Volume 62, Supplement

FEBS LETTERS

4 February 1976

CLINICAL ENZYMOLOGY E. SCHMIDT and F. W. SCHMIDT Div. o f Gastroenterology and Hepatology, Medical School, Hannover, FRG

History Clinical enzymology was born in 1908 when Wohlgemuth discovered the increase in alpha-amylase activity in serum and urine in acute pancreatitis [ 1]. Apparently, the time was ripe: the coincidence with Garrod's concept of the 'inborn errors of metabolism' (1909) is conspicuous [2]. However, diagnostic enzymology developed only slowly, despite the discovery of the increase in lipase activity concomitant with amylase activity in serum during acute pancreatitis by Rona in 1915 [3]. Other digestive enzymes were then proposed for diagnostic purposes, mainly in malignancies of the gastrointestinal tract, but they failed to meet the expectations of them. The next step occurred about 20 years later, when Robinson [4] and Kay [5] introduced the determination of alkaline phosphatases in serum for the diagnosis of bone and liver diseases. Again ten years later the clinical value of acid phosphatases in serum was first appreciated by the Gutman's in bone metastases from carcinoma of the prostate [6]. At that time the essential methods for the routine use of enzyme determinations in the serum existed, namely, the introduction of photometers into clinical laboratories by Heilmeyer [7], and the invention of the optical test by Warburg [8]. Further slow progress may have been partially due to the environmental circumstances which impeded investigation and interrupted the communication between scientists of different countries. Otherwise it is hardly conceivable that the significance of Warburg's and Christian's discovery in 1943 [9] of the elevation of glycolytic enzymes in the serum of tumor-bearing animals would have needed five years and more before it dawned upon clinical medicine. The systematic work of Sibley and Lehninger [10], first published in 1949, extended the data published by Warburg and his E 62

co-workers and stimulated investigations in many places. The def'mitive break-through took place simultaneously in the USA and in Italy with the publications on transaminases by Karmen, Wroblewski and La Due [11], and De Ritis and his co-workers [12], respectively. The obvious improvement in the diagnosis of myocardial infarction and acute viral hepatitis led to persistent endeavours in the investigation of a wide variety of enzymes and their diagnostic value. These in turn resulted in an exponentially growing literature on the subject, which reached rather unmanageable proportions in the late sixties. Out of the bulk of enzymes then on trial, no more than a good dozen emerged as choice diagnostic aids. Some of the enzymes listed in table 1 are now measured in virtually all hospitals and in many medical offices throughout the world. With others, however, there are considerable regional differences of usage: ICDH [13] for example, seems to be an enzyme of Anglo-Saxon interest, OCT [14] has hardly passed over the borders of the Scandinavian countries and GLDH [15] is virtually confined to laboratories in Austria and Germany. On the whole, enzyme determinations in serum have attained an assured place in clinical practice, and much empirical knowledge on their significance has accumulated.

Pathophysiology In contrast to the practical significance of enzyme levels in the plasma, the present knowledge of how they come about is still relatively fragmentary. A given enzyme activity in the plasma can be considered as the result of the rates of inflow, distribution and North -Holland Publishing Company - Amsterdam

Volume 62, Supplement

FEBS LETTERS

4 February 1976

Table 1 Name

EC Number

Abbreviation

Acid phosphatase Alanine aminotransferase Aldolase Alkaline phosphatase a-Amylase Aspartate aminotransferase Cholinesterase Creatine kinase Glutamate dehydrogenase (NAD(P)÷) "r-Glutamyl transferase Isocitrate dehydrogenase (NADP÷) Lactate dehydrogenase L-Leucin arylamidase 5-Nueleotidase Ornithin carbamoyltransferase Triacylglycerol lipase

3.1.3.2 2.6.1.2 4.1.2.13 3.1.3.1 3.2.1.1 2.6.1.1 3.1.1.8 2.7.3.2 1.4.1.3 2.3.2.2 I. 1.1.42 1.1.1.27 3.4.1.-

SP GPT ALD AP Amylase GOT CHE CPK GLDH 3,-GT ICDH LDH LAP

outflow, as outlined in the simplified diagram on the fate o f cellular enzymes in the extracellular space (fig.l). T h e starting-point in this process is the concentration o f cellular enzymes within their cells of origin, which vary to a considerable extent, according to the type of cells and their metabolic functions [ 1 6 - 1 8 ] . The cellular enzyme patterns, well known for the majority of tissues, are fairly constant in normal

Liver Cells Synthesis enhanced Degradation reduced Activity increased

Normallp

Q

5-N

2.1.3,3 3.1.1.3

OCT Lipase

circumstances, though affected even physiologically by developmental, nutritional, hormonal and other factors [ 19,20]. Under pathological conditions, additional variations occur by disturbances and reactive changes in the intracellular protein turnover [21]. The enzyme pattern in the serum depends primarily on the concentration gradient between the intra- and extracellular space [22]. The concentration gradients of the enzymes

ExtracelluiarS_~ Intravascular Space

/

, Lymph Ve sseis

)ifflux~

Activity inhibited DegradatiOn enhanced Synthesis reduced

3.1.3.5

k

Interstitial Space

~Bile

"Sofort-Effekt" Phase of Distribution

Tissues ~--)Spleen Liver Endothelial Cells Blood Cells

~-~ Other Tissues

) Gut Phase of Elimination Enzyme Degradation

Fig. 1. Scheme of enzyme release, distribution and elimination. E 63

Volume 62, Supplement

FEBS LETTERS

between the cells and their environment are so steep that their maintenance requires a considerable portion of the energy-yield of the cell. The complex structure of cellular membranes, serving the opposing functions of the preservation of cellular integrity on the one hand, and of the continuous orderly exchange of a variety of substances on the other, are therefore very vulnerable. It is well-known that the release of potassium and other low molecular weight cell constituents occurs at an early stage of cellular injury. It came as a surprise, however, that simultaneously high molecular weight cellular enzymes likewise begin to leak out. The first investigators of enzyme leakage, e.g. Warburg [23] or Zierler [24] showed this clearly by their experiments with cell suspensions and isolated muscle. Despite this evidence a tedious discussion lasted Enzyme

