. Toxic Hepatic Disease. . . . . . . . . . . . 6.2.2. Infectious and Inflammatory Hepatic Disease ................... 331 335 6.2.3. Degenerative Hepatic Disease ................................. 6.2.4. Extrahepatic Biliary Tract Disease ............. . . . . . . . . . . . . 336 336 6.2.5. Neoplastic Hepatic Disease.................................... 6.2.6. Dif€erential Diagnosis of Jaundice by Means of Serum Enzymes . . . 337 337 6.2.6.1. Adult Types of Jaundice ............................... 340 6.2.6.2. Neonatal Jaundice .................................... 7. Alterations of Serum Transaminase in Pathological Skeletal Muscle States .... 340 313

. .

.

.

314

FELIX

WR6BLEWSKI

7.1. Skeletal Muscle Injury.. ........................................... 340 7.2. Skeletal Muscle Disease States.. .................................... 341 8. .4lterations in Serum Transaminase in Other Abnormal States............... 341 8.1. Renal Disease.. ................................................... 341 341 8.2. Biliary-Pancreatic Disease.. ........................................ 8.3. Thrombocytopenia................................................. 342 8.4. Pregnancy ........................................................ 342 8.5. States Associated with Serous Effusions. ............................. 342 9. Alterations of Cerebrospinal Fluid Tmnsaminase in Central Nervous System Disease ............................................................... 342 9.1. Experimentally Induced Pathologic Central Nervous System States.. .... 343 9.2. Clinical Central Nervous System Disease.. ........................... 343 10. Conclusions ........................................................... 344 References......... :. ..................................................... 344

1. Scope of Chapter

The measurement of serum enzymes has proven to be a useful laboratory adjunct to clinical medicine. The clinical significance of pathological alterations in serum acid phosphatase, alkaline phosphatase, amylase, and lipase is generally acknowledged (B2). Following the demonstration in 1953 of the normal range of activity of transaminase in human serum (K2), a sizable experience has accrued attesting to the clinical implications of alterations in serum transaminase activity in diseases of heart, liver, and skeletal muscle. An extensive literature on enzymatic transamination has accumulated since this reaction was first suspected in 1930. Several reviews on the subject of transamination have appeared (B4, C12, C13, H2, H5, M5) and have dealt in detail with the wide scope of transamination, elucidation of the catalytic systems involved, the role of vitamin Ba in transamination, and the importance of transamination in amino acid metabolism. No attempt wiIl be made in the present discussion to dwell in detail on these aspects of enzymatic transamination. Rather, it is the purpose of this review and the scope of this chapter to point up the pertinent experimental studies which bear on the mechanisms of alterations in serum transaminase activity, and to review the clinical observations which have been made in the past several years and which bear on the clinical significance of alterations in transminase activity of serum and other body fluids. 2. Transamination

2.1. NONENZYMATIC

The intermolecular transfer of an amino group from an a-amino acid to

an a-keto acid is an example of a transamination reaction. In some in-

stances, a-amino acids may be oxidatively deaminated by means of 1,2-

315

S E R U M TRANSAMINASE ACTIVITY

dicarbonyl compounds (H2, K17). The first typical nonenzymatic transamination was observed when a 1,2dicarbonyl compound was replaced by an a-keto acid. This resulted in the oxidative deamination of the amino acid (I), but the nitrogen reappeared in combination with the keto acid, (11), which had undergone reductive amination (H3). The mechanism proposed for this type of nonenzymatic reaction included the condensation of the reactants to form a Schiff-base intermediate (111), followed by shift of the double bond (IV), decarboxylation of the amino acid carboxyl group, and hydrolysis to the corresponding aldehyde (V), and new amino acid (VI) (H2). H

t.

R- -NHn AOOH (1)

+ O===C-R’ I

COOH (11)

-Hz0

-----+

c:

H R-

-N=C-R’

$ R-C-N-

LOOH LOOH (111)

it I

-R’

LOOH COOH (IV)

2.2. ENZYMATIC

As early as 1909, the conversion of amino acids to a-keto acids and of a-keto acids to amino acids had been demonstrated in several animal species (K10, N2). In 1930, it was shown that when glutamate was added to minced pigeon breast, glutamic acid disappeared without the simultaneous increase in ammonia, urea, uric acid, or amide nitrogen (Nl). The observed increase in succinate suggested that the amino group of the glutamic acid was transferred to “some reactive carbohydrate residue” to form another amino acid. Several years later, others observed an increase in the rate of disappearance of oxaloacetate when glutamate was added to pigeon breast muscle (A5, Bl). A new concept in amino acid metabolism was introduced in 1937 when enzymatic transamination was described (B5-B8). It was observed that, when using pigeon breast muscle as a source of transaminase, the interconversion between amino and keto acids could take place by means of an intermolecular transfer of the a-amino group from an amino acid to an a-keto acid (the “reactive carbohydrate residue”) without the intermediate participation of ammonia. Enzymatic transamination, unlike the noncatalyzed transamination, results in oxidative deamination without decarboxylation. It was suggested that the tissue enzyme which catalyzed transaminating reactions be called aminopherase (B4).

316

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WR6BLEWSKI

Although originally it was thought that any a-amino acid, except glycine (B5, B9) , could participate in enzymatic transamination only with a-ketoglutaric acid, it became apparent that one member of a transaminating pair must be either a dicarboxylic keto or dicarboxylic amino acid, since no amino group transfer had been observed between monocarboxylic keto and monocarboxylic amino acids. However, the transamination reaction may be achieved by the addition of a dicarboxylic amino acid to a monocarboxylic keto acid, or by the addition of a dicarboxylic keto acid to a monocarboxylic amino acid (B10). A mechanism analogous to that proposed for the nonenzymatic transamination has been suggested for the enzymatic exchange (B4, K11, K12). It was suggested that at least two enzyme systems were active in catalyzing transamination reactions. One was said to be active in catalyzing reactions specifically involving glutamic acid or a-ketoglutaric acid, while the other affected reactions involving aspartic acid or oxaloacetic acid (K4). A partially purified enzyme preparation, called glutamic aminopherase, which catalyzed amino group transfer involving I,(+)-glutamic acid and a-ketoglutaric acid, was prepared. It was relatively stable when stored as a dry power and had a maximal activity a t pH 7.5. It had been prepared from pigeon breast muscle, pig heart, and rabbit muscle (K18). Another preparation, aspartic aminopherase, was obtained from pig heart, and its activity was directed toward the catalysis of transaminations involving L(+)aspartic acid and oxaloacetic acid. This enzyme preparation was found to be accompanied by the glutamic aminopherase system (K19, K20). Both systems were noted to be thermostable and were inactivated by adsorption, dialysis, or salting out. A thermostable coenzyme present in boiled muscle extracts reactivated all but the thermally inactivated enzyme (K20). A variety of chemical agents influenced the activity of glutamic aminopherase (K19). In 1940, an enzyme preparation obtained from pigeon breast muscle and pig heart muscle was noted to exhibit some of the properties of both aspartic aminopherase and glutamic aminopherase, thereby suggesting that only one enzyme existed. The differences noted previously were related primarily to activity rather than specificity (C10, C14). The name transaminase was suggested for this enzyme. With the utilization of tissue extracts, it was shown that transamination is largely limited to two reactions ((310, C15). L(+)-Glutamic

acid

L( +)-Glutamic

+ oxaloacetic acid

acid

a-ketoglutaric acid

+ pyruvic acid

a-ketoglutaric acid

+ L(+)-aapartic

+ L( +)-danine

acid ( 1 ) (8)

SERUM TRANSAMINASE ACTIVITY

317

Indications of the existence of a coenzyme activator for the apotransaminase were supported by the evidence obtained from experimental studies of vitamin Be deficiency. Deficiency of vitamin Ba was associated with lowered levels of transaminase activity, and the addition of pyridoxal phosphate to the tissue or cell preparations resulted in a t least partial restoration of enzyme activity (A4, L5, S3). Subsequently, it was shown that transamination occurs in model systems when a mixture of pyridoxal and glutamate, or pyridoxamine and a-ketoglutaric acid, is heated (58). In addition, it was noted that purified transaminase preparations contain Be activity which increases with purification (Sl). With crystalline pyridoxamine phosphate, it was possible to replace pyridoxal phosphate as a coenzyme for transamination with purified pig heart apoenzyme (M6). After a suitable incubation period, equal concentrations of pyridoxal phosphate and pyridoxamine phosphate produce equivalent activation. Evidence has been presented to the effect that most pyridoxal phosphate is bound at nonfunctional sites and that no conversion of pyridoxamine phosphate to pyridoxal phosphate takes place prior to the addition of substrates. It would appear that conversion probably occurs after the addition of substrates and after apoenzyme-coenzyme combination (M6). Recent studies have confirmed earlier work on the activation of transaminases by pyridoxal phosphate and have shown as well that many of the newly discovered transaminases also require this coenzyme (M5). With the utilization of pyridoxal phosphate, it was demonstrated that muscle homogenates are capable of catalyzing the transfer of a-amino groups from 25 different amino acids and that this activity was present in pig heart, liver, and kidney tissue homogenates (C2). In 1952 it was established that transaminase activity was not limited to pigeon breast muscle and pig heart, liver, and kidney, but was present as well in varying activities in eight organs of the rat (A7, C15). Inhibition of transamination has been shown to result from silver, mercury, copper, zinc, cyanide (0.05 M results in 80% inhibition while 0.001 M results in 30% inhibition), quinhydrone (0.001 M results in 70% inhibition), and p-benzoquinone (0.001 M results in 100% inhibition). The following substances were noted to have no appreciable effect on the rate of transamination : carbon tetrachloride, toluene, capryl alcohol, ethanol or acetone (10-15%), urethane, 0.02 M sodium arsenate, selenite, fluoride, iodoacetate, bromoacetate, 0.01 M pyrophosphate, cysteine (0.02 M ), glutathione (0.05 M ) , ascorbate (0.01 M ) , ferrocyanide, ferrous sulfate, methylene blue, ferric chloride, semicarbazide, hydroxylamine (0.02 M ), phenylhydrazine, chloride (0.1 M ) , bromide, iodide, nitrate, bicarbonate, acetate, sulfate, phosphate, sodium, potassium, ammonium, magnesium, manganese (0.01 M ) aluminum (0.04 M ) and lead (0.001 M ) (B4, B8, C8, C9, C11, C12, C14, Vl).

