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Electrophoresis of serum lactic dehydrogenase
272
ELECTROPHORESIS
OF SERUM
LACTIC
DEHVDROGENASE
INTRODUCTION
The measurement of serum lactic dehydrogenase (LD) has proved to be a useful means of following the course of a variety of diseasesr-3. However, an elevated serum LD is in itself of limited value in establishing the specific site of tissue damage because of the wide distribution of the enzyme. A solution to this difficulty was suggested by recent studies with starch block 4, agar gel 5 and continuous paper electrophoresisS which indicate that serum LD is separable into 3-5 fractions. Differences in the number of fractions found and in their relativeactivities apparently produce characteristic electrophoretic patterns in myocardial infarction, hepatitis and perhaps leukemia. Unfortunately, the complexity of the methods used in these investigations limits their usefulness in clinical laboratories. The present report describes a simplified procedure in which serum is subjected to electrophoresis on agar for 25 min. Pyruvate and reduced diphosp~op~ri~ne nucleotide (DPNH) are applied to the agar and incubated 30 min; the position of the LD fractions is determined by inspection under ultraviolet light. METHOD
Serum
is electrophoresed on agar on microscope slides by a modi~catio~ of procedure6F7. All reagents are prepared in water redistilled in glass. A uniform r-r.5 mm layer of o.S(?:h agar (Baltimore Biological Laboratories) in barbiturate buffer PH 8.4, G = 0.05 is deposited on microscope slides and aged 12 h before use. A r&cm transverse slit is cut in the agar about 30 mm from what is to be the anode end of the slide. The edge of a piece of hard filter paper is put into the slit, left there IO-GO see and as it is being ~rithdrawn, 0.005 ml serum run into the slit. Up to 3 agar slides are prepared in this manner and placed with their agar surfaces down on the agar blocks of an electrophoresis cell (Fig. I) which permits the use of petroleum ether as coolant as suggested by WlEME 5. When 3 slides are run simultaneously, adequate separation of the LD fractions is obtained in 25 min with the voltage held constant at 160 with a Spinco Duostat. ‘The current remains at 40-45 mR unless escessive heating of the agar occurs. Any tendency for the current to rise is controlled by allowing some of the petroleum ether to evaporate, aided if necessary by a fan. WIEME’S
-- _~_.
.
* Supported by grants from the Muscular Dystrophy Cancer Institute of Canada.
Association
of Canada and the Sational
SERUM LACTIC DEHYDROGENASE
273
The substrates are applied to the agar slides as follows. A 25 x 75 mm sheet of thin, hard filter paper such as Whatman 50 is moistened with a minimum of a solution of t g;/o sodium pyruvate and go rng% DPNH in barbiturate buffer, PH S.4, u = 0.05 which is stable for a week at 4”. A sheet of the moistened paper is applied to each agar slide as soon as it is taken out of the electrophoresis cell; the resulting preparation is placed in a moist incubator at 37” for 30 min. The paper is then stripped from the agar and the latter viewed under an ultraviolet lamp covered with a Wood’s filter. Dark bands, representing the areas in which the greenish fluorescent DPNH has been oxidized to the nonflLlores~ent DPN, indicate the position of the lactic dehydrogenase fractions 8. If desired, the position of the LD bands relative to the serum protein fractions can be established by staining the latter with amido bIack7.