Activities

4 February 1976

for years concerning whether enzyme elevations in the serum must be considered as always being indicative of cellular necrosis. Just the contrary could be learned from clinical experience, e.g. in acute hepatitis or in right heart failure. Moreover, one glance at the pathological enzyme patterns in the serum reveals that they very seldom reflect exactly the pattern of the afflicted tissue, but are distorted by various factors [22,25-29]. Some of these factors have been elucidated, mainly by studies on the isolated perfused liver. This model offers several advantages; the anatomical integrity of the organ is maintained, and many functional properties are known to persist for an adequate time; and the experimental conditions can be controlled and varied without disturbance form the reactions of the whole body. The significance of the intraceUular

in t h e M e d i u m

Reference

Values

~ Mollmin. x l O O g Liver

LDH I 37"c

I

GLDH

25. 1

/ ~

TO2 - C onsumption j 37.C J3013sLactate / Pyru vale ( MedJu m )

3TC

23 iS

U/L

60

120

180

23

240 Min

5 U/L

GOT

175

-

60

120

180

f

24OMin

°o

I. ~'*IIL

GPT

140 37"1

150

7"

,,o

,.o.4oM,

o/

Pyruvate CMedlurn~

O.S

I00

100

80

75

60

S

40

oo

,,o

,o0,.oM,°

Lactate ( Medium )

6/

IO

120

125

Is mMoI/L'

8

0.3

,/~

23C

r~//~

37"C

4

25

3

2

20

~ 23"C

23"C S

60

120

180

240 MIn

5

60

120

180

240 MIn

S

60

120

180

240 Min

Fig.2. Effect of temperature. Fed rats: flow-constant perfusion; (o), 23°C (n -- 10); (e) 37°C (n = 2). E 64

S

60

120

180 240 MIn

Volume 62, Supplement Enzyme

FEBS LETTERS

Activities

UlL

in t h e M e d i u m p Mollmln • lO0g Liver

UIL LDH

/

~

Ar

4 February 1976

4~[ n 14

Reference Values mllhour

O2-Consumptlon

1.4

250

1.2

/

200

Ar 0.8

150

02

Ar

0.6

IOO

50

5

60

120

180

240 M i n

60

) MIn

120

180

240 MIn

UJL GOT

75

3S

Lactate I Pyruvate (Medlurn~

Ar

3O 25

5C

2O O2

1S 25 10 S

60

120

180

240 MIn

0 MIn

GO

120

180

Ar - 02 240 Min

Fig.3. Effect of anoxia during perfusion. Fed rats: 23°C; (o), 4 h O~ (n = 10); (o-A), 1 h O2 - 1 h Ar - 2 h 02 (n = 4). localisation of enzymes could be demonstrated: the leakage of mitochondrial enzymes and isoenzymes is delayed and is much less than that of the cytosolic ones [26,30-33]. It ceases again with about one third of these enzymes still in place, when the disintegration of the dead cells begins, whereas the efflux of cytosolic enzymes goes on up to equilibrium with the environment [34]. Comparison with morphology shows that the release of these mitochondrial proteins is in fact a sympton of necrobiosis. Thus the relation of cytosolic and mitochondrial enzymes in the serum can be used diagnostically for the estimation of the severity of single cell damage, independent of the absolute enzyme levels which are a measure of the number of ceils involved and thus of the size of the damaged area of tissue [22,35]. The molecular weight, which also affects the release rate of enzymes,

is of minor diagnostic importance; only insofar as for a given pathological state the release rate of small enzymes is higher than that of the large molecules. So the former are more sensitive indicators of cellular injury in general than the latter [30]. The various causes of the 'leakiness' of the cells result in three major deficiencies: (1) Disproportion between requirement and supply of oxygen. (2) Disproportion between requiremen t and supply of metabolizable substrates. (3) Incapability of the cell to meet its requirements of energy by utilizing the supply of oxygen and substrates. Fig.2 demonstrates how relative low oxygenation due to an increased requirement with elevation of temperature leads to an augmented release of enzymes from E 65

Volume 62, Supplement

FEBS LETTERS

4 February 1976

Table 2 Effect of the nutritionel state on the isolated rat liver after 4 h o u r s of hemoglobin-free perfusion at 23°C

Glycogen (mg/gliver) Lactate/pyruvate (liver) ATP/ADP (liver) Oxygen c o n s u m p t i o n (~umol/min × 100 g liver) Glucose (perfusion m e d i u m ) (mmol/l)

Enzyme

Fed rats (n = 10)

Fasted rats (n = 6)

44.7 ± 2.7 4.2 ± 0.8 4.06 ± 1.09

0.41 ± 0.18 4.3 ± 0.5 4.03 ± 1.15

0.84 +- 0.23

0.89 ± 0.27

6.6

5.9

-+ 0.6

Activities in the Medium

± 0.28

Reference

Values

Mol/min xlOO Liver

LDH

I

GLDH

Lactate / Pyruvate(Medium SO

7

st

~

10

fed 10

fed

60

120

180

240 Min

U~L

$

~0

120

180

240 MIn

60

120

180 240 Min

UIL GOT

GPT

50

~

mlllg

40

Gly cogen ( L iver) rnM ~1/I

+

starved starved 40

T

30

~

~

30

Glucose (Medium)

10

so 40

r

l

-

30

),/

s~lr~

20

fed 10

I0

fed

5

60

120

180

240 M I n

S

60

120

180

240 Min

10 fed

b

I

starved •

a

t

60

Fig.4. Effect of the nutritional state. Flow-constant: 23°C; (o), fed rats (n = 10); (e), > 15 starved rats (n = 6). E 66