318

FELIX WR6BLEWSKI

The variety of amino and keto acids capable of participating in transamination makes it necessary to distinguish the different catalytic effects. Accordingly, it has become customary to distinguish the enzymes responsible for transamination reactions in one of several ways. In nonreversible reactions, the enzyme is described by a hyphenated term describing the reactants. The enzymes which catalyze reversible transamination reactions are designated by a term referring to both amino acids concerned, i.e., glutamate-aspartate transaminase. Another method of designating enzymes which catalyze reversible transamination reactions is to name them by a 0Units/ml.

(of reporotrd

protein derived from I ml.rerum)

100-

200-

300-

7

OGP-T -GO-T

400-

2.8 2.4

500

--

-

--

P 2*o 1 1.6 -

N

d

-

1.2-

0.4 0.8

'

8.2

v-1

globulin

e

c-2

d-I albumin

hyphenated term describing the favored products of the equilibrium reaction, i.e., glutamic-oxaloacetic transaminase (GO-T) and glutamic-pyruvic transaminase (GP-T). The latter method of designation has been used in the clinical literature regarding serum transaminases, and accordingly will be used in this presentation hereafter. As has been appropriately

319

SERUM TRANSAMINASE ACTIVITY

stated (H2, M5), the final decision on nomenclature of transaminases must await purification and characterization of individual enzymes. Although it now appears that a t least several separate transaminases exist in each of the tissues that have thus far been studied, final characterization of the individual catalytic systems has yet to be achieved (M5). However, the chemical separation and purification of the GP-T and GO-T of heart muscle have been reported by several groups (C3, C4, G3, L4, 0 1 , 52). Both these enzymes have also been separated from each other electrophoretically, using as an enzyme source serum obtained from patients with hepatitis (W3) (Fig. 1). It would appear that at least GP-T and GO-T are separable, distinct, and individual catalytic systems. 3. Methods of Measuring Transaminase Activity of Serum

Although a number of transaminase activities have been demonstrated in human serum (M2), the two serum transaminases currently of clinical import are serum glutamic-oxaloacetic transaminase (SGO-T) and serum glutamic-pyruvic transaminase (SGP-T). Both serum enzymes have been measured chromatographically (K2, K3), spectrophotometrically (Hl, K3, N3, S l l , W13), and colorimetrically (Cl, U1, W9) (Table 1).Other methods AVERAQEACTIVITY OF SGO-T

TABLE1 SGP-T

AND

IN

NORMALHUMANSERUM

Chromatographic Spectrophotometric method method (pmoles glutamate/ml/hr) (units/& serum.)

SGO-T SGP-T

0.622 f 0.191 0.525 f 0.146

Colorimetric method (units/ml serum’)

20 f 7 16.0 f 9 . 0

16 f 8 22 f 12

Units are defined in Section 3.2 of the text. Units are defined in Section 3.3 of the text.

have been utilized for the measurement of transaminase activity in tissue brei (C2, C10, C14, G3). 3.1. CHROMATOGRAPHIC TECHNIQUE

When aspartate or alanine is incubated with a-ketoglutarate and a source of enzyme, the rate of production of glutamate may be taken as a measure of transaminase activity. The amount of glutamate produced after a given incubation period under standardized conditions can be measured by quantitative paper chromatography (A7). The glutamate formed is eluted from the paper, and color development with ninhydrin provides a quantitative determination of glutamate and, thereby, a measure

320

FELIX WR6BLEWSIU

of transaminase activity (T2). Addition of a buffered solution of pyridoxal phosphate in a concentration of 10 pg per milliliter was found to have no measureable effect on the transaminase activity of normal serum (K3). The effect of pyridoxal phosphate on transaminase activity of serums with increased activity was incompletely explored. Recent observations indicate that in some instances abnormally increased serum transaminase activity may be enhanced by the addition of pyridoxal phosphate (W5). Addition of a boiled and filtered extract of rat liver, used as a source of possible activators, similarly was found not to affect the observed transaminase activity of normal serum. No difference was detected between transaminase activity in serum and in plasma from the same donors when oxalate, citrate, or heparin was used as anticoagulant. The SGO-T activity in normal humans varied from 0.41 to 1.36pM glutamate formed per milliliter of serum per hour of incubation, with a mean activity of 0.622 f 0.191. Serum glutamic-pyruvate transaminase activity was found to be between 0.21 and 1.01 pM per milliliter per hour, with a mean value of 0.525 f 0.146. The glutamic-oxaloacetic transaminase activity found in hemolysates of normal human whole blood ranged from 5.0 to 8.7 pM per milliliter per hour, with a mean value of 6.86 f 0.78, while the GP-T activity of hemolysates varied from 1.6 to 3.3 pM per milliliter per hour, with a mean value 2.48 f 0.36. The ratio of GO-T activity to GP-T activity in hemolysates varied between 2.04 and 3.60 with a mean value of 2.70 f 0.40, while in serum this ratio was found to be 0.725 to 1.67 with a mean value of 1.15 f 0.25. In no instance was transaminase activity absent from the examined serums of normal humans or of patients with various disease states (K3). The method of transaminase assay by quantitative paper chromatography of the glutamate produced is sensitive, accurate, and uncomplicated by nonenzymatic transamination (K3). However, the technique is cumbersome, lengthy, and not readily adaptable for the clinical laboratory. It is versatile in that many transaminase systems may be studied (M2) since the formation of glutamate is common to those systems in which a-ketoglutarate is utilized as one of the reactants. 3.2. SPECTROPHOTOMETRIC TECHNIQUES Glutamic-oxaloacetic transaminase activity may be measured spectrophotometrically by utilizing the high ultraviolet absorption of oxaloacetate at wavelength 280 mp to follow the transamination reaction. This is done by measuring the change in optical density as oxaloacetate is produced or consumed. However, the low transaminase activity and high protein content of serum, together with the instability of oxaloacetate, make this spectrophotometric assay difficult to apply to serum transaminase measurements. A spectrophotometric method was devised in which the glutamic-

SERUM TRANSAMINASE ACTIVITY

321

oxaloacetic transamination reaction (3) is coupled to the oxidation of reduced diphosphopyridine nucleotide (DPNH) by oxaloacetate in the presence of an excess of purified malic dehydrogenase ( 4 ) . The oxidation of DPNH, and thereby the transamination reaction, is followed by measuring the decrease in light absorption at 340 mp at which wavelength the reduced pyridine nucleotides have an absorption peak.

+ L( +)-aspartate SGO-T >glutamate + oxaloacetate malic -.>malate + DPN oxaloacetate + DPNH + H Q dehydrogenase

a-ketoglutarate

F

(a) (4)

Serum glutamic-oxaloacetic transaminase activity is expressed as units per milliliter of serum per minute. One unit equals a decrease in optical density of 0.001 under standardized conditions (Kl). At 23"C, the GO-T activity of serums of normal adult humans was found to range between 9 and 32 units per milliliter of serum per minute, with a mean value of 20 f 7 units. Conversion of these units to micromoles per milliliter per hour gives a mean value of 0.57, which falls within the range of 0.41 to 1.36pM per milliliter per hour found by quantitative paper chromatographic assay. The spectrophotometric measurement of SGP-T is accomplished by utilizing a technique analogous to that described for SGO-T (W13). The transamination reaction (6) is coupled to the reduction of pyruvate to lactate by reduced diphosphopyridine nucleotide in the presence of an added excess of purified lactic dehydrogenase (6).

+ L(+)-alanine SGP-T + +)-glutamate + pyruvate lactic DPN + lactate pyruvate + DPNH T dehydrogenase

a-ketoglutarate

L(

2

(6) (6)

Oxidation of reduced diphosphopyridine nucleotide, and thereby the transamination reaction, is followed by measuring the decrease in light absorption a t wavelengths 340 mp. Serum activity is expressed in units per milliliter per minute. One unit equals a decrease in optical density of 0.001 under standardized conditions. Serum glutamic-pyruvic transaminase, measured at 23°C in serums of normal humans, had a mean activity of 16 f 9 units per milliliter per minute. When the mean value of SGP-T activity is converted to pM per milliliter per hour, the value falls within the range of 0.21 to 1.01 pM per milliliter per hour found by quantitative paper chromatography (K3). Several spectrophotometric techniques and modifications for the measurement of SGO-T and SGP-T have been described (Hl, H4, N3, S9, S11).

