235
Clinica Chimica Acta, 68 (1976) 235-240 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7589
ABNORMAL SERUM LACTATE DEHYDROGENASE ISOENZYME A CASE OF LARYNGEAL CARCINOMA AND THYROTOXICOSIS
IN
FUMIKO TANAKA *, NOBUYUKI AMINO, CHOZO HAYASHI, KIYOSHI MIYAI and YUICHI KUMAHARA Central Laboratory for Clinical Investigation, Osaka University Hospital, l-l-50, Fukushima, Fukushima-Ku, Osaka 553 (Japan) (Received October 6,1975)
An abnormal lactate dehydrogenase (LDH) isoenzyme was found in the serum of a patient with laryngeal carcinoma and hyperthyroidism. On electrophoresis it migrated as an additional band between LDH-1 and LDH-2. Followup studies suggested that this higher molecular weight, rather thermostable LDH isoenzyme, might have originated from the cancer tissue, though a possible relationship with the thyrotoxic state cannot be excluded.
Introduction Abnormal electrophoretic patterns of serum lactate dehydrogenase (LDH) isoenzymes have been reported by several investigators [l--9]. Recently we observed an extra band between LDH-1 and LDH-2 on electrophoresis of serum LDH of a patient with laryngeal carcinoma complicated with hyperthyroidism. This paper reports a case history of this patient and some characteristics of this abnormal LDH isoenzyme. The possibility that it originated from cancer tissue is discussed. Materials and methods LDH activities were measured spectrophotometrically by a modification (LDH kit-N, Nipponshoji Co., Ltd., Japan) of the original method of Babson [lo]. Agarose gel (Pol-E Film, Diagnostic Division, Pfizer Inc., New York, U.S.A.) was used for electrophoretic separation of the LDH isoenzymes. Electrophoresis was carried out in a cooled apparatus using barbital buffer (pH 8.6,
* To
whom
reprint
requests
should
be addressed.
236
ionic strength 0.05). The isoenzymes were coloured by the method of Van der Helm [ll]. Serum alkaline phosphatase activity was determined by the method of Kind and King [l2] with slight modifications. Alkaline phosphatase isoenzymes were separated by the method of Ogita et al. 1131 using polyacryl~ide gel electrophoresis. Gel filtration was performed on a Sephadex G-200 column (2.4 cm X 65 cm) in phosphate buffer (pH 7.4, ionic strength 0.17) at a flow rate of S-9 ml/h. Immunoelectrophoresis was carried out as described by Sheidegger [ 14 1. Case report A 55year old male electrical engineer was admitted to this hospital on March 13th, 1975 suffering from inspiratory dyspnea. Except for an attack of malaria at the age of 24, his own previous history and his family history were not remarkable. Tracheotomy was carried out as a first aid operation and 3 weeks later, on April 4th, 1975, after diagnosis of cancer of the larynx, total laryngectomy was performed. The cancer was identified histologically as squamous cell carcinoma. No metastases to the regional lymphnodes were observed. After the operation, he continued to lose weight and complained of palpitation. Although he had no obvious goiter or abnorm~ity of the eyes, he was diagnosed as having hyperthyroidism with high levels of serum thyroxine and triiodothyronine and low cholesterol. Therapy with an anti-thyroid drug was started on April 30th, 1975. The abnormal pattern of LDH isoenzymes was first observed on examination of serum obtained on April 23rd, when the LDH activity was 255 units. The serum alkaline phosphatase level was 11.0 King-Armstrong units and on electrophoresis a small fraction (Alp-O) was seen which did not move from the origin. The erythroeyte sedimentation rate was 25 mm after 1 h. Mild anemia was detected but otherwise routine blood and urine analysis gave normal results. The levels of serum glutamic oxalacetic transaminase and glutamic pyruvic transaminase were normal. The electrophoretic pattern of serum protein and the levels of serum immunoglobulins IgG, IgA and IgM were normal. Anti-thyroglobulin antibody was not detected but anti-thyroid microsomal antibody with a titer of 1 : lo3 was found. Physical and radiological examination showed no evidence of distant met‘astasis. Results The electrophoretogram of LDH isoenzymes of the patient’s serum showed an extra band between LDH-1 and LDH-2 (Fig. 1). The gel filtration pattern of the patient’s serum on Sephadex G-200 is shown in Fig. 2, Two abnormal peaks of LDH activity were detected in front of the main peak, which had the same mobility as normal LDH. The material in these abnormal peaks was found to give the extra band on electrophoresis. No staining for LDH activity was demonstrated after immunoelectrophoresis of the serum against antisera to immunoglobulin IgG, IgA and IgM. The LDH isoenzyme pattern of a lysate of the patient’s erythrocytes did not show either
231
before
after
88 days
7- l-1975 _--._
assayed
after
days
43
5-17-1975
sera were
after
days
4-30-197
* Sample
after
19 days
26
4-23-1975
after
*
*
OF
AND
25.5 25.4 0 0
76.5
69.7
71.0
70.0
at -20°C.