120

180

240 Min

Volume 62, Supplement

FEBS LETTERS

ATP/ADP ratios in the liver [36] (table 2). The histological appearance of the liver tissue is likewise virtually normal. These results among many others show that the release of cellular enzymes is one of the most sensitive parameters for the recognition of minute disturbances of cellular integrity. When increased permeability has been produced previously by a variety of causes, enzyme leakage from the perfused liver was considerably aggravated (fig.5) [36]. In comparing the different types of injury not only quantitative variations can be noticed, but there are also conspicuous qualitative differences of the enzyme patterns in the perfusion medium. These can be attributed on the one hand to different involvement of subcellular structures with the different toxic substances, thus proving again the importance of the intracellular localization of enzymes (fig.6) [36]. On the other hand, the heterogeneity of

the perfused liver. This occurs immediately with mitochondrial (and acinocentral) GLDH, later with partly mitochondrial GOT, and even more delayed with cytosolic GPT and LDH. Though oxygen consumption is twice as high at 37°C as it is at 23°C, the relative oxygen shortage can clearly be read from the continually increasing lactate/pyruvate ratio [36]. Fig.3 shows that temporary absolute lack of oxygen also leads to a steep rise in enzyme activities in the perfusion medium, which persists (with the exception of GLDH) after the restitution of oxygen supply. The significance of the nutritional state of the cells is shown in fig.4. The release rates from the livers of fasted rats are between 2.5-fold and 5-fold higher than from fed rats, though oxygen consumption is similar in both conditions, and extracellular glucose levels are very well maintained by the glycogen-depleted livers. The same is true for the lactate/pyruvate and the Effect of Toxic

Substances

(lmMol/I)

oa

E f f e c t of p r e v i o u s

hst~cl rats 17°C. x:AT Albumlne-Tyrade-S. (n*11]l IAA Monelodolcetete( nl 3);~:KCN' (n=4~; ~: DNp. p-Dinltrold~nol (am-4)~:~)CN,Na-OeNxychelet uIL __°""'*° ~N LDH /[ / ' ~ OLD, 2000(

(n--4~

Injury

to t h e

Liver

Fasted rlts; 37"¢ ; o N o r m a l (n=l) • c h r . T A A (n, 3) o Ic.TAA(n,3)° °c.CC| 4 (n=~l

T

/~&/

4 February 1976

to. ? / ~

0 IAA

[

T

OL0.

ec.CCI 4

15~

~

lO~

1:

o

/"

•

DCH DNP

6

l T ac. IAA

KCN

T

~chr. TAA Normal

"t AT

60

120

180

GOT

240

MIn

T

5 UJL

IAA

60

120

180

240

M|n

5

60

120

180

"/'

GPT

T DNP

~

750

240 Min J

T

IOOG

GOT

~/~

60

120

180

240 MIn

"/'

ac'CCI4 400

GPT

1/,

K H 600 "r

o

IAA

200 40C

/ "

~/:

!~

// ; 60

120

180

240

Mln

$

60

120

180 240

KCN MIn

i

- ....

60

120

180

240 MIn

~S

60

120

180

240 Min

Fig.5. Enzyme activities in the medium. E 67

Volume 62, Supplement Enzyme and

Isoenzyme

FEBS LETTERS Ratios

In t h e

DCH=lmMol/I Na-Desoxycholat (n=4) LD%LDH

4 February 1976 Enzyme Ratios In the Medium

Medium

KCN: 1 mMol// (n=4 I

KCN: lmMol/I (n =4) IAA: lmMol/I McmLoiodo. acetate (n=3)

C'GOT/m_GO T ',']!0

ICDH/G PT

OC.H

2.5

KCN

1600

DCH

I RO0

1200

16 ~TAA 8

acute

~oxJo

OSMg~ C¢l.__d

1.5 acute

AA

I

TAA ,rot Ic

rA~ ;tarvation

4

nt

a

O.S

F*1

Fig.6. 'Membrane lesion' vs. 'necrobiosis'.

Fig.7. Central vs. peripheral or diffuse damage.

enzyme distribution throughout the hepatic lobule affects the release rates in proportion to the cell damage in the periphery or in the central area of the lobule (fig.7). This also shows the different patterns with lack of oxygen or glycogen depletion [36]. Both connections can be seen either histologically or deduced from the sites of action of the injurious agents. Although at present the mechanisms of enzyme release are still unknown, the complexity of conditions and processes involved is beyond doubt. Subtle diagnostic enzymology presupposes some insight into this subject. After being released, cellular enzymes enter body fluids. Viewed from this aspect, the releasing cells can be divided into five groups: cells with direct contact to the intravascular space (e.g. blood cells), cells with direct contact to the intravascular and the interstitial space (e.g. spleen cells), cells with direct contact to the interstitial space only (e.g. muscle cells), cells with direct contact to the interstitial and the transcellular space (e.g. renal tubular ceils) and finally, cells with direct contact to all three compartments (e.g. liver cells, which therefore have been chosen as an example in fig.l). Those cellular enzymes which are released into the transcellular space (urine, bile, intestinal contents, etc.) emerging from the cells on the inner and outer sur-

faces of the body, can be left out from further consideration because they are lost as regards measurement in serum. For the remaining portion, the initial course is different but finally they become distributed all over the extracellular space. This distribution occurs slightly faster from the intravascular to the interstitial space, than in the reverse direction via the lymphatic vessels. Nevertheless, in both ways distribution is rapid. It accounts for the first rapid phase of the well-known biphasic disappearance of injected enzymes (fig.8) [37,38,52,56]. The investigation of

E 68

~ 1.o-~

t)og no.8

Dog no. 27

Wakim u. Fleisher 3 LGb.Ch'n.Med.51,85 , (1963)

o,8 a6.

\ .\

I

~ 0,#.

/I/

/o ~"o-~.o~ ~o"..Q ~.o

~ 0,2- q,

t4~ fIII

F/ o . . o ~

iI

~'0.~.

Hours

Fig.& Fate o f enzymes in body fluids. (-- e) Activity in serum. (o-o) Activity in lymph.