322

FELIX

WR6BLEWSKI

Unlike quantitative paper chromatographic methods, all the spectrophotometric techniques measure serum transaminase by estimating the rate of the enzymatic reaction rather than any one of the end-products of transamination. The spectrophotometric measurement of SGO-T and SGP-T is simple, rapid, and accurate. However, these methods require enzyme reagents which are somewhat expensive, vary in stability, and may not be generally available. 3.3. COLORIMETRIC TECHNIQUES

The colorimetric method estimates serum transaminase activity by measuring the amount of pyruvate formed under standard conditions (Cl, Fl, W9). A technique used for the assay of GO-T in animal tissues has been modified and adapted to the measurement of SGO-T and SGP-T (TI). Measurement of the SGO-T activity involves the conversion of the formed oxaloacetate to pyruvate by aniline citrate. The pyruvate reacts with dinitrophenylhydrazine to form pyruvatedinitrophenylhydrazone, which is extracted by toluene. When the toluene solution of pyruvatedinitrophenylhydrazone is treated with strong alkali, a colored compound results, and this may be measured colorimetrically. The intensity of the color is proportional to the amount of pyruvate, and the quantity of pyruvate reflects the degree of SGO-T activity. Essentially, the same principles are involved in the estimation of SGP-T, except that pyruvate is formed directly by transamination, and therefore conversion of oxaloacetate by aniline citrate is omitted. One unit of SGO-T or of SGP-T is defined aa the activity of 1.0 ml of serum that results in the formation of chromogenic material equivalent to 1 pg of pyruvate under standardized conditions. The amount of pyruvate that is formed during the course of the colorimetric assay is used as an indication of serum transaminase activity. Spectrophotometrically, the measurement of serum transaminase activity depends on the rate of the transamination reaction rather than the amount of a product formed during the enzymatic reaction. Actually, the two methods measure different aspects of transamination and are therefore not entirely comparable. One unit of transaminase activity colorimetrically is equivalent to approximately one unit spectrophotometrically in the normal range; in the abnormal range of serum transaminase activity, one colorimetric unit approximates 1.5-2.0 spectrophotometric units. The mean SGO-T activity of serums of normal individuals determined by the colorimetric techniques is 16 f 8.0 units, with a normal range of 4 to 40 units, while that of the SGP-T is 22 f 12 units, with a normal range of 1 to 45 units. The principal advantages of the colorimetric method for serum transaminase assay are that it requires reagents which are relatively stable and readily available and that the final measurement in the assay is made at 490 mp and therefore does not require ultraviolet spectrophotometry.

SERUM TRANSAMINASE ACTIVITY

323

However, the colorimetric assay is lengthier than the spectrophotometric methods and, in addition, is somewhat less accurate, although under most circumstances it is suitable for routine clinical use. The stability of these serum enzymes is such as to facilitate their clinical usefulness. Specifically, freezing or lyophilization of serum fails to influence fierum transaminase activity; serum and plasma have equivalent activities; storing at room temperature for 24 hours or at 4°C for 5 days does not significantly alter serum transaminase activity. Food ingestion may influence serum activity, but the changes described are within the normal range as defined (W2). Accordingly, SGO-T and SGP-T determinations can be done without regard to the fasting state (C6, K3). The mechanism for excretory and/or secretory handling of serum transaminase is unknown, but the presence of the enzyme in small amounts in urine and in large amounts in bile suggests that renal and biliary routes may contribute in this regard (C6, W5). It is pertinent, however, that oliguria and/or azotemia are not necessarily associated with elevated serum transaminase activity (Ll, WlO). 4. Tissue Distribution of Transarninase Activity

4.1. ANIMALTISSUES In 1952, it was confirmed that transaminase activity is not limited to pigeon breast, pig heart, liver, and kidney, but is present as well in varying activities in eight organs of the rat. Glutamic-oxaloacetic transaminase was maximally present in heart homogenates; to a lesser extent, the enzyme was demonstrated in skeletal muscle, lung, brain, liver, spleen, prostate, and testis, in decreasing order (A7). Previous reviews have compiled the available data on the distribution of GO-T and GP-T in animal tissues (B4, C13, M5). The tissue activities vary among different tissues, with distinct species differences. In all instances in any one tissue, the activity of GO-T is greater than that of GP-T. In the case of GO-T, the greatest activity is observed in extracts of skeletal, diaphragm, and heart muscle, and liver. Glutamic-oxaloacetic transaminase is distributed in homogenates of dog heaa muscle, skeletal muscle, liver, kidney, brain, testis, and lung in decreasing order, with cardiac muscle homogenates containing approximately 300,000 units and lung homogenates containing approximately 6500 units per gram of wet tissue. Dog serum has a normal range of activity from 5 to 50 units per milliliter (N4). The normal range of serum activity for the mouse is 50-125 units per milliliter and for the rat 100-375 units per milliliter (F5, L2, M12, N4). 4.2. HUMANTISSUES

The distribution of transaminase in normal adult tissue homogenates is presented in Table 2 (W13). The impressive amounts of transaminase

324

FELIX WR6BLEWSKI

activity of tissue homogenates as contrasted with the relatively minute activity of serum suggested that tissue injury might be associated with increments in serum transaminase activity. Recent studies on the distribution of the transaminases have been carried out using a large variety of tissues, and these reports have been tabulated in a review (M5). In addition to seeds, microorganisms, teeth, and other preparations, GO-T and GP-T activities have been studied in tumors and found to be about the mme as or lower than in normal tissues which have been used for comparison (B4, C16, C17, E2, K8). TABLE2 TPE DISTRI~UTION OF GLUTAMIC-OXALOACETIC AND GLUTAMIC-PYRUVIC TRANSAMINASE ACTIVITY IN NORMAL HUMANADULTTISSUES Tissue Heart Liver Skeletal muscle Kidney Pancreas Spleen Lung Serum

GP-T GO-T Units X lO-’/gram wet tissue homogenate 156 142 99 91 28 14 10

7.1 44

.02

4.8 19 2 1.2 .7 .016

5. Alterations of Serum Transaminase in Cardiac Disease

The fact that a maximum activity of GO-T was associated with cardiac musculature suggested that necrosis of heart tissue might be reflected in changes in the enzymatic activity of the serum. It was observed originally in 1954 that during the first several days following human transmural myocardial infarction, SGO-T is increased above the normal range (K3). Thereafter, this observation was extended and confirmed (C5, C6, G2, K5, K6, K7, K15, K16, L1, L3, L6, M8, S10, W6). Alterations in SGO-T activity associated with myocardial infarction have been studied experimentally by three techniques (Gl, N4, P1, R2) discussed in Sections 5.1.1. and 5.1.2.

PRODUCED PATHOLOGICAL CARDIAC STATES 5.1. EXPERIMENTALLY 5.1.1. Transmural Myocardial Infarction

Simulating the pathological process of human transmural myocardial infarction, coronary occlusion by coronary artery ligation in the closedchest dog was produced, and it was found that alterations in SGO-T cor-

SERUM TRANSAMINASE ACTIVITY

325

related with electrocardiographic and other observations (N4). Following myocardial infarction experimentally produced in dogs, SGO-T was consistently elevated. The amount of rise of the enzyme in the serums as well as the duration of the rise is roughly proportional to the extent of infarcted heart muscle. These observations were strikingly similar to those previously reported following myocardial infarction in man. The sensitivity of SGO-T as a reflection of cardiac muscle necrosis is demonstrated by the fact that infarcts less than one gram in size resulted in significant but short-lived elevations of SGO-T. Myocardial ischemia of 45 minutes duration failed to result in increased SGO-T. When the dogs died from experimentally produced infarction, and/or were sacrificed a t varying periods following coronary ligation, normal and infarcted cardiac tissues were assayed for GO-T (N4). The fact that the activity of GO-T in infarcted muscle is appreciably less than that in the adjacent normal muscle of the same heart, and the observation that the GO-T activity in infarcted muscle diminishes proportionately with the age of the infarct, strongly suggest that the mechanism of elevation of SGO-T is one of release of intracellular enzyme into the blood stream following death or loss of cellular membrane integrity (M7, N4, R2). Using an experimental technique by which plastic spheres were embolized into the coronary arteries of the closed-chest dog, myocardial infarction of predetermined extent was produced (A2, A3, Gl). It was observed that almost a linear relationship existed between the size of the estimated infarct at autopsy and the peak rise in SGO-T activity. This peak rise usually occurred 9 to 24 hours after infarction. As little as 10% infarction of the cardiac muscle mass produced significant elevations of SGO-T. When myocardial necrosis was produced by intravenous injection of papain into rabbits, elevations of SGO-T activity were also observed (54). Alterations in SGP-T are seen following experimentally produced myocardial infarction in dogs. The mechanism postulated for SGP-T increase is the same as that hypothesized for SGO-T, and rests similarly on the observations that infarcted muscle contains less GP-T than adjacent normal cardiac tissue of the same animal, and that the older the infarct the less GP-T activity is demonstrable in the necrotic tissue. However, the increase in SGP-T following experimental myocardial infarction is consistently smaller than the concomitantly measured increase in SGO-T presumably because canine cardiac musculature contains appreciably less GP-T than GO-T per gram of tissue (R4). 5.1.2. Subendocardial or Focal Myocardial Infarction

