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5-1-1969

Clinical applications of LDH isoenzyme determinations

Ronald M. Wachter University of Nebraska Medical Center

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Recommended Citation Wachter, Ronald M., "Clinical applications of LDH isoenzyme determinations" (1969). MD Theses. 136. https://digitalcommons.unmc.edu/mdtheses/136

This Thesis is brought to you for free and open access by the Special Collections at DigitalCommons@UNMC. It has been accepted for inclusion in MD Theses by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. The Clinical Applications of LDH Isoenzyme Determinations

by

Ronald M. Wachter

A THESIS

Presented to the Faculty of

The College of Medicine in the University of Nebraska

In Partial Fulfillment of Requirements

For the Degree of Doctor of Medicine

Under the Supervi si on of C. A. McVlhori er, l\i. D.

Omaha, Nebraska

February 1, 1969 1

INTRODUCTION

The appearance in the world literature of increasing num­

bers of reports dealing with studies of human enzyme determin­

ations attests to the importance experimenters and clinicians

alike are ascribing to enzymology. Zimmermann (96) states:

"Enzymology, which was born a century ago, has grown vigorously

during the past few decades. More than 700 organic catalysts have

been identified in the tissues of living organisms. Approximately

50 of these enzymes have been identified in the plasma to which

they {,L.in access from injured cells, or perhaps from intact cells.

Interest of clinicians in serum enzymes began over 30 years ago

with the demonstrated diagnostiC usefulness of alkaline and acid

phosphatase, and amylase levels. Despite the clinical usefulness

of these Darameters of disease and the demonstration, during the

next two decades of a number of other enzymes in the serum, clin­

ical and investigative interest in serum enzymology remained spo­

radic until 1953. The demonstration in that year of a transaminase

in normal serum and the subsequent observations that increased levels

of this enzyme faithfully reflect disease of the heart, liver, and

other organs, led to an explosive increase in interest in serum

enzymes.

"The use of serum enzymes as diagnostic aids has been largely

empirical, but the values observed in clinical and experimantal

circumstances permit speculative analysis of the factors that con-

trol serum enzyme levels in normal and diseased subjects. Enzyme

levels are determined as 'activity' rather than 'amount' of enzyme protein, because the relative concentration of coenzymes, poten- tia tors, and inhi. bi tors may modify the apparent level ...... An 2

elegant development in serum enzymology has been the effort to

distinguish the tissue of origin of a serum enzyme which is

ubiquitously distributed in tissue by studying its isoenzymes,,"

\'Vi th thi,s brief historical background of enzymology in general,

and the reference to the recent development of isoenzymology, in the

lines that follow will be a closer examination of the different

molecular forms of a particular functional enzyme, lactic dehydro­

genase (LDH) ..

- 3 DEFINITION, STRUCTURE AND CHEMICAL ACTIVITY

Markert (39), in reviewing the literature, noted that the following enzymes exist in separate molecular forms within single specific tissues: esterase, ribonuclease, pepsin, chymotrypsin, trypsin, lysozyme, cytochrome C, xanthine dehydrogenase, malate dehydrogenase, and lactic dehydrogenase. Because of this, the various investigators noted a need to extend the classification of enzymes beyond that based on substrate specificity alone. They proposed the term 'isozyme' to describe the different molecular forms in which proteins may exist with the same enzymatic specificity.

In the process of glycolysis lactic dehydrogenase (LDH) is the enzyme which facilitates in a reversible manner the conversion of lactate to pyruvate and the concomitant conversion of diphospho­ pyridine nucleotide (DPN) to reduced DPN (DPNH). Since glycolysis is a function carried on by all living cells, LDH is present in these same cells, although its concentration may vary widely from tissue to tissue.

One of the earliest reports of discovery of the multiple molec­ ular forms of LDH came in 1950 when Meister (44) observed in the electrophoresis of crystalline beef-heart lactic dehydrogenase, a faster moving component (A), to which he accounted the whole enzymic activity, and also a slower moving one that amounted to about 15%.

In 1957 Sayre (64) postulated that "LDH may consist of a single molec­ ular species which can be observed as a number of components repre­ senting different states of aggregation as a result of sulfhydryl or other urotein interactions~ In the same year Vesell (76) reported finding three peaks of LDH activity in electrophoretically sep- arated human sera. Most workers are now agreed that there are five electrophor- etically distinct forms of LDR, which are numbered according to their rates of electronhoretic migration. Some workers designate the fastest (+) migrating fraction (heart fraction) LDR-l and the slow- est (-) (liver, skeletal muscle) LDH-5, while others utilize the reverse designation. This has added great confusion to the subject.

For purposes of clarity this paper will adhere to the nomenclature recommended by the subcommittee on isoenzymes of the international

Union: "when multiple forms of an enzyme are identified by electro- phoretic separation, they should be given consecutive numbers, the form having the highest mobility towards the anode being nu.1!lbered one."(4) See figure 1.

ANODE FLOW OF CURRENT CATHODE (+ ) ( - )

HEART LIVER

ISOENZYME

SERUM GLOBULIN a 1 8

Figure 1. Electropherogram illustrating LDH isoenzyme nomenclature. (After Batsakis)(4).

Markert and Appella (38) studied two main electrophoretic bands and found the constituents of each had a molecular weight of 134,000.

This was later confirmed bv others (16,84). The two bands differed slightly in their Michaelis-Menton constants, charge per molecule and isoelectric pnints.

Cahn and associates (9) reported that most vertebrates have two 5 chief categories of T,DH--one predominates in heart, the other in muscle4 In starch gel electrophoresi s the most posi ti vely moving portion is the heart type, the most negatively moving the muscle type. These two types were found to have widely differing amino acid composition (9, 16), electrophoretic mobility on starch grain and gel, substrate inhibition kinetics and analog oxidation ratio, and they were found to elicit antibodies in the rabbit that were non-cross reactive (9).

The fact that only five bands are found in most well-documented careful studies suggested there must be some fundamental significance in this number, which could be explained by the following hypothesis:

LDH from heart is a tetramer, composed of four identical subunits

(HERR); LDR from muscle is also a tetramer, composed of four iden­ tical subunits (1~~~). Rand M are probably elaborated by two dif­ ferent genes. If both Rand M genes are active in the same cell, then the primary gene products recombine in some manner in groups of four, yielding five different molecular species: HRRR, RHRM, HR!rr~f

H~,1lJ\f[M, and MIV!MM. The order of arrangement only affects the antigenic specificity--the subunits all act independently with respect to sub­ strate inhibition and rate of reaction with DPN analogs. This explains the five bands (9, 16, 40, 58, 65). Some of these same workers (2,9) found that one of the isoenzymes from crystalline LDH prepared from beef heart could be dissociated into four inactive subunits of equal molecular weight (34,000 .:t 2,000) by treatment with guanidine hydro­ chloride. This lent further sup:)ort tetramer hypothesis. Later,

Markert (40) showed that a tetramer could be dissociated into mono- mers by freezing in 1 M NaCl; on thawing, reassociation into five functional tetramers in the expected proportions of 1:4:6:4:1 occurred .. 6

Further su:)port of this hypothesis came from Nance and a~-::\socia tes (45)

in the study of a Brazilian fa,nily whose members were found to have an

erythrocyte LDR abnormality, all readily explainable by assuming a

point mutation leading to an alteration of polypeptide chain M.

Sub-bands of LDR isoenzymes have been artificially produced

(17,37,58) by various chemical means and the explanation of their

production also lends SUP90rt to the tetrameric hypothesis of L~R

isoenzyme composition.

Antigenic properties were investigated by several workers (21,

46,52). Plagemann and associates (52) demonstrated antibody to rabbit LDH-l inhibited only trace amounts of LDR-5, and the isoenzymes intermediate between the slow and fast migrating forms were inhibited to an intermediate degree. Gregory and associates (21) demonstrated

selective inhibition of glycolysis by antibody to LDH in homogenates of rabbit liver, rabbit Vx-2 carcinoma and mouse Ehrlich ascites carcinoma. Sodium oxamate was also shown to inhibit LDR in both an­ aerobic and aerobic glycolysis. Ng and associates (46) demonstrated an anti-LDR which inhibited glycolysis and protein synthesis in tumor culture cells but not in normal tissue cells, suggesting that their anti-LDH may have been selective for malignant cells.