Volume 62, Supplement

FEBS LE'I~ERS

enzyme elimination from the circulation began early with the application of tissue extracts and more or less purified enzymes [38,41]. Here the fundamental work of Fleisher and Wakim [37,42-44,56] must be mentioned. They studied the rates of disappearance of transaminases and showed the great difference between GPT and GOT, and between the cytosolic and the mitochondrial isoenzyme of the latter. Additionally they tried to find out about the site of the elimination of circulating enzymes by a series of considered experiments. However, the question of whether enzymes gradually loose their activity, thus escaping detection and only simulating disappearance, or whether their molecules are in fact removed from the circulation, remained open. The problem could be solved by the use of radioactively labelled enzymes which would remain traceable after inactivation and even after resumption into cells or excretion. With regard to in rive experiments labelling of e-amino groups with [14C]acetate [45,46] was found to be more suitable than labelling by 131iodine [47-51]. Not only is the yield of catalytically active enzyme after labelling many times better, but, aside from the expected minor alteration of the electrophoretic mobility, kinetic and molecular properties remain unaltered; further, the stability is excellent in vitro as well as in vivo. The latter, especially, is an indispensable quality in injection experiments [30]. Many of the conclusions of Fleisher and Wakim and others who worked with unlabelled enzymes could be confirmed. In addition, proof was furnished that enzymes are eliminated in a catalytically active form; after distribution, the catalytic activity and the radioactivity of an injected enzyme decrease in parallel. This is true in serum as well as in lymph, and it demonstrates that elimination takes place from both the intravascular and the interstitial space (fig.9) [521. The half-lives of cellular enzymes in the circulation seem to have molecule-specific and species-specific properties. This was shown in animals as well as in peracutely ill patients. The half-life of an individual enzyme is also independent of sex, age, most disease states and its actual level in the plasma [53]. Recently, however, evidence has been reported that very high doses of some drugs may have an effect on the halflife [54].

4 February 1976 mU/mt dpm/ml

or

10000~

r,O.O6

serum

100~ ÷k~.0.29~3

~-~+

r.~Ts- ~

÷

radsoact.

...... in

÷lymph

/

/

100(

) .~, "-. r

P"'e " L " ~

- ~ , ~ , , , , w , , , . ~ e r ~ y m e act. .a....e~ • •- ~ in

kz.0.2777

r,ag5

tOO. 20

,~0

60

#0

fO0

120

~0

t6Omin

Fig.9. Radioactivity and enzyme activity in serum and lymph in the dog (Hs4) after i.v. injection of [~4C]LDHs.

There is no correlation between half-life and molecular weight, as can be read from the very different disappearance rates of the LDH-isoenzymes which have virtually the same molecular weight (fig.10) [54,55]. However, the half-lives may depend, at least partially, on the charge of the enzyme molecules. Substitution of the side chains of LDH-5 with polar groups results in a shorter half-life, whereas substitution with non-polar groups leaves it unaltered or retards elimination. (riga 1) [54,55]. The site of t'mal excretion or elimination of circulating enzymes has been under investigation and discussion for more than 20 years. Former studies have shown, that neither the removal of liver or spleen nor of the kidneys or the intestinal tract affects the elimination rates [38-40,42,56,57]. It was then believed that enzyme elimination might be a function of the reticule-endothelial system on the basis of indirect evidence before the era of labelled enzymes [37,42,44,58]. With 14C-acetylated LDH it was found that not the reticule-endothelial system but the parenchymal cells of most tissues are able to incorporate circulating enzyme molecules. The rate of incorporation was shown to be higher in organs with a rapid protein turnover than in organs with a less active protein metabolism [52]. E 69

Volume'62, Supplement

FEBS LETTERS

4 February 1976 10. Jll

10

Jll

LDH3

L DH,~

H WZ: 92.19~ 7,66

HWZ." 50,10"12,30 n=3 160 m i n

2"0 "

•

n=3

40 " gO "

/~3

£0"

2b

,OOmin

"

6VO " lOOmin

(~0

lo. IIZL_

10

5.

LDHI

LDH 2 HWZ : 2A?,19-*7,,48 n=3 "

2"o

"

~o"

HWZ: 315,30"-39,34

n=5

•

dO " 8"0 " l(}Omin

"

6'0 '

dO " lbOmin

Fig.10. Half-life of LDH isoenzymes in the circulation o f the rat.

~0, U/I

10. u/I

5,

~

•

CH2cH2(co)20 "~"~'~ HWZ"I?'g5+1'27

,

20

1~ 10_U/l

.

.

.

.

40

,

60

•

~0

5

.

.

F E 70

60

dO

20 l OI~~l

I "~ CH'CH'(CO)~19 54 "~, HWZ..2s,7=7±Z54

40

~ "~dc.:~c.2c.~c%.o

utyric acid anhydride

~,N,~ (c"jc"2c°J2° ~,~'~HWZ: Id,=27tO,76

I00 rain

Mnleic acid anhydride

20

tO~U/ l Proplonic acid anhydride

lx Succinic acld anhydride

40

60

5J I

•

80

.

100 m i n

I ~ l k (CH3C0)20 5.1 , ~ w z :2zo5"-3,~

20

40

60

80

,

.

20

.

40

.

.

.

60

.

.

.

O0

100 rain

10~-Valeric acid anhydride

Acetic acid anhydride

100min

HWZ: 20,~3t0,76

~

t 5

100 rain

20

40

60

dO

100

rain

Effect of acylation of the e-amino groups by different c o m p o u n d s on the half-life of LDHM4 in the circulation o f the rat.

i

g

.

1

1

.

Volume 62, Supplement

FEBS LETTERS

4 February 1976

-I!!! [ °,,,=

-I!!![=°,,, '2°°°1~I

IIII 111

f l ~O-/~TIWTYIN

± t

LLe~°°°'~T~'e°u'N

'°°°121LI I ILI I ILI El I'11111"1,1~ II El I'1LI [11~ ~ o • =H~

- = H~

I[~ ,~,,~

I--ICONTENT OF THE~STROINI'ESTINAI.tRaCT

Fig.l 2. Concentration of radioactivity in dog organs 160 min after i.v. injection of [t~C]LDH~ (dpm/g organ). Fig. 12 shows the concentration of radioactivity in various tissues after injection of labelled LDH-5 as compared to its concentration in the blood and to the average body contents. The sequence of contributory organs is different when the size of the organ is accounted for and the respective radioactivities are expressed a percentage of the original dose applied (fig. 13). It came as a surprise that the cells of the reticule-endothelial and the lymphatic system, e.g. the Kupffer cells in the liver or the lymphocytes are

not accentuated in autoradiography, but the parenchyma of the organs and among blood cells the granular leucocytes only are accentuated [52]. A considerable portion of the injected radioactivity, but virtually no enzyme activity is found in urine and bile within the first 8 hours after injection of LDH. It can be concluded from experiments in dogs and rats that these findings in urine and bile are attributable to non-dialyzable split-products of the enzyme molecule after partial degradation in the littoral parenchymal cells of urinary and biliary tract, which exhibit a considerable accumulation of % I 2

~

HALFLIFE LDH-I I13"-60 ~

G

P

T

~7 "-10

H

~, ~ ~ 5

CPK

I~

y

15

6

lid Fig.13. Radioactivity distribution after 160 rain in organs and body fluids in dogs; expressed in percent of injected dose (Hs2 and H~).