Myocardial ischemia of varying durations has been produced in dogs by the use of temporary occlusion of coronary arteries, and the degree of

326

FELIX WR6BLEWSRI

ischemia was correlated with electrocardiographic changes, SGO-T and SGP-T alterations, and heart tissue GO-T and GP-T activity (N5, R3, W5). With a few exceptions, myocardial ischemia, in the presence of confirmatory electrocardiographic changes without histologic evidence of necrosis, resulted in no significant elevation in the two serum enzyme activities. However, when ischemia resulting from experimentally produced coronary insufficiency was prolonged to such an extent that morphologic evidence of tissue necrosis appeared, SGO-T and, inconsistently, SGP-T were increased above the normal range. In most c a m , experimental canine pericarditis was accompanied by no significant rise in SGO-T and SGP-T activity (Al, N5). A similar lack of change in the serum enzyme activities was observed during the course of experimentally produced pulmonary infarction (Al). 5.1.3. Other Types of Cardiac Tissue Injury Viral myocarditis produced experimentally in rabbits was associated with cardiac tissue necrosis and resulted in elevations in SGO-T activity in proportion to the extent of necrosis. When viral pericarditis was produced experimentally, no elevations in SGO-T were observed (Pl). This is in agreement with the observation that in most cases, pericarditis produced experimentally in dogs was accompanied by no Significant rise in SGO-T (N5). 5.2. CLINICAL CARDIAC

DISEASE STATES

5.2.1. Myocardial Infarction The impressive amount of GO-T in human cardiac tissue, as opposed to the relatively small amount of enzyme in an equivalent amount of serum, results in significant elevation of SGO-T activity when the enzyme is released from necrotic cardiac tissue following acute myocardial infarction. Other enzymes present in great enough amounts in heart tissue may be expected to behave similarly (W6). Transmural myocardial infarction in the human adult is associated with a rise in SGO-T which is manifested approximately six to twelve hours after the estimated time of coronary occlusion (C6, C18, El, K5, K6, K7, K16, L1, L3, M8, 55, S10, W4, W6). The rise in serum enzyme activity reaches a peak within 24 to 48 hours, returning to the normal range by the fourth to the seventh day after infarction (Fig. 2). The peak elevations noted following myocardial infarction are two- to Bteen-fold greater than the normal levels for SGO-T. The peak rise and duration of abnormal serum enzyme activities appear to be proportional to the size of the infarction and/or the degree of myocardial necrosis. If serum sampling is inadequate during the first to the

327

SERUM TRANSAMINASE ACTIVITY

third day following coronary occlusion, the maximal elevation may be missed and thereby incorrectly estimated. The relationships between the peak SGO-T rise, the duration of abnormal activity, and the size of the infarct are in keeping with the experimental evidence in dogs as well as with the clinical observation that a poor prognosis is implied when SGO-T activity exceeds 300-350 units at the peak rise after infarction (K6, W6). A small myocardial infarction, however, with slight tissue necrosis and a small rise in SGO-T does not necessarily imply a favorable prognosis.

T L Y L R A T U C C C U ~ ~ ~ IDI ioo 1004 sao sao NJ saz--wouu CUUL U A N 8 4 8 0 7 S I s 7 ~ W . 6 8 ~ 8 874 2 W8C h1oOo) 1.a ai as 7.9 114 i d r3 w 5s ez 34 CYN N f667 00 SO SS6S6SST 69 M0186S62 69 70 csn Innlhvt 7 se ao so 50 t Mlco.Q)LANIo &HEST+ PAIN H P U L Y O N A R V LDCYA

3

m

1

2

1

4

1

1

1

1

1

1

1

1

20

1

U

T SD

2

7 2 S*bS

m

6

6

61

u

W

I

58 I8 I

1

24

1

1

08

1

0

1

0119

FIQ.2. Serial alterations in SGO-T and other laboratory parameters in a patient who incurred an acute anterior myocardial infarction. For comparison, the concomitant changes in serum lactic dehydrogenase activity are shown. Following myocardial infarction, the rise in SGO-T activity is not influenced by, nor correlated with shock, blood pressure, heart failure (L7), location of the infarction, anticoagulants, digitalis or its derivatives, quinidine, age, sex, color, body temperature, sedimentation rate, leucocyte count, or urinary output (K16, L1, M4). The rise in SGO-T following infarction is not necessarily related to the configuration of the electrocardiogram. When the electrocardiogram is not diagnostic of myocardial infarction or is obscured by previous myocardial infarction, bundle branch block, WoH-Parkinson-White syndrome and/or other electrocardiographic aberrations, the rise in SGO-T activity in a clinical setting suggestive of infarction is an especially helpful diagnostic tool (C5, G2, K15, K16, W6). Secondary rises in SGO-T activity have been observed in patients who

1

328

FELIX WR6BLEWSKI

present, following acute myocardial infarction, clinical stigmata consistent with extension of the infarction (C5, Ll). Changes in SGO-T activity may contribute to delineating the process of myocardial necrosis in patients with substernal pain, not only with suspected coronary occlusion, but also in the presence of coronary insufficiency without occlusion. It appears that SGO-T activity remains within normal limits in patients with status anginosus or coronary insufficiency, in spite of accompanying transient ST segment and T-wave abnormalities. When, in these clinical settings of acute coronary insufficiency, SGO-T activities are elevated, it is suggestive that ischemia of cardiac muscle has been accompanied by and/or followed by myocardial necrosis (N5, R5). 5.2.2. Carditis, Pericarditis, Acute Cor Pulrnonale and Others In most instances of pericarditis of various causes, pulmonary emboli with and without pulmonary infarction (G2, K16), cardiac arrhythmias (W5), and rheumatic carditis (M3, N6), no significant or consistent elevations in SGO-T activity have been observed.

r TRANSAMINASE UNITShl , _ _

so 0 ‘ONSET OF PAIN

OAYS

FIG.3. Serial alterationsin SGO-Tand SGP-T in a patient who experienced an acute posterior-wall myocardial infarct.

In most instances of acute transmural myocardial infarction and prolonged coronary insufficiency associated with focal myocardial necrosis, SGP-T activity is not increased (W14). The lack of significant alteration in SGP-T under most of these circumstances is presumably due to the relatively low GP-T activity as compared with high GO-T activity of heart tissue homogenates (Fig. 3). However, when an infarct is sizable, the amount of GP-T liberated is great enough to alter the SGP-T above the normal range. Therefore, increased SGP-T is observed in association with myocardial infarction only when the resulting cardiac tissue necrosis is great enough to cause a rise in SGO-T that is roughly equivalent to 150

SERUM TRANSAMINASE ACTIVITY

329

spectrophotometric units; in all such instances the SGO-T elevation is appreciably greater than the concomitantly elevated SGP-T, unless hepatic tissue injury due to prolonged anoxia or drug toxicity contributes to the SGP-T increment. 6. Alterations of Serum Transaminase in Hepatic Disease

6.1. EXPERIMENTALLY PRODUCED PATHOLOGICAL HEPATICSTATES Viral hepatitis produced experimentally in mice has been associated with an increase in SGO-T (F5) and SGP-T (F4) activity. A relationship appears to exist among the rises in SGO-T activity and the size and virulence of the viral inoculum, the blood virus titer, and the degree of liver necrosis (F5). The serial alterations of SGO-T in viral hepatitis in mice

Before lnsti llation of CClq

After 2 Inst I llations of CCI, After 4 lnsti llationr of CCI,

SOOC

After 6 lnrti llations of CCI,

FIG.4. Alterations in SGO-Tactivity in rats who received varying amounts of carbon tetrachloride via intragastric intubation.

are paralleled by changes in SGP-T activity, which are increased proportionately to a greater extent above the normal range for mice than is SGO-T activity (D2, F4). The injury of hepatic tissue accompanying the partial hepatectomy of mice is associated with elevations in the serum transaminases (F5).The hepatocellular injury resulting from acute toxic hepatitis experimentally produced in rats with carbon tetrachloride was shown to be reflected in the SGO-T alterations :the amount and duration of increased SGO-T activity was noted to be proportional to the amount of toxin administered and to the extent of liver cell damage (M12, Fig. 4). Hepatic GO-T was not significantly decreased even when SGO-T was concomitantly and markedly elevated (M9). Minimal and possibly insignificant decreases in hepatic GO-T, as well as GP-T, were observed during