Biochemical studies (1) have demonstrated that various isoen­ zymes differed in their capacity to oxidize I-lactic acid, d-J.actic acid, d-l-o\-hydroxy butyric acid, d-I-j)(.-hydroxy caproic acid and d-l-~-hydroxy valerie acid.

The techniaues USe -,or identifying the LDH isoenzymes have in­ cluded the electrophoretic rate of migration in solid media and the relative heat stability_ Electrophoretic demonstration of isoenzyme· activity is accomplished by conversion of neotetrazolium to purple 7

AGAR DIAPHORASE GEL LACTATE HYORAZINE NEOTETRAZOLIUM DPN

(-)

ELECTROPHORETIC MIGRATION

AGAR GEL

PYRUVArEIDPNH \ (j>i(,) {LDHl,. STARCH GEL ~LACTATE DPN

Figure 2. Schematic representation of the reactions involved in localizing LDH isoenzymes after they have been separated by electrophoresis in a starch gel. (After 1&lrkert)(39).

formazin by LDH production Qf DPNH, as illustrated in Figure 20

Wroblewski (88) reported that the LDH from human heart muscle apneared to be more heat-stable than LDH from other tissues, and went on to propose a simple heat-stable test for isoenzymes. He found that ~DH-5 was almost entirely inactivated after incubation at 57°C for 30 minutes; LDH-l was not inactivated after incubation at 65°C for 30 minutes; and that intermediate forms of LDH were inactivated after incubation at 65°C for 30 minutes. Actual enzyme activity determinations are made by recording the rate of change in the characteristic absorption spectrums of reduced DPN at 340 mu. This activity may either be measured by the declining optical density as

DPNH is diminishing and pyruvic acid concomitantly is being reduced to lactic acid, or by the increasing optical density as DF~h is in-

creasing 1 and the lactic acid concomitantly is being oxidized to 8 pyruvic acid. The former reaction is favored by the equilibrium of the system, but the latter reaction permits the use of more stable reagents.(5) Strandjord and associates (71) also propose utilizing the heat-stability test for diagnosis of certain disease states.

Isoenzyme activity may also be altered by cold temperatures,as

Zondag (97) found a total loss of activity in the slower moving o isoenzymes when the specimens were stored at -20 C under certain conditions. SERuM AND TISSUE PATTERNS OF LDH ISOENZYJvIES

An interesting facet of LDH isoenzymes is the fact that

each tissue :in the body'elaborates these multiple molecular forms

in its own unique pattern; this discovery has been renorted by

numerous workers. (12, 13, 14, 16, 34, 51, 53, 54, 55, 61, 70,

77, 84, 85, 87, 88, 90, 92) Pfleiderer(5l) found through study

of extracts of numerous human organs of different age that in

the early stage of life all tissues show nearly the same dis­

tribution, exhibiting a m~um of activity in Band 3, and that

during their development a change to the state of the adult

tissues occurs. Cahn and associates (9) reported that during

embryonic development the LDH enzymes of chicken breast muscle

shift from enzymes related to H type (LDH-I) through several

intermediate enzyme types and appear in the adult as pure M

type (LDH-5).

The predominance of LDH-l in aerobically functioning tissues

and the predominance of LDH-5 in anaerobically functioning tiss­

ues was noted by Cahn and associates (9), Pfleiderer and Wachsmuth

(50) and Fondy and Kaplan (16)~ and led to the formulation of a hypothesis that the predominance of LDH-5 in anaerobically met­

abolic tissues was due to its better efficiency in high con­

centrations under those conditions thEn LDH-l. Goldman (18)

states that LDH-5 is better able to convert pyruvate to lactate

in the presence of high pyruvate concentration, thus sustaining glycolytic activity through the replenishment of NAD, which is required as a hydrogen acceptor in the earlier oxidation of triosephosphate. Research of others indicate the only pathway 10 in malignant tumors for non-mitocyondrine oxidation of reduced

IlIAD formed from the oxidation of triosephospha te may be through

pyruvate reduction. There appears to be a change in LDll type

to an enzyme which is geared for pyruvate reduction. In aerobic

tissues where high substate concentrations do not develop, LDH-l

performs more efficiently. Studies of malignant cells show a

characteristic high rate of aerobic glycolysis resulting in high

rates of lactic acid production.(16~ 18, 19, 20, 22, 61) Graff

and associates(20), found that tumor culture cells resort to

glycolysis only when the extracellular glucose concentration

exceeds a threshold level of approximately 5 mg% or less, and

become consumers, rather than producers, of lactic acid when

the glucose concentrations fall below the threshold, which suggests

that glycolysis is the cells' defense against a high influx of

glucose. Gorozhanskaya and associates (19) found negligible

amounts of oxygen and high content of carbonic acid in their study

of malignant effusions. Fandy and Kaplan(16) found that synthesis

of M subunits (LDH-5) is stimulated by low oxygen tension and

synthesis of H subunits (LDH-I) was not affected.

Vesell's (77) discovery of predominance of LDR·-l in mature human erythrocytes (which are highly anaerobic tissue cells) was

in contradiction with the above mentioned hypothesis and led to

the formulation of a second theory (78) consistent with the first,

but more general, holding that each LDR isoenzyme fulfills dis­

tinctive roles by virtue of its restriction in various regions of the cell. Subcellular localizaion of the isoenzymes depends on their different kinetic properties, as well as on the metabolic organization of the particles with which they associate. (4) 11

Table I in the auuendix is an attempt to sumarize various

workers' demonstrations of the heterogeneity of LDll activity in

human tissues. Figures 3 and 4 illustrate the findings of one

worker in his investigations of serum and tissue LDH isoenzyme

patterns (88)"

Human tIssue 5:L _ I. Thyroid 5:[ ______~_~=_ Cardiac muscle 5:L .. '-- Lymph node

Adrenal 5°f01 I - Figure 3. LDH isoenzyme >. composition of watery ex­ - 50[.. - Lung tracts of normal human :~ o []-E -u ...... tissues.(After Wroblewskj 0 and Gregory) (88) 0 0 ....0 5:L ___ Pancreas -0 'E Kidney Q) 5:~_~1 CJ ~ tf 5:Loll_ Spleen 50 Skeletal muscle 0 .cJ••• (leg) lOOt 50 Liver 0 ---- IY./'//?I LD~ LD:~ LD3 L0'2 LDll ~ C7 Plasma isoenzymes of lactic dehydrogenase

I Heart muscle is characterized by a predominance of LDH-l, fol-

lowed in order by LDll-2, LDH-3, LDH-4 and LDH-5. Liver shows a preponderance of LDH-5 followed to a much lesser extent by LDH-4, 12

LDH-3, LDH-2 and LDH-I. Skeletal muscle resembles liver in its i80- enzyme pattern. Kidney shows a co-dominance of LDH-l and LDH-2, followed by LDH-3, LDH-4 and LDH-5. Lung exhibits prevalence of

LDH-~ and to a lesser extent LDH-2 and LDH-4 are seen. Pancreas shows a predominance of LDH-3, followed closely by LDH-l and LDH-2.

In granulocytes LDH-5 predominates. Erythrocytes are richest in

LDH-l and LDH-2. Starkweather and associates (67) found large amounts of LDH-5 are associated with the erythroblast nucleus, and gradually decreasing quantities of this isoenzyme as the cell mat- ures; thus LDH-5 may be a measure of erythrocyte age. Prostate, prostatic fluid, bone marrow, thyroid, adrenal, spleen, brain, cer- vix, skin and stomach isoenzyme patterns are also presented, and the reader is referred to Table I in the appendix for these isoen- zyme patterns as well as the literature references of each.