7 8

-t LDH-5

0.'5

10 *- 2

J

Thour

Fig. 14. Enzyme elimination in human: plasma disappearance rates,

E71

Volume 62, Supplement

FEBS LETTERS

only the alterations o f inflow. Diagnostic enzymology in general is founded on this assumption. Considering enzyme patterns in the serum, the different half-lives o f the respective enzymes contribute a great deal to the distortion o f the original tissue pattern. For the interpretation o f enzyme patterns under diagnostic aspects the analysis o f the distortion is just as weighty as e.g. the notion o f the tissue patterns, the normal values in the serum, or the reliability of the methods. The use, which can be made o f the understanding of the prerequisites and processes forming enzyme patterns in the serum is outlined and exemplified in fig.15 [35].

radioactivity in autoradiographic pictures [52]. Even low molecular weight enzymes, e.g. amylase, are eliminated from the circulation b y the kidneys into the urine in the catalytically active form only in small proportions. Most enzyme activities in bile and urine, originate from the littoral cells themselves and not from the plasma [ 3 8 , 3 9 , 5 9 - 6 1 ] . The ideas about enzyme elimination are still fragmentary [62]. Nevertheless, the well-deffmed different half-lives of enzymes in the plasma are o f major diagnostic siRnificance (fig.14) [53]. Considering an individual enzyme, the constancy of its disappearance rate allows one to assume that modifications o f its actual level in the plasma reflect

Example UII 800 600 400 2OO Imnmm_

o_

H OPK U/l" Acinoperipheral

Acinocentral ~ liver cell ~ I~, I damage M

liver -cell ~3000 2o00 damage

4000

lU ._._tu L_LDH,__G_P.T_,.,t_c_I:~ A.DH . l_.

Acut~ vira.i he 9at

U/I

D _I.CDH_A__D...H. .

GPT

.

.

.

4 February 1976

.

.

Basis

Application in serum

Enzyme patterns of tissues

Recognition of the organ of origin by organ-specific ratios of enzymes

Distribution of enzymes in the tissues

Recognition of the localization of cell damage by the ratios of enzymes with different distribution in the tissue

IntraceUular localization of enzymes

Recognition of the severity of single cell damage by the ratios between cytosolic and mitochondrial enzymes

Alteration of the enzyme patterns of the organs with protracted cellular damage

Recognition of the stage of a chronic disease by the ratios of enzymes with altered activity to enzymes with unaltered activity in the tissue

.

Acute liver

p000

1500

I

500 ~ ; L_...GO_! G' .

_

Liver cirrhosis

Chronic U/li hepatitis'

~00i 25

GOT j

E 72

GPT GLDH LDH :2

GOT

GPT

GLDH LDH :2

Volume 62, Supplement

FEBS LETTERS

4 February 1976

U/i! Acute viral.•............... Chronic hepatitis hepatitis [

2000]

5O0 ' GOT C4:'T GLDH LDH

t .............................................

u/t;

. . . . . . . . .

1600!

~

a°°il

Half life of enzymes in the extracellular space

Recognition of the stage of an acute illness by the ratios of short-living to long-living enzymes

Reactively enhanced synthesis °f mem" brahe-bound "eholestasis-indicating . . . . enzymes concomitant with disturbed cell permeability

Recognition of the degree of ch°lestasis and °f reactive changes by the ratios between cholestasis-indicating" enzymes and other cell enzymes

Impairment of the synthesis of secreted enzymes

Recognition of the reduction of the functional liver mass by the decrease of plasma-specific enzymes

GOT GPT GLDH LDH

Viral hepatitis 1 week 3 week

!,:i!

! (:11

4-

Anicteric viral hepatitis

~

~

OO

Recognition of the extent and the acuteness of cell damage in a distinct organ by the height of enzymes

[ .......................................

['i:,:t ~ [J[ [ " 11 400 _ .. ~ ..... r7 ] . - . . . . ~ GLDH LDH I...................................... GOT GPT GLDH LOH [i GOT GPT ................................ Oil

Release rates of enzymes

U i

[. . .GPT . . . . . . .GLDH . . . . . . . . ),-GT . . . . . . . . . .AP ... Reactive U/l 100 hepatitis

25

Early occlusion of bile duct~.-.

t iF']

~ ~] ! ~',/I f ii~']

~

l.~.~]

[. . .GPT . . . . . . .GLDH . . . . . . . . 7-GT . . . . . . . . . AP ....

Liver cirrhosis

_~

.....

Fig. 15. Application of pathophysiology to enzyme diagnostics in serum.

Diagnostic

application

Any diagnostic method is expected to indicate, reliably, sensitively and specifically, deviations from the normal state at a negligible risk to the patient and demanding as little as possible of him. These demands are largely met by enzyme determinations in the serum. The reliability, sensitivity and specificity of the respective methods are fair to excellent, and the patients are only minimally inconvenienced. From the diagnostic point of view enzyme leakage is in itself a very sensitive sign of disorder. The range of

pathological changes compatible with life is far greater than with other clinico-chemical parameters. A hundred times normal levels are not uncommon and even the thousand-fold has been found (fig. 16). Thus, possible gradations between the extremes and the normal are unusually free, giving much facility to differentiation. The diagnostic specificity of the alterations of enzyme activities in the serum is, however, much less, at least at first sight. Among the enzymes used in practice, there are very few organ specific ones and no disease-specific ones at all. E 73

Volume 62, Supplement

FEBS LETTERS GOT

q-Globulins

U/I Acute severe intoxicatio

4 February 1976

g/l

I

5000 Right heart failure

Acute viral hepatitis 100

Acute toxic hepatitis

500

Acute exacerbation in chronic hepatitis Early obstructive

jaundice Chronic aggressive hepatitis Active cirrhosis

I00

Hepatic metastases Chronic alcohol-

toxic hepatitis Chronic persistent hepatitis

50

Fatty liver

reactive hepatitis Slight fatty infiltration

E Chronic active i ~ hepatitis ~ Active cirrhosis | ~ Acute hepatitis ~ Hepatic metastases ~ Chronic persistent heDatitls Normal range

Normal range

I0

!