Fig. 1. Plexiglas cell for agar electrophoresis on microscope slides. A, microscope slide with agar surface down; B, petroleum ether: c, o.sy* agar in barbiturate buffer pH 8.4, ionic strength o.05: D, same buffer. RESULTS
All sera studied had no fewer than 3 and no more than 5 LD bands. Protein staining showed that the band I coincided with the E,-globulin, band 11 with the as-globulin, band III with @-globulin and bands IV and V, when present, with the y-globulin. Confirmation that the dark bands did in fact represent regions of LD activity was obtained using a photometric method similar to that of WIEME~. In this procedure, the agar slide after incubation with pyruvate and DPNH, was scanned optically at 340 m,u to identify the regions in which DPNH had been oxidized. The results showed the sites of DPNH oxidation on the slides to coincide with the dark bands seen under ultraviolet illumination. In both methods, DPNH was oxidized only in the presence of pyruvate, proving that the bands were specifically due to LD activity in serum. When viewed in a dark room under ultraviolet light, the bands varied from a barely visible darkening of the greenish-white fluorescent background to an intensely dark area. The intensity was found to correlate well with the photometrically determined rate of DPNH oxidation. However, these preliminary tests with approximately 20 sera suggested that subjective factors in judging the intensity of the LD bands might limit the reliability of the method. This proved to be much less of a problem than expected as is shown in the following experiment. Ten serum specimens were divided in 30 replicate samples, coded and submitted randomly over a period of two weeks to an analyst who subjected them to the electrophoresis and drew a diagram of the LD bands seen on each slide. The decoded diagrams indicated that each specimen of serum yielded the same number of bands in replicate tests. It was also found in this experiment and in a subsequent series that the sera in Clin.CiliM..4&z, 6 (1961) 272-275
certain diseases produced consistent patterns, of which examples are shown in Table I. In normal serum only 3 bands of moderate intensity were usually seen, although a fourth band could occasionally be detected. In the 5 cases of myocardial infarction, all of whom had an elevation of the total LD activity of the serum, bands I and II were very intense, band III was only moderately intense and bands IV and 1’ were absent. In contrast, bands I, II and III were of normal intensity in 0 patients with viral hepatitis but band IV and especially band V, was very intense and remained so until the total serum LD activity had dropped to normal as the disease regressed. Obstructive jaundice, such as that caused by a bile duct stone in No. 4, Table I, was associated with a normal LD ele~trophoretic pattern. However, band V was quite intense in the serum of a subject who post ~~~~~rnwas found to have severe cardiac cirrhosis (No. 5, Table I). A pattern identical to that found in case j and in all other patients with active hepatocellular disease could be produced by adding homogcniztbd liver to serum before electrophoresis.
TYPICAL
EXAMPLES
OF ELECTROPHORESlS
Total LD
uctiuity * I
Sormal
2
Myocardisl
3
Hepatitis
_I
Obstructive
j
Cardiac
0
Carcinoma
7 Pernicious
-.
OF SERI:hl
-. .-. -1’e-ry’-Ghse
LACTIC
.-Ictiuitv of LD bamis -pm’.. Iuterzse Moderate
(300 infarct
jaundice
cirrhosis
anemia _.__-.---.~ * Determined with
1oo 1 It)0
I, II v
IV
IS0
4?o
__.~ a commercial
_“_ _. .-Ibss)tt .._ .____._.
I, II, III
IV, v
III
IV, v
I, IT,
111
IV, \;
I, II, 111
qzo
i ShO
UEIIYDROGENASE
1, 11, 111
I, If, III,
T‘
Iv III
I, II kit (Sigma
v
Chemical
Co., St. Louis,
I\
_
1v, v
.._
Mo.)
The LD pattern in patient 6, Table I, is typical of the findings in 5 cases of advanced carcinoma with liver metastases and elevated total serum LD levels. Bands I and II were often as intense as in patients with myocardial infarction and bands IV and V occasionally as marked as in hepatitis. However, band III, which in all other conditions was only moderately intense, was very intense in all 5 cases of advanced carcinoma. The LD pattern in pernicious anemia is of interest in view of the high total LD levels found in this and other types of megaloblastic anemialo. In the 4 cases examined, bands I and II were very intense, band III only moderately strong and bands IV and V both absent (No.?, Table I). Since a similar pattern was produced by adding hemolysate to serum, it is possible that elevated serum LD level in pernicious anemia has its origin in the increased rate of hemolysis characteristic of this condition. DISCUSSION
The LD electrophoretic patterns in myocardial infarction, hepatitis and obstructire jaundice described above are in agreement with those reported by previous workers who used more complex, quantitative methods*s5, 7, 9. These findings are of limited practical value since the conditions mentioned can usually be differentiated
SERU.MLr\CTIC DEHYDR3SEN.GE
275
readily by established clinical and laboratory methods. However, the proposed method may prove useful as a simple means of assessing the contribution of hepatic parenchymal damage to an elevated serum LD level. WIEME’S observations51 9 and our results indicate that intense LD activity in bands IV and V, particularly in the presence of only moderate activity in the other fractions, may be a relatively specific indication of hepatocellular damage. This conclusion is supported by the similarity of the LD pattern produced by adding liver homogenate to serum before electrophoresis. The observation that the serum LD pattern in pernicious anemia can be reproduced by adding hemolysate to serum before electrophoresis suggests that the pattern seen in pernicious anemia may have its origin in the increased rate of hemolysis known to occur in this condition. This argument is weakened by the finding that the pattern in pernicious anemia is identical with that in myocardial infarction. The occurrence of the same LD patterns in two unrelated conditions, myocardial infarction and pernicious anemia, indicates that these and other LD patterns described above are not necessarily tissue specific. Attempts to apply the present technique to glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been unsuccesful to date. However, glutamic oxalacetic transaminase and malic dehydrogenase are readily demonstrated with the present method, but the variations in their electrophoretic patterns in disease have not been explored as yet.