'Normal human serum exhi bi ts a predominance of LDH-2 (37-55% of total LDH activitY/' followed in descending order by LDH-l

(23-45%), LDH-3 (8-24%), LDH-4 (1-8%) and LDH-5 (0-4%). For more details refer to Table I in the appendix.(25,43,59,74,75,85,87)

A normal diurnal variation has also been demonstrated by Stark- weather and associates (70) as shown in Table II of the appendix.

Here they found a general rise in total LDH activity from the 8 A.N!.

B. A., 50, if. Normal adult

Total plasma LD = 300 units I ml. Figure 4. Serum L~H isoen­ >- zyme composition of a nor­ mal individual. (After Wro~­ lewski and Gregory)(88)& irJ ...... -----'-c=J- ...... -~a.::.::.::;.::.::;a.-.~~---I.~~ a. - LD~ LDJr LD3 LDZ Plasma isoenzymes of lactic .~ehydro9.enase 13 basal state; ryeak total activity (double the basal rate) occurred at 6 P.M., as did LDH-5, while LDH-l activity was at its lowest level at this time. LDH-2 changes paralleled those of LDH-I; LDH-3 and LDH-4 were much less affected. 14

ALTERL'i.TIONS IN THE NORMAL SERUM ISO''';NZYFE PATrl'ERN

Enzymes are proteins and as such, are synthesized in their cells of origin from amino acids, and somehow find their way into the plasma. Analysis of plasma, serum or tissue homogenate is not car- ried out to determine the amount of an enzyme in terms of weight, but rather the activitl of the enzyme is determined. Besides being dependent upon the method of assay, this measured activity is also dependent upon tll" .\:Jresence, absence, or rela tive concentrations of inhibitors, activators, co-factors, substrate, and finally the enzyme itself. Therefore, any alterations in the activity of the enzyme may be due to alterations in anyone or more of these factors.

Normal breakdown and turnover of tissues is thought to contribute to most of the enzyme activity in normal serum.(4) Figure 5 111u8- trates the factors influencing measured serum enzyme activity.

FACTORS INFLUENCING

MEASURED SERUM ENZYNfE ACTIVITY

ENZYME DEGRADA.TION ENZYME FQRMATION ~ ENZYME LEVEL IN CELL ~ CONCOMITANT PERMEABILITY OF SPECIFIC i DAMAGE TO ----....;)~ CELL ~ftEMBRANES<"'IIi1:'.----- TISSUE OTgER TISSUES ~ INJURY

EN ZYME LEVEL IN SrmUM.

ELIl'HN ATION IN DEG RA,IlA. TION URINE & BILE IN S'SRUM

INHIBITOR~ ACTIVATORS . 1 NIETHOD CJF' CO-FACTORS' MEASURED ACTIVITY ASSAY SUBSTRATE~ ~~~~~~~~~~~ Figure 5. Factors influencing measured serum enzyme activity. (Mod- ified from Batsakis and Hauss.)(4,23) 15

Alterations in cell membrane permeability aupear to playa large part in enzyme release. Permeability can be varied physiologically, and changes may be reversible or irreversible.(4)

Tissue injury has long been known to result in serum enzyme elevations, and on the surface it would appear easy to attribute these elevations to the altered membrane permeability resulting from the in,jury. That other factors may have an important effect on serum enzyme activity puzzled Wroblewski in 1958 (93) and later (91) he used the example of glutamic oxaloacetic transaminase (GOT) in cardiac tissue to present his point. He agreed that the high activ­ ity of human cardiac tissue compared with the relatively lower ac­ tivity of an equivalent weight of serum appears to explain in part the observed significant elevation of serum GOT values after myo­ cardial infarction. However, not all enzymes present in large amounts in the cardiac tissue as compared with equivalent amounts of serum are noted to increase in the serum of patients experien­ cing myocardial infarction; i.e. tributyrase and cholinesterase.

Experimants with intravenous injection of GOT and LDH into dogs results in a rapid rise in serum GOT and LDH values, with a return of these enzyme activities to normal within 1-4 hours. In the case of

LDH, when serum enzyme activity rises to ten times that of normal, it returns to normal range of serum activity in 1 hour. At this rate of serum enzyme degradation and/or elimination, the LDH content of the infarcted heart muscle would not appear to be sufficient to maintain the serum enzyme activity level at 3 to 6 times normal serum value for a period of 5-7 days. Human cardiac muscle con­ tains ap;Jroximately 250, 000 units of LDH/ gm. of wet tissue; with comparable techniClues the serum contains approximately 400 units/Illl. 16

One gram of heart tissue would cause an LDH activity of 100 units/mI.

in 2.2 liters of serum. An LDH increase o~ 1500 units/mI. often

observed in patients with myocardial infarction presupposes the nec­

rosis of 15 gm. of cardiac muscle. In order to maintain a serum

level of 1500 uni ts/ml., 15 gm. of heart muscle would have to be

destroyed per hour, or 360 gm. /day. If the serum LilH level were

maintained at about 1500 units or more for 3 days, which is seen

frequently clinically, 10SO gm. of heart muscle would be required,

whereas a normal heart weighs only about 300 gm. Thus some other

mechanism must be involved to account for the prolonged enzyme

elevations seen.

The report of Hill (27) in 1954 of elevated LDH serum levels

in neoplastic disease has led to investigations of the mechanism

of such an elevation.(3,4l,47,4s,62,81) Mahy (41) reports earlier workers presented evidence that enzymes are removed from the blood

by the reticuloendothelial system. His work and others'(3,62,4s) with mice infected with certain strains of Riley virus showed that

the resultant LDH serum elevations were most likely due to decreased

clearance rates from the ~asma. The possibility that in other disease states the enzyme serum clearance rate may be decreased certainly is worthy of some consideration.

The role of necrosis in enzyme elevation was further studied by'Hroblewski (93) and';Iest (81). They both found increased levels of LDH in the absence of necrosis of malignant tumors in rats, suggesting that increased synthesis, altered membrane permeability or some other factors must ~lay a role in the elevation of LDH.

Hause (23) has reported that enzyme elevations in cases of surgery, infectious diseases, and nulmonary 6mbolism react in the 17 the same manner as in myoc::lrdial infarction; quantitative and tem­ poral differences may exist~ but the reaction remains the same-­ nan-specific. These enzyme changes occur along wi tIl other comrilon non-specific symptoIDs--rise in temperature, leukocyte increase~ changes in the CBC, serum protein, UA, increased erythrocyte sed­ imentation rate, blood sugar and rest nitrogen elevation and other symptoms which together Hauss has termed the Il acute syndrome. 1t

If the foregoing is true, if there are so many variables to consider to account for serum enzyme elevations, can any clinical use be made of enzyme determinations? tlTroblewski (89) speaks well to this question: IlMost if not all enzymatic reflections are non­

specific in that more than one etiologic factor and more than one anatomic site can account for similar quantitative and or serial enzyme changes. In addition, the multiplicity of enzymes present in body' fluids, as well as the influence of other body fluid constituents, such as inhibitors, antienzymes, activators, competitors, drugs and others, may account for apparent artefacts included in the extensive enzyme activity of the body fluid.

However, the concomitant measurement of several enzymatic activ­ ities and clinical correlation makes it possible at times to pin­ point the type and site of a pathologic process. In addition, in some instances, serum enzyme changes have permitted recognition of disease such as hepatitis in the prodromal phase of the malady; in other instances, such as drug toxiCity, serum enzyme alterations have indicated pathologic effects at a time when other labOratory parameters have been uninfl1Jenced; in the case of suspected myo­ cardial infarction associated with bundle branch block of other masking electrocardiographic aberrations, serum enzyme changes may 18 contribute pivotally to the resolution of the diagnostic problem.