I

5

I Fig.l 6. Degree of elevation of cellular enzymes in serum as compared to that of other plasma constituents, exemplified by GOT activity vs. 7-globulin concentration in fiver disease. Table 3 The diagnostic specificity can be greatly enhanced yet by benefiting from the notions of physiology and pathophysiology of enzymes at hand [ 1 2 , 2 2 , 6 3 - 6 5 ] . From the enzyme patterns of tissues the 'leading enzymes' for the recognition of the affected organ can be derived [65]. As can be read from table 3 scarcely one single enzyme is sufficient, but in many cases advantage must be taken from the different E 74

'Leading enzymes' for organ diagnosis Liver and biliary system Heart Skeletal muscle Bone Blood Pancreas Prostate

GPT, GLDH, 7-GT, CHE, (AP) CPK, GOT, LDH (Isoenzyme 1) CPK, ALD, (GOT) AP, SP LDH Amylase, Lipase SP (tartrate inhibitable)

Volume 62, Supplement

FEBS LETTERS

4 February 1976

GOT:

~ 50 U/I organ?

GPT:

~ 20 U./I organ?

~ 100 U/I liver/biliary tract

~ 1500 U/I sketeletal muscle

CI~K:

~ 15 U/1 organ?

~ 250 U/I heart

LDH:

~ 150 U/1 organ?

~ 2509 U!l blood cells

AP:

~ 120 U/I organ?

~ 500 U/I bones? liver/biliary tract?

7-GT

~ 75 U/I liver

~ 15 U/1 bones

~ 750 U/1 liver/biliary tract

Fig.17. Enzyme ratios in serum for the diagnosis of the injured organ.

ratios of more or less unspecific or only group-specific enzymes and isoenzymes in the various organ systems. Fig. 17 gives an example how to reach organ differentiation, proceeding from the assumption that the activity of GOT in the serum is only about three times elevated, which is a rather ambiguous value. In reality, organ diagnosis is by no means the main object of enzyme determinations in the serum; it cannot be, because of the limitations of their applicability. They are rather useless in diseases of those organs, which are not listed on table 3, e.g. diseases of the kidneys, the respiratory tract, the endocrine glands, etc. [ 6 6 - 7 2 ] . In the diseases of these organ systems enzyme alterations in the serum also occur sometimes. But only the secondary affliction of organs, which are accessible for diagnostic enzymology in general, are thus manifested. This is especially true in malignancies: they exhibit characteristic enzyme patterns in the serum only in the case of metastases in the liver or the skeletal system. Even carcinoma of the prostate cannot be reliably detected by the estimation of the prostatic isoenzyme of acid phosphatase before the tumor has penetrated the capsule of the gland [73]. This is, however, partly due to unsatisfactory assay methods.

As for other small organs, e.g. heart or pancreas, only acute cellular damage leads to distinct elevations of enzymes in the serum. This confines the application of enzyme determinations virtually to myocardial infarction on the one hand and acute pancreatitis or the acute relapses of chronic pancreatitis on the other. Among the disorders of the hemopoetic system, it is primarily untreated pernicious anemia with its extremely high LDH activity, which is a rewarding object of enzyme diagnosis in the serum. With the diseases of skeletal muscles and bones elevated enzyme activities may arouse suspicion of an illness or can serve to find carriers of hereditary disorders, but they don't contribute much to differential diagnosis [66-72]. On the other hand, in nearly all diseases mentioned, periodical enzyme measurements for the follow-up of the course, for the evaluation of desirable and undesirable effects of treatment, peculiarly of drugs, and for the early detection of complications involving other organs, can be carried out easily and advantageously [74]. Up to now, diagnostic enzymology flourishes most in hepatology. Here, several favorable prerequisites coincide: the size of the organ, the absence of a E75

Volume 62, Supplement

FEBS LETTERS

basal membrane around the hepatic sinusoids, and the multifarious enzyme equipment of the parenchymal cells. The former two qualities account for the sensitivity by which even minute hepatocellular damage is indicated by the elevation of cellular enzymes of hepatic origin in the serum. Thus enzyme determinations are most suitable as screening tests for hitherto subclinical hepatic disorders, such as chronic inflammations and toxic injuries, be that alcohol or industrial poisons or drugs with hepatotoxic sideeffects. Fig. 18 demonstrates the high incidence of abnormal findings with the four 'screening' enzymes in chronic inflammatory diseases of the liver [72,75]. The rich enzyme equipment of the liver permits differential diagnosis to be advanced into considerable details. That is why nearly all the examples in fig. 15 are taken from liver pathology. An adequate set of enzymes for differential diagnosis of the disorders of the liver and the biliary tract contains some, whose extracellular increase indicates disturbances of cellular permeability, (e.g. GOT, GPT, GLDH, LDH,) others, whose elevations in the serum indicates not only raised permeability, but enhanced synthesis of these membrane-bound enzymes [76-82]. This cellular reaction is frequently provoked by or combined with cholestasis, which can be evaluated by a concomitant rise of, for example, 7-GT and AP even in the nonjaundiced patient. The third group of enzymes used in hepatology comprises CHE and clotting factors Chronic hepatitis (all types) n = 154 GOT GPT 3,-GT Normal activity: \5% 8%/ 8% \

CHE 35%

V

/

2%

V

0.6%

Cirrhosis (all types and stages) n = 131 GOT GPT "r-GT Normal activity: \6% 28%/ 12% \

CHE 11%

V

4% .

V

0.7%

Fig.18. Sensitivity of a small enzyme pattern with chronic liver diseases. E76

4 February 1976

which are synthesized in the liver and secreted into the plasma. The decrease of these plasma-specific enzymes indicates reduction of the synthesizing and secreting capacity or of the number of liver cells or of both [64,68,70,72,74]. By enzyme determinations in the serum in liver disease various functions can thus be examined and information as to the extent, the severity, the stage and the type of cellular damage can be derived by analysing the distortion of the enzyme pattern in the serum (see fig.15) [35,83]. Clinical experience and comparison with other, especially morphological findings have shown characteristic enzyme patterns to belong to distinct liver diseases, their successive stages and their different etiology. Fig. 19 gives an example for the differentialdiagnostic procedure starting again with the ambiguous GOT activity of about 50 U/1. Although the given numbers are schematized and deviation may be considerable in individual cases, the diagnostic accuracy is as high as 80-90%. This is not worse than with other more taxing and more risky diagnostic methods.

