H. J. VAN
H. A. ZONDAG,
H. A. PH. HARTOG
AXD M. W. VAN Pvovinciaal
(Received December zgth, 1961)
Lactic dehydrogenase (LDH), like several other enzymes, has proved to be a mixture of different components which are very similar in biological activity but different in electrophoretic behaviour. The pattern of distribution of these isoenzymes varies in different tissues, and the fact that disease of an organ can cause characteristic changes in the plasma isoenzyme composition, reflecting the isoenzyme composition of the tissue involved, affords new diagnostic possibilities (WIEME~, WROBLEWSKI et aLz, PLAGEMANN et aL3). Unfortunately the complexity of the methods used has limited their usefulness in the clinical laboratory. The introduction of a relatively simple form of “enzyme electrophoresis” by VAN DER HELM *, 5 led us to investigate whether this method is suitable for routine use in the clinical laboratory, particularly in assessing the diagnosis of myocardial infarction. For this purpose enzyme electrophoresis was carried out in sera from 45 patients with clinically verified or suspected myocardial infarction. Total LDH (KINGS), SGO-T and SGP-T (REITRIAN AND FRANKEL~) determinations were carried out on the same blood samples. If possible, repeated determinations were carried out on different days during the course of illness in sera from patients with proven myocardial infarction, so that 86 estimations were made in 25 patients suffering from this disease. To investigate whether the LDH isoenzyme pattern of myocardial infarction is also present in the serum in patients suffering from other diseases, enzyme electrophoresis was also done on 85 other patients, including 70 with various non-cardiac diseases. NETHOD
Electrophoresis is carried out in agar gel during 30 min at 140 V with 8 ,ul of serum in a 5-mm slit, using the technique on microscope slides as described by WIE;\~E~. Two or three samples can be subjected to electrophoresis on the same slide. The slide is then incubated for 2 h at 37” in a medium of the following composition, which is freshly prepared before use. Stock solution I, 3.6 ml. Stock solution II, 0.1 ml. Diphosphopyridine nucleotide, 4 mg. Stock solution I (pH 7.4-7.5). Sodium lactate solution (&71%) 1.0 ml; sodium cyanide 50 mg; disodium hydrogen phosphate dihydrate I.8690 g ; potassium dihydrogen phosphate 0.2722 g; nitroblue tetrazolium 25 mg; water to go ml. Stock sohtion II. Phenazine methosulphate I mg/ml in distilled water (stored in the dark).
It is convenient to place the slide upside down on two (colourless) matches on a glass plate, and to pipette the incubating fluid under the agar layer. In this way less than 4 ml of the incubating fluid is sufficient. After incubation the slide is washed with water, and the proteins are fixed with a mixture of 70 ~01s. ethanol, 5 ~01s. acetic acid, and 25 ~01s. distilled water. The plate is subsequently dried while covered with filter paper and densitometry is carried out, followed by planimetry. The activity of the fractions is expressed in percents of the total area. Because of the high LDH activity within the erythrocytes, especially of fractions I and z, haemolytic serum samples cannot be used in this investigation. RESULTS
In normal human sera 4 or 5 LDH fractions are found. The fastest fraction (I) always lies between the albumin and the crl-globulin, the second on the fastest part of the al-fraction, the third between PI- and &globulin, the fourth on the y-globulin front and the fifth on the slowest part of the y-globulin. The LDH isoenzyme pattern of normal human serum is given in Fig. I, while the mean percentage amounts and
Fig. I. Normal LDH
pattern (two TABLE
Normal values (20 donors) Fmctiovl Mean (in %) Range (in %)
Myocavdial Mean (in %) Range (in %)
infarction I o-6
(20 cases) I
the ranges found in 20 normal subjects (blood donors) are given in Table I. The LDH isoenzyme distribution in the serum of a patient after myocardial infarction is shown in Fig. 2. The first and, to a lesser extent, the second fraction are greatly elevated. The mean of the different LDH fractions and the ranges in 25 patients with clinically verified myocardial infarction at a time when the total LDH activity was at its highest, are also given in Table I. In order to follow the course of the disease repeated determinations were often carried out. The regular course which was obtained in some cases is shown in Table II ; it can be seen that the total LDH activity did not rise to a very high level in this
H. J. VAN DEEl HELM et al.
particular patient and also that fraction I remained relatively low. This was the patient with the lowest percentage of fraction I after proven myocardial infarction. Demonstration of this case was chosen because even in this patient a rise of fraction I during the first few days was observed, followed by a gradual decline on the fol-
Fig. 2. Myocardial infarction.Top:
about I h after infarction. TABLE
about 24 h after infarction.
MIY0C~RDI.M. INFARCTION Patient AZ.