The use of body fluid enzymes to supplement cytologic techniques is an addition to the armamentarium for the study of patients with oncologic diseases •..•. " 19

LDH ISOENZYME .~LTERATIONS IN DISEASE STATES

Initial enzymatic studies in disease states showed gross elevations in LDH, SGOT, and others. (92, 83, 82, 47, 79, 74, 63,

31, 30, 28, 24, 6). As was stated before, all organs contain

LDH, and organ injury produces an elevation in serum LDH; how­ ever, this elevation is non-specific. Cohen (10) proposed that if some characteristic feature of LDH could be identified that would betray its source, the locus of specific organ injury might be established. With the discovery of the multiple molecular forms of LDll, identifiable by electrophoretic separation, heat fractionation, and other means, such a characteristic feature was found. Cohen's work, plus that of others discussed below, showed that the serum pattern of LDll isoenzymes following organ injury is usually characteristic of that organ. 20

ISOEN'ZYMES AND NfYOCAI-mIAL INFARCTION

Cardiac muscle has a predominance of LDE-l and 2, and thus injury to this tissue results in increases in serum LDH-l and 2.

This increase has been noted within hours (87, 90, 91) of the onset of the acute attack, is maximal before the fifth post- infarction day, and remains elevated for 5-10 days after all total plasma enzyme activities have returned to normal range.

(87, 90) Typical serum LDH isoenzyme patterns in patients with acute myocardial infarction are reported by Reusch and Bunch (59) as shown in Table III of the appendix. In all there was a predominance of LDH-l. In two cases SGOT and total LDE changes were subtle, but isoenzyme analysis clearly demonstrated a shift from the normal predominance of LDH-2.

Wroblewski and associates (90) demonstrated the serial LDH isoenzyme changes in acute myocardial infarction as shown in

Table IV of the appendix. They presented two patients whose LDH-l abnormalities persisted for 19 and 20 days. These abnormalities are further illustrated by the report of Nutter and associates (49).

See figure 6.

SERIAL CHA.NGES IN LDH-l TN ACUTE MYOCARDIAL INl<'ARCTION I

Day 1 2 3 4 5 6 7 8 9 10 11

Subjects 8 18 22 22 23 22 22 '22 18 16 12

Abnormal 5 17 22 22 22 22 21 21 17 15 10

Normal 3 1 0 0 1 0 1 1 1 1 2 r-- I ! ! Figure 6. Data showing serial LDH-l changes in acut'e myo-

cardial infarction. (After Nutter)(49). 21

starkweather (70) studied LDH levels serially on 25 con-

troIs monthly for 9 months and found insignificant variations in

individual enzyme levels even though a wide range of activity was

• observed for the group. Therefore it appeared to him that normal

serum LDH ranges as derived from statistical data could lead to

erroneous clinical interpretati~n. For example, if a person with

a normal LDH level of 0.192 units shows a level of 0.384 units,

statistical data would place it within normal range when actually

a two-fold increase has occurred. Since baseline determinations

are usually not available, he proposes one should get three fast-

ing samples and evaluate the changes. The examples in figure 7

illustrate this point.

Basoline 4th day Difference serum serum LDH LDH

infarction .360 .130 til' .144 lin .082 "" .178

Figure 7. Chart show"ing small but significant isoenzyme changes

from base line levels. (After Starkweather)(70).

In some instances, myocardial infarction may not be accom-

panied by diagnostic serum enzyme changes, but analysis of the

LDH isoenzyme pattern helps pin down the diagnosis, as illustrated

by the case in figure 8. (74) 22

3!.~L __- Figure 8. Case illustrating insignificant total LDH changes

but significant isoenzyme alterations. (After Van Der Hel~74).

The serial determination of LDH isoenzymes may also be of help in diagnosing myocardial infarction which has occurred

several days before medical attention is sought, as illustrated by figure 9. (74)

Acute Attack 8 Days Before Rosuital Admission

9 12

Figure 9. Chart showing LDR isoenzyme alterations in a patient

suffering a myocardial infarction eight prior to medical attention.

(After Van Der Helm) (74).

Thus it would appear from the data presented that prolonged changes in LDF-l occur in uatients with acute myocardial infarction and that the serial analysis of LDH-l is a sensitive aid in the 23 diagnosis of acute myocardial infarction.

It must be Dointed out that in some cases of acute myo- cardial infarction, there has been noted a rise in LDH-5; this change has been ascribed to the presence of severe congestive heart failure, causing increased serum contributions from the hepatic cells. (87, 73)

Of particular importance is the negative value of the "zymo- gram.II Thus LDH isoenzymes can be used to differentiate between myocardial infarction and other conditions not readily distin- guishable from myocardial infarction. Cardiovascular diseases with normal fractions of LDH-l and LDH-2 include: congestive heart failure, in all but severe cases,; acute pericarditis; paroxysmal atrial fibrillation with stenocardia; chronic angina pectoris, heart block with Stokes-Adams seizures; acute venous thrombosis of the lower extremity; and pulmonary embolism. (74,

87, 11, 73)

Starkweather and associates (70) attempted to prognosticate the clinical course of the patients with myocardial infarctions on the basis of enzyme studies. They found that there was no correlation between serum LDH levels and mortality •. They did, however, find some relationship with survival and an elevation in LDH-5 within 24 hours post-infarction_" Twelve such individuals exhibited a rapid rise in that fraction within 24 hours and did not recover from the attack. It is usual to attribute such rises in LDH-5 to some degree of congestive heart failure; however in these twelve patients, post mortem examination showed no con- gestion of the liver, and the source of the LDE-5 was not found in these cases. 24

Although the agar gel electrophoresis method of determination is the one used by a great number of workers, it is somewhat cumbersome. Wroblewski (88) realized this early and proposed a simple heat-stable test for determination of LDH isoenzymes.

This determination takes advantage of the fact that different

LDH isoenzymes have different thermolability properties •• lso­ enzymes which migrate electrophoretically with the gamma glob­ ulins are relatively thermolabile ••• lsoenzymes which migrate toward the anode (rapidly migrating) are relatively thermostable.

LDH which remains after incubation at 65° C for 30 minutes has been defined as "heat stable LDH" and represents LDH-l activity.

This quick method of isoenzyme determination in the diagnosis of myocardial infarction has several proponents. (5, 71, 88) 25

ISOENZYMES Hi BEP1\.TIC DISEASE

HEP./\.TITIS ''lieme and Van Maer-eke (85) noted elevations in rat sera of LDH-5 after experimental induction of liver necrosis with carbon tetrachloride. Trujillo and assooiates (73) noted in four cases of infectious hepatitis an elevation in total LDll in all, elevated LDH-5 in all, elevated LDE's 2, 3, 4 in two of the four, and no abnormality in LDH-I. Vesell (77) noted most of the LUH activity in sera of patients with hepatitis was in

I,DH's 4 and 5. 1Jfatoole and Novak (43) found in the stud;,\, of four cases of acute infectious hepatitis an elevation in LDH's 4 and

5. Cohen (10) reports an LDll isozymogram in a oatient with hepatitis: LDH-l=18%; LDH-2=27%; LDH-3=9%; LDH-4=8%; LDH-5=38%.

CIRRHOSIS Matoole and Novak (43) studied 35 cases of Laennec's cirrhosis and found the most frequent enzyme alteration to be elevations in LDH's 5 and 4. Trujillo and associates (73) found

LDH-I elevations in 9 of 15 patient s with cirrhosis, and J,Dll-l elevations in 2.

BILIARY OBSTRUCTION Wright and associates (87) studied 10 patients with biliary obstruction and found absolute increases in LDll-4 and LDll-5 with the elevations of LDH-4 being greater than those of LDll-5. This is in contrast to the marked relative in- creases in LDH-5 seen in hepatocellular damage. Trujillo and associates (73) and :N1atoole and :Novak (43) merely found elevations in LDH-5 or LDH's 5 and 4.