A simplified procedure for demonstrating the electrophoretic pattern of serum LD has been described. In normal serum only 3 LD fractions are usually present, but in certain diseases as many as 5 LD bands can be identified. Differences in the number of bands present and in their relative activity produce characteristic patterns in subjects with myocardial infarction, hepatitis, pernicious anemia and carcinomatosis. However, the results are not entirely tissue specific, since identical patterns are produced in such unrelated conditions as pernicious anemia and myocardial infarction. REFERENCES 1 L. P. WHITE, Ne, E@. J. Med., 255 (1956) 984. 2 H. J. ZIMMERMAN AND H. G. WEINSTEIN, J. Lab. Cliu. Med., 48 (1956) 607. 3 M.C. BLANCHAER,P.T.GREEN, J.P.M.4CLEAN AND AM.J.H~~~~~~~~~, Blood,13 4 E. S. ELLIOT AND A. G. BEARN, J.Cli)z.1nzwst.,37 (1958) 672. 5 R. J. \\'IEME,Clin.Chinz.9cta. 4 (1959) 46. B B. R. HILL, .4un. N.1'. -4cad. Sci., 75 (1958) 304. ’ R. J.\V1E~E,clin. Chim.Acta,4 (1959) 317, 8 T.\VIELAND AND G. PFLEIDERER, Biochewz. .?., 329 (1957) s R. J. \VIEME, .4&a Gastro-Entevolog. Belg., 21 (1959) 09. lo I'.HELLER,H.G.~~EINSTEIN,M.WESTANDH.J.ZI~MERMAN,J.
(1958) 245.
IIL. Lab.Cliu.Med.,
55 (Ig6o)425.
Cli% Chim. Acta, 6 (1961) 272-27j
ELECTROPHORESIS
OF SERUM
LACTIC
DEHVDROGENASE
INTRODUCTION
The measurement of serum lactic dehydrogenase (LD) has proved to be a useful means of following the course of a variety of diseasesr-3. However, an elevated serum LD is in itself of limited value in establishing the specific site of tissue damage because of the wide distribution of the enzyme. A solution to this difficulty was suggested by recent studies with starch block 4, agar gel 5 and continuous paper electrophoresisS which indicate that serum LD is separable into 3-5 fractions. Differences in the number of fractions found and in their relativeactivities apparently produce characteristic electrophoretic patterns in myocardial infarction, hepatitis and perhaps leukemia. Unfortunately, the complexity of the methods used in these investigations limits their usefulness in clinical laboratories. The present report describes a simplified procedure in which serum is subjected to electrophoresis on agar for 25 min. Pyruvate and reduced diphosp~op~ri~ne nucleotide (DPNH) are applied to the agar and incubated 30 min; the position of the LD fractions is determined by inspection under ultraviolet light. METHOD
Serum
is electrophoresed on agar on microscope slides by a modi~catio~ of procedure6F7. All reagents are prepared in water redistilled in glass. A uniform r-r.5 mm layer of o.S(?:h agar (Baltimore Biological Laboratories) in barbiturate buffer PH 8.4, G = 0.05 is deposited on microscope slides and aged 12 h before use. A r&cm transverse slit is cut in the agar about 30 mm from what is to be the anode end of the slide. The edge of a piece of hard filter paper is put into the slit, left there IO-GO see and as it is being ~rithdrawn, 0.005 ml serum run into the slit. Up to 3 agar slides are prepared in this manner and placed with their agar surfaces down on the agar blocks of an electrophoresis cell (Fig. I) which permits the use of petroleum ether as coolant as suggested by WlEME 5. When 3 slides are run simultaneously, adequate separation of the LD fractions is obtained in 25 min with the voltage held constant at 160 with a Spinco Duostat. ‘The current remains at 40-45 mR unless escessive heating of the agar occurs. Any tendency for the current to rise is controlled by allowing some of the petroleum ether to evaporate, aided if necessary by a fan. WIEME’S
-- _~_.