Present problems and o u t l o o k

After the rapid progress of clinical enzymology during the '50s and '60s, recently, a feeling of saturation and even symptoms of fatigue have been noticed in this intermediate field between applied biochemistry and medicine. Its achievements can be considered to be fairly satisfactory only with hepatology. A predominant concern of today's clinical enzymologists is the improvement of routine and reference assay methods and their standardization on national and international levels. A second field of 15resent investigation deals likewise with methodology, but rather with its fundamentals. The principle of determination of enzymes by their respective catalytic activities, so dependent upon the conditions of assay - though generally specific and sensitive enough - might be replaced in future by utilizing the antigenic qualities of the enzyme proteins for measurement. At present, assays of this type are in use either for isoenzyme differentiation or for enzymatic analysis of other compounds,

Volume 62, Supplement

FEBS LETTERS GOT:

~ 50 U/1

GPT:

~ 150 U/I

4 February 1976

GOT/GPT ~ 0.3

Lb.

-150

00 u/1

subsiding acute hepatitis?

infectious mononucleosis

obstructive jaundice? drug icterus?

AP:

~ 150 U/I viral hepatitis?

~ 500 U/I obstructive jaundice?

alcohol-toxic hepatitis?

"y-GT:

~ 100 U/1 viral hepatitis

drug icterus?

~ 300 U[1 alcoholtoxic hepatitis

GLDH:

~ 5 U/I ~ 50 U]I drug icterus obstructive jaundice

Fig.19. Enzyme ratios in serum for the diagnosis of the type of liver injury.

e.g. hormones. Probably, enzyme immunoassays in a restricted sense will facilitate or even make possible to enlarge the diagnostic set by such enzymes, which would give significant information in disease states, but whose activity determinations are too tricky for routine. The search for further enzymes which might complement the enzyme patterns used hitherto - tiny in view of the real number of enzymes - constitutes a third area of work. Under clinical aspects, only such enzymes that are likely to supply new information and fill genuine gaps in today's diagnostic possibilities are worth investigating. Therefore, success in this field presupposes endeavors in biochemistry, physiology and pathophysiology as well. More than the metabolic function of an enzyme is taken into consideration, the cellular origin, its site of function, its molecular forms and, last but not least, its behavior in distinct disorders, are the main points of interest. It appears

that by now the easily accessible enzymes are exploited for medicine. The investigation of special enzymes, e.g. of the cell surface, of the replication apparatus, or of the antibody formation system, will, of course, be difficult, but seems promising. There is no reason for fatigue or resignation, on the contrary, a challenge for co-operation in further developmental work.

References [1] Wohlgemuth, J. (1908) Biochem. Z. 9, 1. [2] Garrod, A. E. (1909) Inborn errors of metabolism. Frowde, Hodder and Stoughton. [31 Rona, P. (1915) in: Handbuch der biochemischen Arbeitsmethoden. (E. Abderhalden ed.) Vol. 8, p. 301, Urban and Schwarzenberg, Berlin. [41 Marland, M., Hausman, F. S. and Robinson, R. (1924) Biochem. J. 18, 1152. E 77

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[51 Kay, H. D. (1929) Brit. J. exp. Path. 10, 253. [6] Gutman, A. B. and Gutman, E. B. (1938) J. din. Invest. 17,473. [7] Heilmeyer, L. (1933) Medizinische Spektrophotometrie. Fischer, Jena. [8] Warburg, O., Christian, W. and Giese, A. (1935) Biochem. Z. 282, 157. [9] Warburg, O. and Christian, W. (1943) Biochem. Z. 314, 399. [10] Sibley, J. A. and Lehninger, A. L. (1949) J. biol. Chem. 177,859. [11] La Due, J. S., Wroblewski, F. and Karmen, A. (1954) Science 120, 497. [12] DeRitis, F., Coltorti, M. and Giusti, G. (1955) Minerva Med. 46, 1207. [13] Sterkel, R. L., Spencer, J. A., Wolfson, S. K. jr. and Williams-Ashman, H. G. (1958) J. Lab. clin. Med. 52, 176. [14] Reichard, H. (1957) Scand. J. clin. Lab. Invest. 9, 103. [15] Gerlach, U. (1957) Klin. Wschr. 35, 1144. [16] Greenstein, J. P. (1947) Biochemistry of Cancer. Academic Press, New York. [17] Delbriick, A., Schimassek, H., Bartsch, K. and Biicher, Th. (1959) Biochem. Z. 331,297. [18] Schmidt, E. and Schmidt, F. W. (1960) Klin. Wschr. 38, 957. [19] Knox, W. E. (1972) Enzyme patterns in Fetal, Adult and Neoplastic Rat Tissues. Karger, Basel. [20] Rechcigl, M. jr. (ed.) (1971) Enzyme Synthesis and Degradation in Mammalian Systems. Karger, Basel. [21] Schmidt, E. and Schmidt, F. W. (1970) Enzym. biol. clin. 11, 67. [22] BiJcher, Th., Schmidt, E. and Schmidt, F. W. (1958) Transact. 9th Middle East Med. Ass. Beirut. [23] Warburg, O. and Hiepler, E. (1953) Z. Naturforsch. 7b, 193. [24] Zierler, K. L. (1957) Amer. J. Physiol. 190, 201. [25] Schmidt, E., Schmidt, F. W. and Wildhirt, E. (1958) Klin. Wschr. 36, 280. [26] Rees, K. R. and Sinha, K. P. (1960) J. path. Bact. 80, 297. [271 Cain, H. and Assman, E. (1960) Klin. Wschr. 38, 433. [28] Bruns, F. H., Brosswitz, E., Dennemann, H., Horn, H. D. and Noltmann, E. (1961) Klin. Wschr. 39, 342. [29] Bengmark, S. and Olsson, R. (1962) Proc. Soc. exper. Biol. 109, 258. [30] Schmidt, E., Schmidt, F. W., Herfarth, C., Opitz, K. and Vogell, W. (1966) Enzym. biol. din. 7,185. [31] Schmidt, E., Schmidt, F. W. and Otto, P. (1967) Clin. chim. Acta 15, 283. [32] Villa, L., Dioguardi, N. and Agostoni, A. (1967) Klin. Wschr. 45, 44. [33] Schmidt, E. (1968) in: Praktische Enzymologie (Sehmidt, F. W. ed.) p. 93, Huber, Bern. [34] Bohley, P. and Hanson, H. (1964) Z. physiol. Chem. 337, 16.