330

FELIX WR6BLEWSKI

the course of viral hepatitis in mice, a t the time when SGO-T and SGP-T activities were impressively elevated (F4). On the other hand, more recent experiments utilizing homogenates subjected to ultracentrifugation suggest that rat liver GO-T and GP-T located primarily in the mitochondria1 and supernatant fractions decrease within 24 hours of carbon tetrachloride administration, reaching their lowest tissue activities at 72 hours and returning to the normal values encountered prior to exposure to the toxin within a week. These tissue enzyme changes paralleled inversely the serum enzyme changes observed concomitantly. Serum glutamic-oxaloacetic transaminase increased sharply within 24 hours and reached a maximal level 72 hours after administration of carbon tetrachloride to rats. By the end of one week, both SGO-T and SGP-T had returned to normal. In chronic hepatic injury studied in rats with repeated carbon tetrachloride administration, SGO-T and SGP-T were elevated appreciably less than in experimentally produced acute injury despite the remarkably great decrease in liver transaminase. These observations would suggest that, in experimentally induced acute hepatic injury, serum transaminase alterations possibly might be related to release of intracellular enzymes from injured and necrotic liver cells. In chronic hepatic injury, however, it would appear that the sizable decrease in liver tissue transaminase is not simply due to efflux of intracellular enzyme. It has been suggested that a disturbance in synthesis of enzyme protein may play a role (A6). Cirrhosis and hepatic tumors, produced experimentally in rats by the chronic administration of butter yellow, have been shown to be accompanied by elevated SGO-T activity (C17, M11). Common duct occlusions produced experimentally in dogs have resulted in elevations in SGO-T activity which returned to normal within a week following the relief of biliary tract obstruction (F3). During acute and chronic liver injury produced experimentally in rats, it has been noted that the pyridoxal content of liver was diminished and paralleled the hepatic tissue transaminase changes previously noted (A6). However, administration of pyridoxal phosphate and pyridoxine to rats with liver disease, or the addition of the coenzyme to hepatic homogenates prepared from these animals, failed to increase the tissue transaminase activity. This suggests that decrease in GO-T and GP-T activity of liver in experimental rat liver damage is chiefly due to diminution of the apoenzymes (A6). 6.2. CLINICAL HEPATICDISEASESTATES

Acute hepatic disease in humans has been noted to be associated with rises in SGO-T and SGP-T activity (C7, D3, W10, W13, W14). In most instances, the quantitative and serial changes in these two serum enzymes

SERUM TRANSAMINASE ACTIVITY

331

are sufficiently characteristic of the various types of liver disease to assist in diagnostic differentiation (W14). 6.2.1. Toxic Hepatic Disease The largest elevations in SGO-T and SGP-T have been observed in acute toxic hepatitis due to carbon tetrachloride and in patients with acute infectious and/or homologous serum hepatitis (C6, D3, W10, W14). Exposure to carbon tetrachloride results in elevations of both serum enzymes within 24 hours, reaching peak levels as high as 27,000 units. With cessation of exposure to the toxin, SGO-T and SGP-T fall precipitously toward normal. The alterations in SGP-T parallel those seen in SGO-T activity but are usually greater in the case of SGO-T than in the case of SGP-T. Toxic hepatitis due to chloropromazine (S6, W12), salicylates (M3, W5), azaserine (W5), pyrazinamide (W5), and other agents is usually associated with smaller elevations of serum transaminases than have been observed in carbon tetrachloride toxic hepatitis. Continued increments in the serum enzymes are observed with continued administration of these drugs when they prove t o be hepatotoxic; discontinuance of the hepatotoxic agent results in a rapid decrease of the serum transaminases toward normal. 6.2.2. Infectious and Inflammatory Hepatic Disease Acute liver cell injury, as seen in acute infectious and homologous serum hepatitis, results in impressive increments in the two serum transaminases (C7, D3, W3, W10, W12, W13, W14, W15). Although the changes in the activity of these enzymes parallel each other, the rise of SGP-T usually exceeds that of SGO-T activity (Fig. 5). It appears that the rise in serum transaminases in viral and homologous serum hepatitis begins during the prodromal phase of the disease, reaching a peak elevation which is 10-100 times greater than the normal serum activity a t the time the patients are the sickest, as adjudged by fever, malaise, anorexia, nausea, vomiting, and hepatic tenderness. With subjective and objective evidence of improvement, a fall in both serum transaminases toward normal occurs. The natural, uncomplicated course of infectious hepatitis is associated with a gradual increase in the activities of both serum transaminases to a peak, followed by a gradual decrease in serum enzyme activity toward the normal range during the recovery phase (W3). When complications occur during the course of hepatitis, the added stress appears to influence the disease, and this is reflected in a secondary superimposed rise in SGO-T and SGP-T activity. Ambulation during recovery from infectious hepatitis is sometimes associated with a small rise in serum transaminase. If the rise following ambulation is 50 or more units, return to rest discipline is advised, in which case the SGO-T and SGP-T activities usually resume their decline

332

FELIX WR6BLEWSKI

toward normal (W3). Relapses of infectious or homologous serum hepatitis are associated with secondary rises in SGO-T and SGP-T activity (Fig. 6). Unresolving hepatitis is associated with persistently elevated serum transaminases at the time the serial alterations would be expected to return toward normal (W14). The failure of SGO-T and SGP-T activity to return to the normal range suggests the development of chronic hepatitis and/or postinfectious cirrhosis. The serial alterations of SGO-T and SGP-T in

FIG.5. Alterations in SGO-T,SGP-T,and other laboratory parameters in a patient with acute infectious hepatitis.

the course of acute infectious hepatitis follow a characteristic pattern; deviations from this usual course of enzyme alterations may suggest associated complications, relapses and/or chronicity of the hepatic infection. It appears that the serial and quantitive changes in SGO-T and SGP-T during the course of hepatitis reflect the clinical state of the patient more accurately than conventional liver function tests. In this regard SGO-T and SGP-T are thought not to reflect liver cell function per se, but rather

333

SERUM TRANSAMINASE ACTIVITY

to represent the reaction to acute liver cell injury. Accordingly, the serum transaminase alterations are not necessarily correlated with conventionally employed tests of liver function; changes in serum transaminase appear not to be an index of liver cell function. The sensitivity of serum transaminase as a reflection of liver cell injury may account for the observation that in acute hepatitis, SGO-T and SGP-T are elevated in the prodromal and clinical phase of the disease at a time when tests of liver cell function may be unaltered. Observations during the course of an epidemic of acute infectious hepatitis in a closed environment indicated that elevations in serum transaminase more sensitively reflect subclinical hepatitis than do conventional pdmittd

rR-ltkd

Discharged

i

Dischorgld-

I

o SGO

0

SCP

Tmnonlmrr Udk/ml

TdolBilirubh m 9 a X 23.5 CIphalin Cbccubtim 4+ T h m l Turbidity unih 1.1 AIL. Phoaphaku 6.units 9.9 DSP Rekntbn Y. TOWRok(n/Albumin 9m.X I S / Told CI~deshrol/F~w mpm.% 1

DAYS 0

30.4

5.2

11.1

4.2

3.3

2.4

3+

4+ 2.3 l4.5

3+

3+

3+

1.6 8.1

1.7 6.5

1.7 5.3

7.9

10.4

1.9 1.8 Nip. I+ 1.8 1.8 5.2 4 4

30 6.W4.8 358/267 ~

20

~

~

40

~

60

1

80

6 W4.8 336/105 238/104 1

1

100

1

6 8/47

120

1

1

140

FIG.6. Alterations in SGO-T, SGP-T, and other laboratory parameters in a patient with acute homologous serum hepatitis. After discharge from the hospital, the patient was readmitted with a recurrence of the homologous serum hepatitis.

liver function tests (W15). Examination of individuals during the course of an institutional epidemic of acute infectious hepatitis, utilizing liver function tests as well as transaminase determinations as criteria, yielded 5 classes of patients: (a) asymptomatic individuals with normal SGO-T, serum bilirubin, and thymol turbidity; (a) asymptomatic individuals with transiently abnormal SGO-T (up to 100 units), normal serum bilirubin, and normal thymol turbidity; ( c ) asymptomatic individuals with abnormal SGO-T (up to 350 units), normal serum bilirubin, and abnormal thymol

1

1

334

FELIX WR6BLEWSKI

turbidity; ( d ) symptomatic individuals with abnormal SGO-T (up to 350 units), hyperbilirubinemia, and abnormal thymol turbidity; (e) symptomatic individuals with clinical icteric hepatitis. These individuals were categorized respectively as (a) normals, (b) contacts, (c) nonicteric hepatitis individuals, and (d) subicteric hepatitis individuals. The final group of

FIQ.7. Serial alterations in SGO-T, SGP-T, and other laboratory parameters during the course of nonicteric hepatitis associated with infectious mononucleosis.

individuals consisted of those persons who developed icteric clinical infectious hepatitis for which they required hospitalization. These and other epidemiologic observations afforded a view of the course and epidemiologic behavior of a closed-environment epidemic of infectious hepatitis, and