HEPATIC METASTASES AND 1° HEPATOMA In 14 cases of hepatic metastases and 1 case of 1 0 hepatoma, Trujillo and associates (73) found elevated total LDll in 14 of 15, elevated LDH-5 in all 15, elevated LDH~2, 3, 4 in 12 of 15, and LDH-l elevations in 10 of 15. In all the :9redominant isoenzyme was LDH-5. 26

Thus, in most cases of liver disease studied the LDH ab­ normality most often encountered was that of elevation of the predominant isoenzyme found in hepatic cells, LDH-5. 27

ISOENZYMES nr PULMONt:cRY E!,;LBOLISM

In 1961 Wacker and associates (19) 'oroposed a !!triad for

the diagnosis of pulmonary embolism and infarction" which in­

cluded serial measurements of serum LDH, SGOT, and bilirubin.

In 11 patients they renorted consistently elevated LDH, and

consistently normal SGOT in the first four days nost embolism.

Twelve of the 17 had significantly increased bilirubin. Batsakis

and Briere (4) also reported consistent elevation of serum LDH

in patients with pulmonary infarcts, but could not improve diag­

nostic accuracy with concomitant serial determinations of SGOT and bilirubin. They noted that pulmonary emboli in a hospital

population usually occur as complications of some other disease

and the associated disease not infrequently influences enzyme

levels. Their most successful method of diagnosis of pulmonary

embolism was with the use of CPK (creatine phosphokinase) deter­

mina tions o.r',DH isoenzyme determinations. Isoenzyme; al terations

included an increased total activity pattern and a relatively

greater prominence of LDH-3. These changes were attributed to a

combination of effects of hemorrhage (LDH-l and LDH-2) and pul­

monary necrosis (LDH-3). On the second day following infarct,

the majority of patients studied also showed an increase in LDH-5

related to accompanying congestive heart failure and anoxemia of

the centrilobular portions of the liver.

Tru~illo and associates (73) studied 26 patients with pul­ monary embolism and found normal total LDH in H3 patients, and only minimal increases in the remaining eight. However, he did

find alterations i"1 ~}Oe LDH-2, 3, 4 fraction. (Their method of

LDH isoenzyme determination involved heat fractionation in 'i'lhich 28

the activities of LDH-2, 3~ and 4 could not be differentiated.)

See figure 10.

I SERIAL EIi;ZYflIiE CHANGES IN PUHIOlJARY EJVIBCrliISNI I Day 1 2 3 4 5 6 7 8 ! 9 10 11 Ho. Subjects 3 12 19 23 25 24 26 25 124 221 15

t::; LDH-l Abnormal I 2 4 8 3 2 7 J 7 6 4 LDH-2,3,4 Abn. 2 5 10 8 8 12 12 16 12 11 I 7 I JDH-5 Abn. 1 3 9 12 12 10 ; 11 12 12 8 6 ! j ~ I Figure 10. Daily enzyme data in 26 patients with uncomplicated

pulmonary embolism. (After Trujil1o)(73).

Although the data from this study do not demonstrate high- ly consistent LDH-2, 3, 4 alterations, these workers found the main value of LDH isoenzyme determinations in pulr:lOnary embolism to be the ability to exclude myocardial infarction and to appraise other disorders as possible sources of enzyme alteration.

Cohen and his workers (11) studied 19 cases of pulmonary embolism. Twelve of those were found to have normal serum total

L1JH; four of these had elevated LDH-I, and eieht were normaL

Seven had elevated serum total LDH; three had elevated LDH-3; three had elevated LDH-l and 2, and one had elevations of LDH-

1,2, and 3. They concluded that the LDH abnormalities that developed appeared to be related to associated disorders of the liver or heart.

From the above it would appear that pulmonary embolism may or may not be accompanied by elevations in total LDH or LDH-3, but that the main use of LDH isoenzymes would be to exclude myo- cardial infarction. 29

LDH ISOENZYJ\IES IN ANEMIA

In 1958 Zimmerman and his workers (95) reported elevated serum levels of LDH in patients with megaloblastic anemia, sickle cell disease, hemoglobin S-C disease, S-thalassemia, and Hemoglobin

A-S or A-C disease. These alterations were thought to represent hemolysis, hepatic necrosis, or inherently high LDH concentrations in primitive marrow cells.

Wright and associates (87) studied four patients with un­ treated pernicious anemia, 1 with paroxysmal nocturnal hemo­ globinuria, 2 with sickle cell anemia, and 1 with idiopathic hemo­ lytic anemia, and found almost equal absolute increases in LDH-l anct I.DH-2 and a concomitant reduction in the remainder of the fractions. This pattern is similar to patterns of red blood cell hemolysa tes reported earlier. !J.'hus it would appear that any hemolytic process would result in increases in LDH-l and LDH-2 in the serum.

Starkweather and associates (67) reported large amounts of

LDH-5 were associated with erythroblast nuclei, and that grad­ ually decreasing amounts of this enzyme were found as the cells matured, suggesting that LDH-5 may be an indicator of erythrocyte age; the LDH in aged erythrocytes was found to be LDH-l. They propose that when erythrocyte isoenzymes from patients with hematologic disease are assayed, increases in LDH-5 indicate ac­ tive or hyperactive erythroid tissue, and absence of this fraction suggests nonfunctioning tissue. An increase in the relative per­ centage of LDH-l suggests the presence of an older cell popUlation.

Heller and co-workers (24) studied serum isoenzymes in patients with megaloblastic and blood loss anemias and found evi- 30 dence to supnort the hypothesis that the high plasma levels of

enzymes originated in the bone marrow.

Thus, in the presence of hemolysis there are elevations of

LDH-l, in megaloblastic or hyperplastic marrow conditions there are elevations of LDH-5, and in aplastic marrow conditions there

is essentially no LDH-5. 31

ISOENZYIViES IN MUSCULAR DISORDE-qS

Normal skeletal muscle has a predominance of LDH-5 and thus one would predict some alterations of this enzyme in skeletal muscle disorders~ Wright and associates (87) report marked transient increases in LDH-5 in nine patients suffering crush­

ing trauma, hip fractures, or undergoing extensive surgical procedures involving muscle.

The effect of exercise on the serum enzyme composition was studied by Swaiman and Award (72). It was postulated that heavy exercise would result in muscle cell damage which in turn would result in enzyme release. However, after studies on 11 men during and after strenuous and prolonged exercise, only slight increases in aldolase, CPK, SGOT, and LDH were found, and in some no changes or even decreases in enzyme activity were found.

Starkweather and associates (69) studied the effect of artificially induced hemostasis by placing a tourniquet on the patient's arm for five minutes, after which blood was with­ drawn and a three fold increase in LDH was found. Fifteen minutes after the pressure release, the serum LDH had almost returned to base line levels, but an elevation in LDH-5 remain~d.

Emery (15) studied carriers and patients with Duchenne type pseudohypertrophic muscular dystrophy. He found no significant differences in the serum LDH isoenzyme patterns between healthy children and affected children; also he found no differences between normal women and carriers. However, the LDH isoenzyme pattern of muscle extracts from affected children was very different from the controls. LDH-5 appeared to be completely 32 absent; this was true in all sta?8S of the disease irrespective of the grade of severity.

Brody (8) ancl Kelly and associates (36) also re?:)orted find- ing virtually no LDH-5 activity in sera of pattents with Duchenne- type muscular dystrophy_ However, Brody studied many muscular and neurologic disorders. His findings are summarized in figure 11.

LDH-5 ABNORIVLil.LITIES IN VARIOUS lIiWSCULAR AND NEUROLOGIC DISORDER.S

Clinical Diagnosis No. of Patients Total l~o. wi th Decreased of LDH-5 Patients

Muscular Dystrophy 12 32

Amyotrophic Lateral Sclerosis 6 51

Infantile Spinal Muscular Atrophy 5 5

:Myotonic Dystrophy 4 8

Idiopathic Inflammatory Myopathy 4 18

Dermatomyositis 1 4

Peripheral Polyneuropathy, Idiopathic 3 22

Diabetic Polyneuropathy 1 6

Neuromyopathy(?) Due To Carcinoma 1 3 Spinocerebellar Degeneration 2 7

Charcot-:Marie~Tooth Disease 2 18

Benign Congenital Hypotonia 1 3

Motor Retardation 1 1

Figure 11. Summary of LDH-5 abnormalities in various muscular

and neurologic disorders studied by Brody (8).