.
* Supported by grants from the Muscular Dystrophy Cancer Institute of Canada.
Association
of Canada and the Sational
SERUM LACTIC DEHYDROGENASE
273
The substrates are applied to the agar slides as follows. A 25 x 75 mm sheet of thin, hard filter paper such as Whatman 50 is moistened with a minimum of a solution of t g;/o sodium pyruvate and go rng% DPNH in barbiturate buffer, PH S.4, u = 0.05 which is stable for a week at 4”. A sheet of the moistened paper is applied to each agar slide as soon as it is taken out of the electrophoresis cell; the resulting preparation is placed in a moist incubator at 37” for 30 min. The paper is then stripped from the agar and the latter viewed under an ultraviolet lamp covered with a Wood’s filter. Dark bands, representing the areas in which the greenish fluorescent DPNH has been oxidized to the nonflLlores~ent DPN, indicate the position of the lactic dehydrogenase fractions 8. If desired, the position of the LD bands relative to the serum protein fractions can be established by staining the latter with amido bIack7.
Fig. 1. Plexiglas cell for agar electrophoresis on microscope slides. A, microscope slide with agar surface down; B, petroleum ether: c, o.sy* agar in barbiturate buffer pH 8.4, ionic strength o.05: D, same buffer. RESULTS
All sera studied had no fewer than 3 and no more than 5 LD bands. Protein staining showed that the band I coincided with the E,-globulin, band 11 with the as-globulin, band III with @-globulin and bands IV and V, when present, with the y-globulin. Confirmation that the dark bands did in fact represent regions of LD activity was obtained using a photometric method similar to that of WIEME~. In this procedure, the agar slide after incubation with pyruvate and DPNH, was scanned optically at 340 m,u to identify the regions in which DPNH had been oxidized. The results showed the sites of DPNH oxidation on the slides to coincide with the dark bands seen under ultraviolet illumination. In both methods, DPNH was oxidized only in the presence of pyruvate, proving that the bands were specifically due to LD activity in serum. When viewed in a dark room under ultraviolet light, the bands varied from a barely visible darkening of the greenish-white fluorescent background to an intensely dark area. The intensity was found to correlate well with the photometrically determined rate of DPNH oxidation. However, these preliminary tests with approximately 20 sera suggested that subjective factors in judging the intensity of the LD bands might limit the reliability of the method. This proved to be much less of a problem than expected as is shown in the following experiment. Ten serum specimens were divided in 30 replicate samples, coded and submitted randomly over a period of two weeks to an analyst who subjected them to the electrophoresis and drew a diagram of the LD bands seen on each slide. The decoded diagrams indicated that each specimen of serum yielded the same number of bands in replicate tests. It was also found in this experiment and in a subsequent series that the sera in Clin.CiliM..4&z, 6 (1961) 272-275
certain diseases produced consistent patterns, of which examples are shown in Table I. In normal serum only 3 bands of moderate intensity were usually seen, although a fourth band could occasionally be detected. In the 5 cases of myocardial infarction, all of whom had an elevation of the total LD activity of the serum, bands I and II were very intense, band III was only moderately intense and bands IV and 1’ were absent. In contrast, bands I, II and III were of normal intensity in 0 patients with viral hepatitis but band IV and especially band V, was very intense and remained so until the total serum LD activity had dropped to normal as the disease regressed. Obstructive jaundice, such as that caused by a bile duct stone in No. 4, Table I, was associated with a normal LD ele~trophoretic pattern. However, band V was quite intense in the serum of a subject who post ~~~~~rnwas found to have severe cardiac cirrhosis (No. 5, Table I). A pattern identical to that found in case j and in all other patients with active hepatocellular disease could be produced by adding homogcniztbd liver to serum before electrophoresis.
TYPICAL
EXAMPLES
OF ELECTROPHORESlS
Total LD
uctiuity * I
Sormal
2
Myocardisl
3
Hepatitis
_I
Obstructive
j
Cardiac
0
Carcinoma
7 Pernicious
-.
OF SERI:hl
-. .-. -1’e-ry’-Ghse
LACTIC
.-Ictiuitv of LD bamis -pm’.. Iuterzse Moderate
(300 infarct
jaundice
cirrhosis
anemia _.__-.---.~ * Determined with
1oo 1 It)0
I, II v
IV
IS0
4?o
__.~ a commercial
_“_ _. .-Ibss)tt .._ .____._.