SERUM TRANSAMINASE ACTIVITY

335

suggested that asymptomatic individuals with nonicteric and subicteric hepatitis may possibly communicate the disease without themselves being recognized as having clinical hepatitis. The serum transaminase changes observed in the prodromal phase of hepatitis and in individuals with nonicteric and/or subicteric types of acute hepatitis permit a better understanding of the epidemiologic course of the disease and facilitate the diagnosis and thereby the management of individuals with subclinical or otherwise unrecognized hepatitis. Whether alterations in SGO-T in the serum of blood donors may contribute to the detection of those individuals who serve to transmit homologous serum hepatitis must await further study. Infectious mononucleosis usually is accompanied by normal SGO-T and SGP-T activity. However, when this malady is complicated by hepatitis, there is a rise in SGO-T and SGP-T at a time when the liver functions may be normal or inconclusively affected as measured by conventional tests (Rl, W14, Fig. 7). The severity of the hepatitis accompanying infectious mononucleosis appears to be related quantitatively to the peak rise in SGO-T and SGP-T, with the latter being greater than the former throughout the course of the elevated serum transaminase activity (Rl). 6.2.3. Degenerative Hepatic Disease

Active or progressive Laennec cirrhosis is associated with elevations in SGO-T and smaller elevations or no increment in SGP-T (W14). The values of SGO-T are in the range of 50 to 250 units (M10, WlO). Biliary cirrhosis is generally accompanied by somewhat greater elevations than portal cirrhosis (M10). Alterations in SGO-T have proved of little value in distinguishing between primary and secondary biliary cirrhosis (K9). Cirrhosis complicated by acute hepatitis has been shown to exhibit SGO-T and SGP-T elevations quantitatively and serially characteristic of acute hepatitis but superimposed on the serum enzyme elevations identified with hepatic cirrhosis (W10). It appears that the serum enzyme alterations may be used to determine whether one is dealing with sudden hepatic decompensation secondary to cirrhosis or with superimposed acute hepatitis in the cirrhotic individual. In the case of sudden hepatic decompensation without hepatitis, no superimposed rise in serum transaminase has been observed. Cirrhosis is the cicatrical phase which follows liver cell injury, presumably from various etiologic insults. Histologic examination of hepatic tissue from a cirrhotic liver may not permit the determination of whether the liver disease is static and cirrhotic or whether continued liver cell injury is present and accompanying the cicatrical sequelae of past cellular injury and/or necrosis. Serum transaminase alterations would appear to permit this differentiation and thereby suggest classification of cirrhosis into four

336

FELIX WR6BLEWSKI

types, depending, in part, on the serum enzyme alterations encountered: (1) inactive and compensated cirrhosis, the state in which liver function tests may be abnormal but serum transaminases are within normal range: (2) inactive but decompensated cirrhosis, the situation in which liver function is markedly disturbed, as reflected by function tests and such clinical parameters as fluid retention and jaundice, but in which serum transaminases are of normal activity; (3)active but compensated cirrhosis, the disease state in which hepatic scarring is present, but in which progressive and continuing liver cell injury exists without hepatic decompensation (liver function tests are abnormal and SGO-T is increased above the normal range) ; (4) active and decompensated cirrhosis with cicatrical changes from previous hepatic tissue injury and, in addition, continuing liver cell injury as well as decompensated hepatic function (liver function tests and SGO-T activity are abnormal in the presence of fluid retension and hyperbilirubinemia) . 6.2.4. Extrahepatic Biliary Tract Disease Extrahepatic biliary obstructive jaundice is characterized by increments in transaminase activity from 40 to 300 SGO-T units and 50 to 400 SGP-T units. Although both enzymes are altered in the same direction, the SGP-T activity usually exceeds the corresponding SGO-T activity in acute extrahepatic biliary obstruction (W14). The serum enzyme activities generally return to normal within a week after relief of the biliary obstruction. 6.2.5. Neoplastic Hepatic Disease Serum glutamic-oxaloacetic transaminase activity has been reported to be as sensitive an index of primary and metastatic cancerous involvement of the liver as serum alkaline phosphatase, but the former is unaffected by the presence of active metastatic bone cancer (W11). It is pertinent that normal SGO-T activity has been reported in most instances of nonmalignant and malignant bone disease in the absence of hepatic involvement, whereas many of these cases were associated with elevations in serum alkaline phosphatase. Therefore, elevations in alkaline phosphatase due to bone disease may be differentiated from those increments due to liver disease, as SGO-T is not elevated in bone disease. This generalization may be of special value when one is in doubt as to whether an elevated serum alkaline phosphatase is secondary to bone or to liver disease (W11). The degree of increased SGO-T activity seen in metastatic cancer to the liver is roughly proportional to the amount of liver cell injury resulting from tumor growth. When SGP-T is increased concomitantly with SGO-T in metastatic liver disease, it is of lower activity than SGO-T (W14).

337

SERUM TRANSAMINASE ACTIVITY

6.2.6. Differential Diagnosis of Jaundice by Means of Serum Enzymes 6.2.6.1. Adult types of jaundice. Although a battery of liver function blood tests may at times be necessary to help define the etiologic explanation of hyperbilirubinemia in the jaundiced patient, i t has recently been suggested that in many instances, the laboratory information will suffice if limited to the determination of the serum total bilirubin, serum alkaline phosphatase, and serum transaminases (W7). Table 3 summarizes the TABLE3 COMPARISON or RANQESOF SERUMENZYME ACTIVITIES I N PATIENTS WITH VARIOUSTYPESOF JAUNDICE Jaundice due to

Serum alkaline phosphatase (units/ml serum.)

Extrahepatic biliary tract obstruction Intrahepatic primary and metastatic carcinoma or lymphoma Acute homologous serum and infectious hepatitis (increasing icteric phase) Cirrhosis Hepatotoxic drugs (excluding carbon tetrachloride) Hemolysis 0

SGOSGPtransaminase transaminase (units/ml (units/ml serumb) serum.)

8.0-45.0

44-288

<

64-400

8.M1.6

43-300

>

26-240

4.0-11.2

460-2140

<

600-2600

4.G15.2 3.5-11.4

45-300 68-370

> <

20-258 176-440

3.0-4.3

32-140

>

20-40

Normal values: 1.5-4 units/ml.

* Normal values: 8-40 units/ml. Normal values: 5-35 units/ml.

alterations in these serum enzymes as seen in patients with icterus. Extrahepatic biliary obstructive jaundice is readily differentiated from that due to homologous serum and infectious hepatitis by the fact that the serum alkaline phosphatase is usually higher in the former than in the latter. As a rule, in the initial or increasing icteric phase of acute hepatitis, both serum transaminases are well over 400 units, while in obstructive jaundice the serum transaminases are usually below 400. In both instances, SGP-T activity is greater than the simultaneously measured SGO-T activity. In addition, the serial alterations in the serum enzymes in obstructive jaundice and in jaundice associated with acute hepatitis are readily distinguishable. Figure 8 depicts the alterations in serum total bilirubin and serum enzymes in a patient with acute extrahepatic obstructive

338

FELIX WR6BLEWSKI

jaundice, and Fig. 9 depicts these alterations in a patient with acute hepatitis. The serum enzyme alterations are quantitatively and serially distinct in the two types of jaundice. Although toxic hepatitis due to drugs may mimic the serum enzyme alterations seen in obstructive jaundice, especially in the case of chlorpromazine, when the alkaline phosphatase may become appreciably elevated, hepatitis due to hepatotoxic agents usually may be distinguished from obstructive jaundice and acute hepatitis.

A SGO-T Normal Range 40 0 SOP-TNormaf Range

Transaminore Units 120

Serum Bilirubin lo

35

’

B Units Ceph Flocc

NO9

NO9

Bile in Urine

+++

Urobilinogen 8.4 5.2 Units 5.4 Days

2

I

I

4

hprration

I

I

6

I

I

e

I

1

10

1

I

12

I,

I

14 Discharge+

I

I

16

FIG.8. Serial alterationsin SGO-T,SGP-T, serum bilirubin, serum alkaline phoaphatase, and other laboratory parameters in a patient with jaundice due to obstruction, by calculi, of the common duct.

When the toxic insult to the liver is stopped by the discontinuance of the administration of hepatotoxic agent, the serum transaminases begin to fall toward normal even though the serum bilirubin and/or serum alkaline phosphatase may remain unchanged or even increase transiently. Intrahepatic carcinoma and lymphoma associated with jaundice present serum enzyme changes similar to those observed in cases of cirrhosis. However, in most instances of active Laennec cirrhosis with hyperbilirubinemia, the serum alkaline phosphatase is normal or only slightly elevated, while in most cases of intrahepatic malignant neoplasm with jaundice, the alkaline phosphatase is appreciably elevated above normal. Postnecrotic cir-

339

SERUM TRANSAMINASE ACTIVITY

rhosis, unlike Laennec cirrhosis, may be associated with an elevated serum alkaline phosphatase activity and consequently may present serum enzyme alterations which mimic those observed in intrahepatic neoplasia with jaundice. Hemolytic jaundice in the adult is usually readily distinguishable, on clinical grounds alone, from other causes of jaundice; in most instances the serum enzymes remain normal except for transient and slight elevations in SGO-T, with no alteration of SGP-T above the normal range.