44 60 60 56
54 40 38 44
16 72 53 ‘7
265 535 660 585
7 9 II ‘3 16
7 I :
450 450 325 355
56 53 47 45 38
42 44 48 52 52
L 3 5 3 7
Tmnmninases SGO-T IO
5 9 9 12
SGP.T 4 3 5 IO
Patient de G. Days after Days aftev admission infarctiolz 0 8 I 9
350 315 290
53 40 34
37 47 ;s -9
6 8 ‘4
2 2 _.
lowing days. Thus, even in cases where fraction I is not greatly elevated, useful information can be obtained if repeated determinations are carried out. This is of special importance if a diagnosis of myocardial infarction has to be made when the initial attack has occurred a few days earlier and when SGO-T and total LDH are only slightly elevated or have already returned to normal values. This is illustrated in Table III. Therefore we conclude that the method, besides being more specific for myocardial infarction, can also be more sensitive than the total LDH and SGO-T determinations, especially when repeated determinations are carried out. When liver Cl&. Chim. A&,
7 (1962) j4o-jad
is present, as for example in cardiac shock or decompensation with confollowing myocardial infarction, apart from fractions I and 2 a rise in the
fifth fraction can be seen. This is demonstrated in Table IV. In this way it is sometimes possible to detect the presence of two foci of damage (in this patient myo-
with liver damage.
Admission: acute myocardial infarction
Decompensation with liver involvement
I4 15 16
65 62 60 60
35 35 32 38 42 4’ 45
3 3 4 3” 3 3
I!I h I
455 365 315 3’5
50 55 52 53 50
Number Congestive failure Acute pericarditis Paroxysmal atria1 fibrillation Chronic angina pectoris
: 1420 U.
: 150 U. LDH
: 140 U.
of patients 5 I
Heartblock with Stokes-Adams seizures Acute venous thrombosis of the lower extremity Pulmonary embolism
2 2 I I
cardial and liver tissue). When the fifth fraction becomes greatly elevated after myocardial infarction (Fig. 3), the percentage amounts of fraction I and z decrease so that perhaps in these more complex cases a calculation of “absolute” values for the activities can be useful. A clinically important application of this technique of enzyme electrophoresis would be in differentiating, if possible, between myocardial infarction and other conditions not readily distinguished from myocardial infarction. The conditions studied, which gave no LDH pattern similar to that of myocardial infarction, are shown in Table V. In this table pulmonary embolism is of special interest. UncomCZin. Chim. Acta, 7 (1962) 540-544
plicated pulmonary embolism is not associated with a rise in transaminases or LDH. Massive embolism, however, can give rise to grossly elevated enzyme-activities of SGO-T, SGP-T and LDH. Isoenzyme electrophoresis in these patients demonstrates that the rise in LDH is caused exclusively by an elevation of fraction 5 (with normal fractions I and 2, excluding myocardial infarction), indicating liver damage (Fig. 4). Conditions which appeared to give rise to LDH patterns which sometimes closely resemble those of myocardial infarction, were some cases of generalized carcinoma (seminomas), haemolytic anaemia and pernicious anaemia.
4. Pulmonary part
embolism with liver damage. SGO-T : 850 E. SGP-T : 290 E. LDH : 4200 E. of the slide the same sample is shown after 30 min at 56”, demonstrating the heat lability of fractions 3, 4 and 5.
The differentiation of these diseases from myocardial infarction, apart from clinical evidence, is nevertheless easy because of the absence of the typical course of the fractions as found after myocardial infarction. In our opinion the method of LDH electrophoresis can be more useful in assessing the diagnosis of myocardial infarction than most current methods in the clinical laboratory. The method is relatively simple, especially for those laboratories where electrophoresis on agar gel is already in use for routine investigation of protein fractions. In comparison with the methods used by WIEME 8 and BLANCHAER 9, this procedure offers the additional advantage that the “isozymograms”, when dry, can be kept indefinitely and are ideally suited for scanning. Thus, the necessity of photographic reproduction or visual inspection under ultraviolet light g is avoided and results are easily expressed in reproducible figures. SUMMARY
In patients with myocardial infarction the utility of a relatively simple method of LDH isoenzyme electrophoresis was investigated. The results obtainedindicate that this laboratory procedure gives important information to the cardiologist. REFERENCES 1 R. J. WIEME, Studies 01%agav-gel electrophovesis, Arscia, Brussel, 1959. 2 F. WROBLEWSKI, C. Ross ANI) K. F. GREGORY, New Engl. J. Med., 263 (1960) 531. 3 P. G. W. PLAGEMANX, K. F. GREGORY AND F. WROBLEWSKI, J.Biol. Chew., 235 (1960)2282,
2288. 4 H. J. VAN DER HELM, Lamet, ii (1961) 108. 5 H. J. VAN DER HELM, CZin.Chim. Acta, 7 (1962) 124. 6 J. KING, J. Med. Lab. Technol., 16 (1959)265. ’ S. REITMAN AND S. FRANKEL, Am. J. Cliti. Pathol., 28 (1957)56. * R. J. WIEME, Clin.Chim. Acta, 4 [Igjg) 46. 9 ilf. C. BLANCHAER, Clin.Chinz. Acta, 6 (1961) 272. Clin. Chin?. A&,
7 (1962) j4O-j44