I, II, III
IV, v
III
IV, v
I, IT,
111
IV, \;
I, II, 111
qzo
i ShO
UEIIYDROGENASE
1, 11, 111
I, If, III,
T‘
Iv III
I, II kit (Sigma
v
Chemical
Co., St. Louis,
I\
_
1v, v
.._
Mo.)
The LD pattern in patient 6, Table I, is typical of the findings in 5 cases of advanced carcinoma with liver metastases and elevated total serum LD levels. Bands I and II were often as intense as in patients with myocardial infarction and bands IV and V occasionally as marked as in hepatitis. However, band III, which in all other conditions was only moderately intense, was very intense in all 5 cases of advanced carcinoma. The LD pattern in pernicious anemia is of interest in view of the high total LD levels found in this and other types of megaloblastic anemialo. In the 4 cases examined, bands I and II were very intense, band III only moderately strong and bands IV and V both absent (No.?, Table I). Since a similar pattern was produced by adding hemolysate to serum, it is possible that elevated serum LD level in pernicious anemia has its origin in the increased rate of hemolysis characteristic of this condition. DISCUSSION
The LD electrophoretic patterns in myocardial infarction, hepatitis and obstructire jaundice described above are in agreement with those reported by previous workers who used more complex, quantitative methods*s5, 7, 9. These findings are of limited practical value since the conditions mentioned can usually be differentiated
SERU.MLr\CTIC DEHYDR3SEN.GE
275
readily by established clinical and laboratory methods. However, the proposed method may prove useful as a simple means of assessing the contribution of hepatic parenchymal damage to an elevated serum LD level. WIEME’S observations51 9 and our results indicate that intense LD activity in bands IV and V, particularly in the presence of only moderate activity in the other fractions, may be a relatively specific indication of hepatocellular damage. This conclusion is supported by the similarity of the LD pattern produced by adding liver homogenate to serum before electrophoresis. The observation that the serum LD pattern in pernicious anemia can be reproduced by adding hemolysate to serum before electrophoresis suggests that the pattern seen in pernicious anemia may have its origin in the increased rate of hemolysis known to occur in this condition. This argument is weakened by the finding that the pattern in pernicious anemia is identical with that in myocardial infarction. The occurrence of the same LD patterns in two unrelated conditions, myocardial infarction and pernicious anemia, indicates that these and other LD patterns described above are not necessarily tissue specific. Attempts to apply the present technique to glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been unsuccesful to date. However, glutamic oxalacetic transaminase and malic dehydrogenase are readily demonstrated with the present method, but the variations in their electrophoretic patterns in disease have not been explored as yet.
A simplified procedure for demonstrating the electrophoretic pattern of serum LD has been described. In normal serum only 3 LD fractions are usually present, but in certain diseases as many as 5 LD bands can be identified. Differences in the number of bands present and in their relative activity produce characteristic patterns in subjects with myocardial infarction, hepatitis, pernicious anemia and carcinomatosis. However, the results are not entirely tissue specific, since identical patterns are produced in such unrelated conditions as pernicious anemia and myocardial infarction. REFERENCES 1 L. P. WHITE, Ne, E@. J. Med., 255 (1956) 984. 2 H. J. ZIMMERMAN AND H. G. WEINSTEIN, J. Lab. Cliu. Med., 48 (1956) 607. 3 M.C. BLANCHAER,P.T.GREEN, J.P.M.4CLEAN AND AM.J.H~~~~~~~~~, Blood,13 4 E. S. ELLIOT AND A. G. BEARN, J.Cli)z.1nzwst.,37 (1958) 672. 5 R. J. \\'IEME,Clin.Chinz.9cta. 4 (1959) 46. B B. R. HILL, .4un. N.1'. -4cad. Sci., 75 (1958) 304. ’ R. J.\V1E~E,clin. Chim.Acta,4 (1959) 317, 8 T.\VIELAND AND G. PFLEIDERER, Biochewz. .?., 329 (1957) s R. J. \VIEME, .4&a Gastro-Entevolog. Belg., 21 (1959) 09. lo I'.HELLER,H.G.~~EINSTEIN,M.WESTANDH.J.ZI~MERMAN,J.
(1958) 245.
IIL. Lab.Cliu.Med.,
55 (Ig6o)425.
Cli% Chim. Acta, 6 (1961) 272-27j