2000

,

1

14

TRANSAMINASL

Normal

1

THYMOL TURBIDITY unit0 3 . 8 4 . ~ 4.6

5.6

47

54

CEFWALIN FLOCCULATI 4+ 4+ BSP RETENTION Y. 1 7 WMlNE P'TASE B.Unit0 1 6 3 2 9 1 5 cna. TotaVFrrnDmX & ? I 88

4+

4+

4+

6.8

86

6?

lo8

114

op

ld.(Rokln/Ubmhgm

'MYS

.696.77.1 3.73.5 3.7

,

,

10

1.I 3.5 ,

L5 3.6

,

20

gg 134

,

I.e 3.T

,

30

eo

3.2

2.4 2.1 1.7

1.8

3+ 3+ 2+ 25 24 5.3 4.6 1 6 ~ r p tom 1 0 1 ITS 63 6 7 6%?51

3+ 05

4*

85

u

59

,

IT^ 80

T.I XL? 9 , 4.0 , 4.1, 46, 4.2 , 3.S, 2

40

m

60.

1.0 2+

1.6

%?6 3.s

I?

60

,

43

To

,

,

00

FIQ.9. Serial alterations in SGO-T, SGP-T, wrum bilirubin, serum alkaline phosphatam, and other laboratory parameters in a patient with acute homologous serum hepatitis.

Most types of surgically amendable jaundice, e.g., extrahepatic biliary tract obstructive jaundice, can be distinguished in the laboratory from medical types of jaundice by the characteristic alterations in serum enzymes. In obstructive jaundice, serum alkaline phosphatase is elevated usually to a level greater than 10 units. Serum glutamic-pyruvic transaminase is increased to a greater extent than the simultaneously measured SGO-T, the former usually to levels less than 400 and the latter to less than 300 units. In all the types of medical jaundice other than acute

340

FELIX WR6BLEWSKI

hepatitis, SGO-T values are greater than the simultaneously determined SGP-T activity. In the case of acute hepatitis, in the increasing icteric phase when SGP-T is greater than SGO-T, the values of the serum transaminases are greater than 600 and 500 units respectively. When acute hepatitis is superimposed on a liver containing metastatic deposits, the SGO-T activity may be greater than the SGP-T activity. This turn of events has been found to be associated with a n abnormal serum protein electrophoretic pattern, including elevated P- and 7-globulins. However, in spite of the greater activity of SGO-T than of SGP-T, the values of the serum transaminases are usually greater than 600 in the initial or increasing icteric phase of the hepatitis. When obstructive jaundice occurs in a patient with metastatic liver disease, SGP-T activity may be greater than, equal to, or somewhat less than the SGO-T activity. However, the serum transaminase values rarely go above 300 units even when serum bilirubin is 30 mg yo (30 mg/100 ml) or more. 6.2.6.2. Neonatal jaundice. Whereas in adults the history and physical examination often supply adequate clues from which it is possible to establish a correct diagnosis for the presence of jaundice, such is not the case with respect to jaundice in the newborn infant. Furthermore, while available laboratory techniques for estimation of liver function usually furnish the necessary data for confirmation of the causative factor of icterus in adults, in newborn infants these same procedures more often fail to supply adequate information from which to derive a diagnostic conclusion. It would appear that serial determinations of serum transaminase activity may be of distinct value in the differential diagnosis of jaundice of unknown origin in the newborn infant (K13, K14). Levels of activity up to 120 units for SGO-T and 90 units for SGP-T must be considered physiological in the early neonatal period. I n the case of neonatal physiological jaundice, and usually in hemolytic states, the levels of activity remain within the normal neonatal range. In the case of very severe hemolysis, there may be a temporary initial elevation only in SGO-T activity which may reach levels of 300 to 400 units. I n the case of neonatal biliary obstruction, there is a sustained increase in serum enzyme activity which may reach levels of 800 units. In the case of acute neonatal hepatitis a sharp rise in enzyme activity during the stage of increasing hyperbilirubinemia is followed by a rapid fall (K14). 7. Alterations in Serum Transaminase in Pathological Skeletal Muscle States

7.1. SKELETAL MUSCLEINJURY Skeletal trauma encountered during the course of experimental surgical procedures on dogs has shown that injury to skeletal muscle is accompanied by a moderate increase in SGO-T activity and a slight increase in SGP-T

SERUM TRANSAMINASE ACTIVITY

34 1

activity (L8). These increments in the serum transaminases appear to be related to the release of intracellular enzymes into the blood stream. Surgical trauma in humans has been observed to result in elevations of seruni transaminase. The alterations of serum transaminase appear within 12 hours after surgery and usually return to normal within 3 to 4 days postoperatively. Elevations of the order of 50 to 200 units have been observed in the experimental situation, and elevations of up to 100 units in the clinical settings. There appears to be a relationship between the degree of muscle trauma and the peak rise in SGO-T (L6, L8). From a study of traumas of the body, including contusions, abrasions, lacerations, fractures, and dislocations, it was concluded that SGO-T may be appreciably increased by traumatic injuries. Although cardiac trauma may account for increased serum transaminase elevations exceeding 500 units, SGO-T activity cannot be used as a specific test of cardiac injury in accident victims inasmuch as persons experiencing body traumas may show SGO-T activity increments unrelated to demonstrable cardiac injury (L6). 7.2. SKELETAL MUSCLEDISEASE STATES In a study of neuromuscular diseases, SGO-T was found to be elevated in progressive muscular dystrophy and dermatomyositis (Ll, S7), and in gangrene of toes (S7). Amyotrophic lateral sclerosis, progressive muscular atrophy, myasthenia gravis, and nerve section were not associated with elevation of transaminase activity . 8. Alterations in Serum Transaminase in Other Abnormal States

8.1. RENALDISEASE

Although acute and chronic renal disease, azotemia, and uremia have not been found to be associated with elevations in SGO-T (C6, L1, W l l ) , no extensive experience with renal infarcts has been reported. In the production of experimental graded renal infarcts in dogs by arterial ligation (P2, R2), it has been observed that SGO-T activity is increased above the normal range in proportion to the size of the infarct. The infarcted renal tissue was noted to contain one third to one fourth of the enzyme activity of the uninfarcted kidney tissue (P2). In a study of patients who incurred body traumas, it was noted that an individual who experienced a kidney laceration had a peak SGO-T of 378 units 1045 hours after the injury (L6). 8.2. BILIARY-PANCREATIC DISEASE

Acute pancreatitis (C7) is inconstantly associated with rises in S O - T and SGP-T. Whether these alterations are reflections of release of enzyme from necrotic and/or inflammatory pancreatic tissue, or are due to transient

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biliary obstruction caused by edema around the common duct, is not clear. In any case, the elevations observed are less than 100 units at the peak, and a return to normal values occurs within 3 to 5 days. When pancreatitis is associated with obstructive jaundice due to persistent common duct obstruction, the serum transaminase activities are elevated, the SGP-T being greater than the SGO-T, and remain so as long as the obstructive phenomenon continues. 8.3. THROMBOCYTOPENIA

Serum and plasma usually contain similar SGO-T and SGP-T activity (K3). However, platelet-free plasma has been reported to have decreased SGO-T activity, and blood platelets have been shown to contain GO-T activity (Ml). No reports on patients with thrombocytopenia have appeared. 8.4. PREQNANCY

An extensive study of SGO-T during various stages and conditions of pregnancy has shown no significant alteration of the serum enzyme above the normal range (B3), although elevated SGO-T has been reported in eclampsia (B3) and pre-eclampsia (B3, CS). From this study (B3) it appeared that the mean activity of SGO-T during uncomplicated pregnancy was somewhat lower than the mean activity of SGO-T in normal, nonpregnant adults. Intra-uterine death and premature placental separation during the course of human pregnancy were not associated with changes in SGO-T or SGP-T activity (M4). The experimental production of intra-uterine death in pregnant mice failed to cause a rise in SGO-T activity (M13). 8.5. STATES ASSOCIATED WITH SEROUSEFFUSIONS

Transaminase activity has been observed in other body fluids, including urine, bile, and serous effusions (W5). No definitive studies of the significance of alterations in these body fluids have appeared, except in the case of serous effusions of the pleural, pericardial, and peritoneal types. I n most instances, the transaminase activity of serous effusions is less than that of the serum activity of the same individual. In isolated examples of tissue injury associated with metastatic cancer within serous cavities, GO-T activities greater than the SGO-T activities have been observed. The significance of these observations requires further study (W8). 9. Alterations of Cerebrospinal Fluid Transaminase in Central Nervous System Disease

Although injury to central nervous tissue (57)is usually unaccompanied by significant alterations in SGO-T and SGP-T, elevations of SGO-T have been observed in instances of extensive cerebral tissue injury associated

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with massive cerebral hemorrhages and thromboses. In these clinical settings, peak elevations as high as 100 units have been reported (L7). However, in most instances of central nervous tissue injury, the serum transaminases usually remain normal or are increased slightly and transiently (M4). The failure of intracellular enzymes to be liberated into the blood stream as a result of central nervous tissue injury is presumed to reflect the influence of a blood-brain barrier (Dl, F2, G4, G5).