He found the selective decrease in LDH-5 is not restricted )3 to patients with muscular dystrophy, but may also be seen in other myopathies and in diseases primarily affecting neurons.

Acquired as well as hereditary disorders were found to be associ­ ated with this phenomenon. Therefore a decrease in LDH-5 is not diagnostic of a particular disease entity, it does not differentiate primary lesions of muscle from neurologic lesions, and it is not indicative of a hereditary rather than acquired disorder. This study did indicate, however, that a deficiency of muscle LDH-5 constitutes a sign of nonspecific abnormality in the neuromuscular system.

The mechanism for this decrease in LDH-5 in abnormal muscle has not as yet been adequately explained. Brody (8) suggests two hypotheses. The observation that the activity of the fast­ moving isoenzymes is preferentailly decreased in the presence of excess lactate suggests that a function of the slow-moving i80- enzymes may be to maintain activity in the presence of accumulated lactate. Thus the slow-moving isoenzymes participate primarily in anaerobic (glycolytic) metabolism and the fast moving isoenzymes participate in aerobic metabolism. Since the experimental de­ nervation in animals has produced earlier diminution of the glyco­ lytic than of the oxidative enzymes, it may well be that the selective reduction of LDH-5 in abnormal muscle may coincide with the selective reduction of other glycolytic enzymes.

A second hypothesis (8) is based on the observation that the isoenzyme pattern of normal human fetal muscle also shows a selective deficit of LDH-5, and that it is possible that low LDH-5 activity in abnormal reflects persistence of or a reversion to a primitive stage of metabolic adjustment. ISOENZYhlES IN DISORDERS OF THE GASTROIhTESTINA1 TRACT

Hendrich and Hule (25) studied the serum LDH isoenzymes

of 16 patients with ulcerative colitis and found no substantial difference from the group of 55 controls. Homogenates of speci­ mens of rectal mucosa from 8 patients during the acute phase of ulceration showed high levels of 1DH-5, while specimens of 4

patients in the healing stage or remission showed lower 1DH-5 values.

Prochazka and associates (54, 55) have done work on experi­ mental lesions of the stomach in rats. In rats with stress in-duced

ulcers, they found increased total LDH activity, as well as elevated LDH-l,2,3, and 4 with decreased 1DH-5; all of which could not be explained. They also performed isoenzyme determinations on tissue homogenates of normal fundic mucosa, fundic mucosa with moderate gastritis, fundic mucosa with extensive gastritis, marginal mucosa of the pyloric peptic ulcer, gastric carcinoma tissue, and normal pyloric mucosa. They found progreSSively decreasing amounts of 1DH-l in the categories previously listed, and found progress­ ively increasing amounts of 1DH-5 in the same categories respec­ tively. 35

LDH ISOEN:6YMFS IK NEOPLASTIC DIS}~ASE

With the massive amount of research being carried out in the field of cancer, it is not surprising that a great deal has been published about LDH isoenzymes by its researchers.

Until Hill and Levi published an article in Cancer Research in 1954, (27) the authors had not been able to find any report of a study of LDH'activity in human serum. These authors noted in a study of 51 patients with neoplastic diseases that 49 of them had elevation of a serum comuonent which catalyzed the I ~ oxidation of reduced DPN and the simultaneous reduction of pyruvic acid to lactic acid; in other words, the serum component referred to as LDH. White (83) also noted this elevation in patients with neoplastic disease, and found that it was more consistently elevated in such patients (16%) than was phos- pbohexossisomerase (48%), aldolase (35%) or SGOT (31%). Most people in his group with high serum LDH activity had obviously widespread cancer. Although Hill (28) later could not sub- stantiate his earlier findings, the consensus remained that serum

LDll activity was elevated in cancer patients. (6, 18, 82, 34)

In 1957 Vesell and Bearn (76) subjected sera of normal and

"abnormal" patients to starch gel electrophoresis and found three peaks of LDH activity corresponding to the alpha-I, alpha-2, and beta serum protein peaks; in three cases of leukemia studied, all showed elevation in alpha-2 activity relative to the alpha-l peak.

Hsieh and associates (30) demonstrated a d.ecrease in serum LDH activity in mice bearing squamous cell carcinomas or rhabdomyo- sarcomas after removal of such tumors, and noted a rise in serum

LDH in the group where the tumors recurred. The question then arises, what causes the elevated serum

LDH activity in patients with cancer? Jacobson and Bishio (33) proposed four hypotheses for the origin of elevated LDH in patients with cancer: 1) Volume of the tumor reached a critical size such that the rate of LDH production by the tumor cells was higher than the normal rate of removal of this enzyme; 2) The tissue cells which were replaced by the tumor were ruptured or in some way altered so as to release IDH and other com;Jonents into the circulatory system; 3) The behavior of normal tissues may be affected in some manner leading to a release and/or increased synthesis of the LDH normally present in the organ; and 4) A factor or virus is often associated with the transmissable tumors and causes an abnormally high LDR. Th'ese hy:potheses have been sunported in whole or in part by other workers (3, 48, 62, 41, 93, 69).

~at kind of LDH is produced by these tumors? Richterich and Burger (61) compared isoenzyme patterns of benign and malig­ nant effusions and found that: 1) In benign effusions (transudates or inflammatory exudates) the isoenzyme pattern reflected the serum pattern and the activity of the slowly migrating isoenzymes was low; 2) In malignant effusions the electrophoretically slowly migrating fractions were elevated in comparison to the correspond­ ing sera; and 3) The activity of the slowly migrating fractions was markedly increased independent of the type of malignancy

(carcinoma of the lung, eso:phagus, pancreas and breast; hyper- ne)Ll'OLLC, reticulosarcoma).

That a high rate of glycolysiS is characteristic of tumor tissue has long been estab1ished (18, 19, 20). Ascites fluids 37 in cancer patients have been found to have a high content of carbonic acid, negligible amounts of oxygen , and lactic acid concentrations two or three times blood levels indicating large amounts of pyruvate reduction. Thus, ascttes cells in cancer cases were found to exist under nearly anaerobic conditions. (19) / ! Goldman and associates also found a predominance of, ltDH-5 in cancerous tissues. They reported that earlier workers indicated that the only pathway in malignant tumors for non-rrlitochondrial oxidation of reduced NAD formed from the oxidation of triose- phosphate may be through pyruvate reduction, and that there appear- ad to be a change in LDH type to an enzyme which is geared for pyruvate reduction. Goldman further states that the slowly mi- grating LDH (M-LDH or LDH-5) nis better able to convert pyruvate to lamtate in the presence of high pyruvate concentration, thus sustaining glycolytic activity through the replenishment of NAD, which is required as a hydrogen acceptor in the earlier oxidation of triosephosphate.:

Studies of tissue homogenates of various carcinomas have demonstrated this shift in isoenzyme pattern to increasel amounts of LDH-5. Carcinomas studied include those of rectum, colon, breast, stomach, lung, prostate, kidney, and thyroid, as well as astrocytomas and malignant tumors of glial origin. (10, 14, 16,

53, 61, 66) Studies of malignant effusions also showed this shift.

(22, 61) studies of leukocytes in chronic lymphocytic leukemia and chronic myelogenous leukemia produced different results; l:.rmphocytes exhi bi ted relatively low levels of LDH-5 acti vi ty (7.

3%), whereas granulocytes exhibited high levels of this isoenzyme

(24.1%). Some workers (68, 87) have found isoenzyme alterations 38

of LDH-3 to be the greatest, both in tissue homogenates and in

sera of patients with neoplastic disease. The only instances of

LDH-5 alterations were in demonstrated cases of hepatic metastases;

when these metastases were shown to be absent, only LDH-3 was

elevated. However, the majority of workers feel that the main

alteration appears to be in the LDH-5 fraction.