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[35] Schmidt, E. and Schmidt, F. W. (1975) in: Klinische Pathophysiologie (W. Siegenthaler, ed.) 3. Aufl. p. 177. Thieme, Stuttgart. [36] Schmidt, E., Schmidt, F. W., M6hr, J., Otto, P., Vido, I., Wrogemann, K. and Herfarth, C. (1975) in: Pathogenesis and Mechanisms of Liver Cell Necrosis (D. Keppler, ed.) p. 147, MTP Press, Lancaster, UK. [37] Wakim, K. G. and Fleisher, G. (1963) J. Lab. din. Med. 61, 86. [38] Dunn, M., Martin, J. and Reissman, K. R. (1958) J. Lab. clin. Med. 51,259. [39] Sibley, J. A. (1958) Ann. New York Acad. Sci. 75, 339. [40] Strandjord, P. E., Thomas, K. E. and White, L. P. (1959) J. din. Invest. 38, 2111. [41] Amelung, D. (1960) Z. physiol. Chem. 318, 219. [421 Fleisher, G. A. and Wakim, K. G. (1963) J. Lab. din. Med. 61, 76. [431 Fleisher, G. A. and Wakim, K. G. (1963) J. Lab. din. Med. 61, 98. [44] Wakim, K. G. and Fleisher, G. A. (1963) J. Lab. clin. Med. 61,107. [45] Rajewski, K. (1966) Biochim. Biophys. Acta 121, 51. [46] Boyd, J. W. (1967) Biochim. Biophys. Acta 146, 590. [47] Boyd, J. W. (1967) Biochim. Biophys. Acta 132, 221. [48] Melani, F. K., Bartelt, M., Conrads, R. and Pfeiffer, E. F. (1966) Z. kiln. Chem. klin. Biochem. 4,189. [49] D'Addabo, A. and Klaus, D. (1961) Acta Isotop. 1, 39. [50] Massarrat, S. and K6bler, H. (1965) Arch. klin. Med. 216, 285. [51] Massarat, S. (1968) Z. ges. exper. Med. 148, 56. [52] B~ir, U., Friedel, R., Heine, H., Mayer, D., Ohlendorf, S., Schmidt, F. W. and Trautschold, I. (1972/73) Enzyme 14, 133. [531 Biir, U. and Ohlendorf, S. (1970) Klin. Wschr. 48, 776. [54] Biir, U., Mayer, D., Ohlendorf, S. and Schmidt, F. W. in preparation. [55] Schmidt, E. and Biir, U. (1975) LAB 2, 5. [56] Fleisher, G. A. and Wakim, K. G. (1968) Enzym. biol. clin. 9, 81. [57] Biir, U. and Ohlendorf, S. (1971) Z. ges. exper. Med. 154, 140. [58] Haug, H. and Mannes, H. (1969) Klin. Wschr. 47, 225. [591 Kemp, E. and Laursen, T. (1960) Scand. J. din. Lab. Invest. 12, 463. [60] Hiatt, N. and Bonorris, G. (1966) Amer. J. Physiol. 210, 133. [61] Appert, H. E., Dimbiloglu, M., Pairent, F. W. and Howard, J. M. (1968) Surg. Gyn. Obstetr. 127, 1281. [62] Posen, S. (1970) Clin. Chem. 16, 71. [63] Schmidt, E. and Schmidt, F. W. (1961) in: Proc. 1st Europ. Symp. Med. Enzymol. Milan 1960, (N. Dioguardi, ed.) p. 100. Karger, Basel. [64] Weber, H. and Wegmann, T. (1964) Atlas der klinischen Enzymologie. Thieme, Stuttgart. [65] Schmidt, E. and Schmidt, F. W. (1975) Diagnostik 8, 427.

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[66] Hess, B. (1963) Enzymes in Blood Plasma. Academic Press, New York. [67] Amelung, D. (1964) Fermentdiagnostik interner Er~ankungen. Thieme, Stuttgart. [68] Henley, K. S., Schmidt, E. and Schmidt, F. W. (1966) Enzymes in Serum. C. C. Thomas, Springfield, lll. [69] Batsakis, J. G. and Russel, O. B. (1967) Interpretative Enzymology. C. C. Thomas Springfield, Ill. [70] Schmidt, F. W. (ed.) (1968) Praktische Enzymologie. Huber, Bern. [71] Haschen, R. J. (1970) Enzymdiagnostik. G. Fischer, Stuttgart. [72] Schmidt, E. and Schmidt, F. W. (1973) Kleine Enzymfibel. Boehringer-Mannheim. [73] Richterich, R. (1963) Dtsch. med. Wschr. 88, 1421. [74] Schmidt, E. and Vido, I. in: Toxische Lebersch~iden (L. Wannagat, ed.) p. 166. Thieme Stuttgart, in the press.

4 February 1976

[75] Schmidt, E. and Schmidt, F. W. (1973) in: GallenwegeLeber (W. Boecker, ed.) p. 32. Thieme, Stuttgart. [76] Kaplan, M. M. (1972) New Engl. J. Med. 286, 200. [77] Gibinski, K., Szaton, R. and Maraszek, J. (1963) Gastroenterologia 99, 237. [78] Id~o, G., Morganti, A. and Dioguardi, N. (1972) Digestion 5, 326. [79] Schmidt, E. and Schmidt, F. W. (1973) Dtsch. reed. Wschr. 98, 1572. [80] Aronson, K. F., Hanson, A. and Nosslin, B. (1965) Acta chit. Scand. 130, 92. [81] Rosalki, S. B. and Rau, D. (1972) Clin. chim. Acta 39, 41. [82] Cohen, M. I. and McNamara, H. (1969) J. Ped. 75, 838. [83] Schmidt, E. and Schmidt, F. W. (1963) Enzymol. biol. din. 3, 1.

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