9.1, EXPERIMENTALLY INDUCED PATHOLOGICAL CNS STATES Experimental studies in dogs revealed a relationship between experimental cerebral infarction and the GO-T activity of cerebrospinal fluid (Wl). Recent studies (Dl, F2, G4, G5) indicate that increased activity of the GO-T of cerebrospinal fluid may occur in a number of neurological diseases. This has been ascribed variously to release of enzyme from destroyed nervous tissue, altered intracellular metabolism, differences in cellular permeability, and decreased elimination of enzymes from the cerebrospinal fluid. I n all the groups of neurological diseases studied, no correlation between the activity of transaminase and the protein content of cerebrospinal fluid has been observed (Dl, F2, G5). I n comparison with the findings in cerebral infarction in man, infarctions produced experimentally in dogs led to considerably greater and more consistent increases in the cerebrospinal fluid transaminase (F2, Wl). These differences have been ascribed t o the timing of the experimental infarct, the method of infarction, and the healthy state of the animal prior to infarction. 9.2. CLINICALCNS DISEASE

The normal range of GO-T activity of cerebrospinal fluid obtained from individuals without central nervous system disease has differed in the various reports delineating these values (Dl, F2, G5). These differences result in divergent interpretations of the changes of enzyme activity observed in pathological states of the central nervous system. One study has shown the normal range of GO-T activity of cerebrospinal fluid t o be 0-20 units and that of GP-T to be 0-18 units (Dl). Each unit is defined as the transaminase activity which will cause a decrease of 0.001 in the optical density of a mixture of reactants under standardized conditions (Dl, K1, 14713). Other investigators, defining cerebrospinal fluid GO-T activity as the number of micromoles of a-ketoglutarate per hour per milliliter, report the mean normal activity as 0.899 f 0.042 pmoles (F2). Another recent report defines the mean normal GO-T activity as 43 f 12 micromolar units, each unit representing the number of micromoles of oxaloacetate produced per hour per 100 ml of spinal fluid a t 37°C (G5). Although the recent reports on the clinical significance of alterations in cerebrospinal fluid transaminase activity differ, the following tentative generalizations and summary seem justified. Glutamic-pyruvic trans-

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aminase activity was normal in most of the patients that were studied but was noted to be elevated prior to treatment in four patients with tuberculous meningitis (Dl). The explanation for these isolated but striking alterations is obscure. Glutamic-oxaloacetic transaminase activity in cerebrospinal fluid is usually correlated with acute and significant injury within the central nervous system from many causes, including thromboembolic, degenerative, infectious, and neoplastic phenomena. The increase in transaminase activity appears to occur at varying times after the onset of the central nervous tissue injury. However, clinically significant central nervous tissue injury has been seen to occur in the absence of increased cerebrospinal fluid transaminase activity. No correlation has been found between serum transaminase activity and the enzyme activity of the cerebrospinal fluid (Dl, G5), nor has any relationship been observed between transaminase activity and leucocyte count, erythrocyte count, protein, glucose, chloride content, or other laboratory parameters of cerebrospinal fluid. From the data presently available, it would appear that the lack of specificity and the relative insensitivity of changes in cerebrospinal fluid transaminase activity limit the clinical usefulness of this enzyme reflection of CNS disease (Dl). 10. Conclusions

Extensive biochemical studies of enzymatic transamination have foreshadowed the clinical implications of transaminase activity in body fluids. Serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase activities are readily measurable by relatively simple techniques. Significant alterations of these serum enzymes have been observed during the course of cardiac, hepatic, and muscular diseases and reflect enzyme changes at the intracellular level of the respective tissues. Although a multiplicity of diseases is associated with serum transaminase elevations, diagnostic aid is afforded when these serum enzyme alterations are correlated with the clinical facts. Assessment of the clinical significance of alterations in transaminase activity in body fluids other than serum will require further study.

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H1. Henley, K. S., and Pollard, H. M., A new method for the determination of glutamic-oxaloacetic and glutamic-pyruvic transaminase in plasma. J . Lab. Clin. Med. 48, 785-789 (1955). H2. Herbst, R. M., The transaminase reaction. Advances i n Enzymol. 4, 75-97. H3. Herbst, R. M., and Engel, L. L., A reaction between a-ketonic acids and a-amino acids. J. Biol. Chem. 107, 505-512 (1934). H4. Hergt, K., and Langin, J. L., Serum transaminase determination: A simplified adaptation t o the Beckman model B spectrophotometer and some clinical applications in the general hospital. A m . J . Med. Sci. 233, 69-76 (1957). H5. Heyns, K., The importance of transamination in metabolic processes. Angew. Chem. 81, 474-482 (1949). K1. Karmen, A., A note on the spectrophotometric assay of glutamic-oxaloacetic transaminase in human blood serum. J . Clin. Invest. 34, 131-133 (1955). K2. Karmen, A., Wdblewski, F., and LaDue, J. S., Quantitative estimation of glutamic-oxaloacetic transaminase activity in human serum. Clin. Research Proc. 1, 90 (1953). K3. Karmen, A., Wr6blewski, F., and LaDue, J. S., Transsminase activity in human blood. J . Clan. Invest. 34, 126-133 (1955). K4. Karyagina, M. K., Formation and breakdown of amino acids by intermolecular transfer of aminograms. VI. Metabolism of L(-) aspartic acid in different animal tissues. Biokhimiya 4, 168-183 (1939). K5. Kattus, A. A., Jr., Watanabe, R., and Semenson, C., Diagnostic and prognostic significance of serum transaminase levels in coronary occlusive disease. Circulation 16, 502-511 (1957). K6. Kattus, A. A., Jr., Watanabe, R., Semenson, C., Drell, W., and Agress, C. M., Serum aminopherase (transaminase) in diagnosis of acute myocardial infarction. J. Am. Med. Assoc. 160, 16-20 (1956). K7. Kessler, G., and Phelps, A., Serum glutamic-oxaloacetic transaminase J . Albert Einstein Med. Center 4, 91-94 (1956). K8. Kit, S., and Awapara, J., Free amino acid content and transaminase activity of lymphatic tissues and lymphosarcomas. Cancer Reaserch 13, 694-698 (1953). K9. Kleckner, M. S., Determination of iron, mucoprotein, transaminase and cholinesterase in the serum in the differentiation of primary biliary cirrhosis, secondary biliary cirrhosis and cholestatic hepatic disease. Clin. Research Proc. 6 , 211 (1957). K10. Knoop, F., Vber den physiologischen Abbau der Sauron und die Synthese einer Aminosaure im Tierkorper. 2. physiol. Chem. 87, 48g502 (1910). K11. Konikova, A. S., Dobbert, N. N., and Braunshteh, A. E., Labilization of the a-hydrogen of amino-acids in the presence of aminopherase. Nature 169, 67-68 (1947). K12. Konikova, A. S., Kritsman, M. G., and TeIe, R. V., Study of the mechanism of transamination by means of deuterium. XVI. Formation and decomposition of amino acids by intermolecular transfer of the amino group. Biokhimiya 7, 86-92 (1942). K13. Kove, S., Goldstein, S., and Wr6blewski, F., Activity of glutamic-oxaloacetic transaminase in the serum in the neonatal period. Pediatrics 20, 584-589 (1957). K14. Kove, S., Goldstein, S., and Wr6blewski, F., Measurement of activity of transaminases in the serums as an aid in diagnosis of jaundice in the neonatal period. Pediatrics 20, 590-600 (1957). K15. Krause, S., and Krause, G., Serum glutamic-oxaloacetic aminopherase (transaminase) determinations: Value in the diagnosis of acute myocardial infarction in the presence of left bundle-branch block. J . A m . Med. Assoc. 161, 144-147 (1956).

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S1. Schlenk, F., and Fisher, A., Note on the purification and properties of glutamic-

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W7. Wrbblewski, F., The significance of alterations in serum enzymes in the differential diagnosis of jaundice. A.M.A. Arch. Internal Med. 100, 635-641 (1957). W8. Wrbblewski, F., The clinical significance of lactic dehydrogenase activity of serous effusions. Ann. Infernal Med. 48, 813-822 (1958). W9. Wr6blewski, F., and Cabaud, P., Colorimetric measurement of serum glutamicpyruvic transaminme. Am. J. Clin. Pathol. 27, 235-239 (1957). W10. Wrbblewski, F., and LaDue, J. S., Serum glutamic-oxaloacetic transaminase activity aa an index of liver cell injury: A preliminary report. Ann. Internal Med. 48, 345-360 (1955). W11. Wr6blewski, F., and LaDue, J. S., Serum glutamic-oxaloacetic transaminase activity aa an index of liver-cell injury from cancer. A preliminary report, Cancer 8, 1155-1163 (1955). W12. Wr6blewski, F., and LaDue, J. S., Serum glutamic-oxaloacetic aminopherase (transaminaae) in hepatitis. J. Am. Med. Assoc. 160, 113Ck1134 (1956). W13. Wr6blewski, F., and LaDue, J. S., Serum glutamic-pyruvic transaminase in cardiac and hepatic disease. PTOC. SOC.ExptZ. BWZ. Med. 91, 569-571 (1956). W14. Wdblewski, F., and LaDue, J. S., Serum glutamic-pyruvic transaminase (SGP-T) in hepatic disease: A preliminary report. Ann. Internal Med. 46, 801-811 (1956). W15. Wr6blewski, F., Jervis, G., and LaDue, J. S., The diagnostic, prognostic, epidemiologic significance of alterations of serum glutamic-oxaloacetic transaminase in hepatitis. Ann. Internal Med. 46, 782-800 (1956).