Clinical application of LDH isoenzymes in patients with new-

plastic disease is useful in several areas. Malignant effusions

demonstrate elevated LDH-5 act~vity and can thus be differentiated

from benign effusions. (60, 61) Following chemotherapy or radiation

therapy, analysis of the effusion could be of prognostic value;

decreases in LDH-5 would indicate effectiveness of the therapy,

and no change or increases in activity of this isoenzyme would

indicate ineffective therapy. (19)

starkweather and associates (69) attempted to correlate

enzymatic aberrations with clinical evaluation of disease. Their results are summarized below:

: Extent of Disease--Clinical vs. Enzymatic Classification i~------1~R~f:;1c~~~y~~:a~r6n_i-N;;~~r -. Clinical I Total LDH and iso- ' Isoenzyme patj Total LDH & Iso, i Classificati0nl enzyme pattern(27) tern only(12)1 enzyme pattern(ql L·-"-"-----'"--·-'---"-----·--"l---"---~------~ "-"--- ! .. ------"---,------I / Extensive ! 25 10 4 Limi ted I 2 2

None of the patients with abnormal total serum LDR demonstrated normal isoenzyme patterns. In the four "clinically extensive" but enzymatically normal patients, the enzyme nattern became abnormal within five days to four weeks after admission to the 39 study. Thus, the use of such ~arameters as a screening test for the early detection of neoplastic disease does not appear to possess adequate sensitivity to be of clinical use.

Elhilali and associates (14) nroposed the use of LDH 1so­ enzymes in the differentiation of hyperplasia and carcinoma of the prostate. Prostatic fluid was examined and prostatic tissue frDm transuretbral resection, prostatectomy and needle biopsy were obtained. A shift to slower migrating isoenzymes was demonstrated in tissue specimans from patients with carcinoma; only normal pro­

static fluid isoenzyme levels were established, although in three cases of carcinoma studied, the isoenzyme patterns were abnormal.

Since it is estimated that 95% of the patients with carcinoma of the postate are disgnosed too late for operative treatment, an earlier method of detection would certainly be advantageous. Such a method is proposed; that is a screening test which would involve a study of the LDH isoenzymes of prostatic fluid. In cases of early carcinoma this would be practical, however in advanced

carcinoma of the prostate, fluid is difficult to obtair.• Another apnlication of LDH isoenzymes would be to monitor changes in the

sera of patients with advanced carcinoma under treatment as an

index of the efficacy of that treatment. (14)

Starkweather and associates (69) proposed the determination of LDH isoenzyme responses following therapy for neoplastic disease as a prognostic guide. Also proposing this plan were many others.

(47, 7, 94) Of 21 patients who showed a decrease in the isoenzymes complement (ascribed by those workers to the host's tumor), 12 improved either objectively or subjectively, 1 was unchanged and

7 grew worse. 40

'rIlE EFFECT 0:3' OJ=L\.L CONTRACEPTIVr:S ON SERUM El~ZYMES

Pulkkinen and 'Villman (56) studied 75 women taking estro­ genic, progestogenic, combination, and sequential oral contra­ ceptives and found no difference between that group and a group of 30 controls. They found that the total LDH activity in the whole groun was decreased; however, estrogenic pills had no effect on LDH; the progestagenic pills lowered the activity of

LDH but did not alte~ the isoenzymes. The duration of adminis­ tration made no difference on the results obtained. Administration of a combination oral contraceptive to both men and W(men pro­ duced similar LDH serum changes, i,e, total LDH activity was low­ ered in 1-2 days following initiation of administration. RECAPITULATION AND CONCLUSION

Lactic dehydrogenase which is present in all living cells, plays an important role in glycolysis by catalyzing the reversible conversion of lactate to pyruvate, producing in the process a molecule of' reduced DPN. Most workers believe there are five electrophoretically distinct molecellar forms of LDI! otherwise known as isoenzymes. Each of the five isoenzymes is a tetramer: that is it is composed of fou' subunits. These four subunits are of two basic types: the n M II or muscle-liver unit and the "H" or heart unit. LDH-I and LDH-5 are homogeneous, containing four

IIMIl and four !tR" units, respectively, whereas LDH-2, LDH-3, and

LDH-4 are combinations of the two basic subunits~

The techniques used for identifying the LDH isoenzymes have included the electrophoretic rate of migration in solid media and the relative heat ~bility. Electrophoretic demonstration of isoenzyme activity is accomplished by conversion of neotetra­ zolium to purple formazin by LDH production of DPNH.

Nomenclature is basea upon electrophoretic migration rates, with the form having the highest mobility toward~ the anode being designated as LDH-I. Thus, the hea.rt fraction is LDH-l and the slowest or liver and skeletal muscle fraction is LDH-5. LDH-l is more heat stable than the other isoenzymes. This property gives rise to a heat stab1e test for isoenzymes which affords separation of LDH activity into three categories:LDE-l; L:!2H-2,3,1+; and. LDH-5.

This activity is determined by measuring the declining optical density as DPNH is diminishing and pyruvic acid concomitantly is being reduced to lactic acid, or by the increasing optical density as the reaction is proceeding in the reverse manner. 42

Each tissue of the body elaborates its own uniaue pattern of

LDH isoenzymes. LDH-l appears to predominate in aerobically func­

tioning tissues and is more efficient there than is LDH-5; LDH~5 predominates in aerobically functioning tissues, where it is most efficient.

Heart muscle is characterized by a predominance of LDH-I, fol­

lowed in order by LDH-2, LDH-3, LDH-4 and LDH-5. Liver shows a

nredominance of LDH-5 followed to a much lesser extent by LDH-4,

IJDH- 3, LDH-2 and LDH-l. Sl

isoenzyme pattern. Kidney shows a co-dominance of LDH-l and LDY"'.z followed by LDH-3, I,DH-4 and LDB-5. Lung shows predominance of

LDH-3, and to a lesser extent LDH-2 and LDH-4 are seen. Pancreas

shows a predomin.ance of LDH- 3, followed closely by LDH-l and LDH-29

In g-ranulocytes LDH-5 predominates. Mature erythrocytes are rich­ est in LDH-l and LDH-2; nucleated erythrocytes are richest in

LDH-5. Prostate, prostatic fluid, bone marrow, thyroid, adrenal,

spleen, brain, cervix, skin and stomach isoenzyme patterns are also presented. Normal human serum appears to be richest in LDH-2, followed by LDH-l, I,DH- 3, LDH-4, and LDH-5.

~mny factors influence the measured serum activity of an enzyme--some of which are: rate of enzyme formation 2nd degradation within a cell; permeability of the cell membrane; rate of degrad­ ation in plasma or elimination in urine and bile; presence, absence and relative concentrations of inhibitors, activators, co-factors and substrate; and finally, the method of assay--all act together to yield the final measured enzyme activity_

The serum pattern of LDH isoenzymes following organ injury is usually characteristic of that organ. IiiLyocardial infarction produces an el.evation in serum LDH-l, which may be demonstrated within a few hours of the acute attack, and persists for 5-10 days after all total plasma enzyme activities have returned to normal. Elevations in serum LDH-5 which have sometimes also occurred have been ascribed to the presence of severe congestive heart failure. LDH isoenzymes can be used to differentiate between myocardial infarction and congestive heart failure~ acute pericarditis, paroxysmal atrial fibrillation with stenocardia, chronic angina pectoris, heart block with Stokes-Adams seizures, acute venous thrombosis of the lower extremity and pulmonary embolism.

In most cases of liver disease (including hepatitis, biliary and fatty nutritional cirrhosis, biliary obstruction, and hepatic metastasesJ the LDH abnormalitj most often encountered was ele­ vation of the predominant isoenzyme found in hepatic cells, LDE~5.

Pulmonary embolism produces inconsistent LDH-2,3,4 alter­ ations and the main use of LDH isoenzymes would be to exclude myocardial infarction.

Isoenzyme studies in anemia reveal LDH-l elevations in the presence of hemolysis, LDH-5 elevations in megaloblastic or hyperplastic marrow conditions, and no LDH-5 activity in aplastic marrow conditions.

In muscle disease studies of muscle extracts show only that a deficiency of LDH-5 constitutes a sign of some nonspecific abnormality in the neuromuscular system.

In neoplastic disease the bigh rates of glycolysis charac- 44

teristic of tunor cells with the concomitant shift to production of LDH-5 which is ~ore suited for pyruvate reduction accounts for

serum elevations of LDH-5 in neoplastic disease. These elevations have not been found until the tumor has reached a clinically detectable size. Therefore, at present the use of L.Dll isoenzyme

screening tests for early detection of neoplastic disease would not appear to be of value. LDll isoenzyme determinations may be useful in several aspects of neoplastic disease, however. Isoen­

zymes may differentiate malignant effu0~ons from benign.

Efficacy of anti-tumor therapy could be determined by monitoring isoenzyme changes. Early carcinoma of the prostate could be detected by testing prostatic fluid or needle biopsy speciment.

Oral contraceptives ap~ear to have no effect on LDB isoen­

zyme patterns, although the total LDH serum activity may be reduced by some preparations.

Fortunately, the applications of LDH isoenzyme determinations are not to the diagnostician vvhat penicillin and cortisone were once thought to be to the clinician. Diagnosis and treatment of ailments of ueop1e have not been reduced to a task capable of being carried out by an automated analyzer and a computer.

Rather, it is a physiCian who, after eliciting a history and examining the patient, selects a group of laboratory determinations

(which in certain instances this paper has tried to show would include LDll isoenzymes) which may help him in confirming or ruling out his clintcal impresstons; and then attempting to ex­ ulain such results on the basts of those clintcal impressions, never losing sight of the :fact that he ts treating a .:2.§-tient and not a laboratory result. That is the clinical aD~lication of LDE isoenzyme determination. 46

APPENDIX

,------LDH I SOEN 2YM": 1:'A'J'TERNS OF NOHlvl11.L HUMAN TIS,3UES ISOENZYME PEAK % 1'C"NL LDH ACTIv~l'Y IN EACH TISSUE REF'. LDH-1 LDH-2 LDH-3 LDH-4 LDH-5 HEART 73 24 3 - - 77 48 35 13 3 1 70 35 26 ;1.2 16 11 85 58 36 3 2 1 90 LIVER 3 8 25 23 41 77 1 3 7 12 77 70 2 4 11 27 58 85 1 3 7 - 74 90 SKELETAL - - 5 16 79 77 MUSCLE 4 7 21 27 41 85 14 16 14 7 43 90 KIDNEY 43 44 12 1 - 77 TABLE I 33 34 24 7 1 70 38 32 15 - - 90 Summary of serum I.UNG 14 34 35 5 12 77 and tissue LDH 5 13 32 30 20 70 isoenzyme pat­ 6 21 11 90 17 32, terns as reported PANCREAS 37 12 49 2 - 77 in the references 27 32 27 9 5 70 32 15 43 - 2 90 cited in the last column" GRUmr,O- 5 10 16 26 43 12 CYTES 4 15 22 14 Lf5 70 RBC'S & 43 44 12 1 - 77 HEl,WLY- 44 39 14 2 - 13 SATES 36 44 15 4 2 70 39 56 - 5 1 85 PROSTATE 12 33 35 18 2 14 3 21 48 24 4 70 PROSTATIC 8 23 24 25 20 14 FWID BONE 4 13 28 30 25 70 MARROW' THY':1.0ID 5 22 35 27 11 70

--,--"~-' ADRENAL 4 18 36 33 9 70

SPLEEN 5 14 29 32 20 : 70 9 27 33 20 5 90 BRAIN 25 29 31 14 1 70 CERVIX 8, 57 21 - 8 90 SKIN - 4 34 37 25 29 STONlACH FUNDIC 31 23 25 17 4 55 i MUC0SA PYLORIC 4 15 31 30 21 55 MUCOSA

SERUM 31 37 21 7 4 43 23 48 18 7 3 85 33 47 16 3 2 87 32 39 24 3 2 59 44 46 8 1 1 74 23 55 15 5 1 25 44 41 12 3 0 75 47

TABLE II

: 7~' ) LDH I SO E}; zniE :)IUT~AL VA~T!\.l'TON \ ,1J ,

HOUR ··TC)1'ii.L LDH I ;~()~N 2Yx/1ES LDH units 1 2 5 .240 27 38 20 8 7

1000 .312 28 38 19 7 8

1200 .288 26

1400 .360 25

• "".. _ ....,< •••• ~.~,_-.~,_."~, H. 1600 .432 22

1800 .528 20 31

2000 .528 19 31 23 10 17

2200 10 11

2400 10 9 0200 d36 8 9 0400 .336 40 18 8 8 0600 .250 38 21 7 8

Table II. LDH isoenzyme diurnal variations in a normal individual. (After Starkweather)(70). 48

TABLE III

TYPICAL SERUM LDH I SOEN ZYM}~ PA'r'11ERNS IN PATIENTS \VITH ACUTE MYOCARDIAL INJ<"ARCTIONS (59) -'"~''"'-''''''''''''''''''''~''.'''"'''''"''''''''''''' PATIENT SGOT ISOENZYMES

units %". 3 4 B.J. 940 3 Tr.

E.J. 250 7

E.S. 234 22

H. :N. 32 8 Tr. L.N. 147 37 416 42 9 Tr. L.J. 88 22 296 37 1 Tr. Tr. o. F. 226 30 ; 491 :55 36 9 Tr. M.R. 198 231 310 58 40 Tr. Tr. Tr. I 1 Tr. Tr. j' 5 . :16- 32 0-6 0-4 ~J~~~~tt-o-~~: t-;=;~i ::0 :i-:~9 ?~: i ~".'. _J Table III. Examples of serum LDH isoenzyme patterns of patients with acute myocardial infarctions.(After Reusch and Bunch)(59)o 49

. TABLE IV

r-,.."...... _._. __~""",","~."-,...."...."....."..""""",=,,",--,,,.,".~ .. ~~."'T" - I SERIAL CIH PG""S IN SERmil LDH Isr)E~;zn~ES ; .. ~AT IEN~~.JiI1'ILJlg.·c1'"l~" i!rX:Q_C_~~RP.I4b ..]NlfAE CT LQlL.J.9Q.L ~ !Day! LDH f % LDH Isoenzymes I GOT . . ;! r--T---'-;r---3-"-'Ij:--"-"~)""'- -1 . - ... """"..... -"'''''_,,___ ,~ 0 ,4~ ,"",,~."_ ~ -+, .... ,.~ ...... "~~~"t'."'-"hl" "," """., "" "".",... ~",..,.,., ,,,,~_.,,-,,,,,. ,u. c,.,. ",' ._..... __ ~ ...... ~.,-" ..... ~_.,_" ~.,"·--~"""~ .. ~""---~-....r""-"-.....,--...... --"'~,-~ 'fl~ Poster:-.',,,.or i,' 6 I 640! 48 27 10 4 4: 22' ~al1 . T , I , - l>u .... \ 8 \ 780 I:j 6 28 1 4 2 14 11 I 760 30 20 12 1] 0 25 15 ' 31 ')0 13 7 6 20 19 : 22 I} 15 q 2. 22 27 . 18 21 15 3 12 '14

Antero­ 33 33 9 0 2 26 septal 61 20 8 0 1 160 Iv~. I . 39 20 13 0 4 20 32 24 20 8 1 25 25 22 13 3 0 18

Antero­ 44 33 9 3 0 84 se'!:ltal 42 31 11 2 3 30 M.1. 26 29 17 7 8 25 14 22 q 5 5 25

Subendo­ 38 cardial 20 M.l. I I .j--~~.--.' Ta ble IV. Examples of serial IJDR isoenzyme changes in pa tients with acute myocardial infarction. (After 'Jro­ blewski and associates)(90). 50

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