---.--____-
65.4 47.5
50.4
73.7
LDH-T
(units)
LDH
LDH-I
activities
storage
260
260
270
255
270
300
days
11
LDH
ACTIVITIES
Total
after
5
SERUM
relative
ON
operation
to
Time
UP STUDY
7 days
I
4-11-1975
3-241975
Date
FOLLOW
TABLE
-__
16.2
75.1
66.7
62.2
67.8
65.1
LDH-2
--__~
ALKALINE
11.0 11.5
7.3 11.2 15.7 19.2 15.6
11.9 17.3 22.9 24.2 25.0
61.8 62.2 69.7 70.5 71.2 ----___
15.0
20.1
16.2
82.8
13.6
12.5
16.6
LDH-5 Total
Alkaline LDH-4
-___
ISOENZYMES
LDH-3
PHOSPHATASE
0
6.2 6.0
0
6.2
4.6
0
0 3.2
2.3
0
0
Alp-3
(units)
5.8
6.3
2.8
2.9
9.2 8.6
Alp-2
activities
Alp-1
phosphatase
1.4
0
0
1.6 1.7
1.6 0.9
2.1
3.5
Alp-0
0.8
0.9
1.0
Alp-4
239
Fractcm
No
Fig. 2. Gel filtration of the patient’s .seum on a Sephadex G-200 column. 3.0 ml of serum were applied to a 2.4 cm X 65 cm column. Elution was performed at a flow rate of 6.6 ml/h 4’C. l -r. protein: l - - - - - -*, LDH activity.
the additional LDH isoenzyme fraction or irregularity in electrophoretic mobility. On electrophoresis of a mixture of equal amounts of normal serum and the patient’s serum, the distribution patterns of activities in the two were superimposed. When the patient’s serum was incubated at 56°C for 30 min, the bands of LDH-4 and LDH-5 disappeared, but the abnormal extra band still appeared. During this heat inactivation treatment the total LDH activities of normal serum and the patient’s serum decreased to 67% and 77%, respectively. Table I shows the results of follow up studies on this patient. The activity of the abnormal LDH isoenzyme, which was tentatively named LDH-T, decreased after removal of the cancer tissue and had disappeared on May 17,1975. When serum obtained at this time was fractionated by gel filtration on Sephadex G200, no abnormal peaks of LDH activity were observed. Another specimen of serum, obtained 11 days before the operation was analysed by electrophoresis after storage at -20°C for 56 days and showed high LDH-T activity (21.8% of the total activity). The appearance of LDH-T was not due to freezing, because a sample of serum from a normal subject did not show any abnormal LDH isoenzyme or an irregular electrophoretic pattern after storage at -20°C for 2 months. Discussion Extra bands of a sixth isoenzyme of serum LDH have been reported by several investigators [2,3,8]. Lundh [2] and Voigt [3] detected an abnormal band between LDH-3 and LDH4 on electrophoresis of sera from a patient with leukemia and a healthy woman, respectively. Nagamine [8] reported an extra
240
band between LDH-2 and LDH-3, attributed to formation of an LDH-immunoglobulin A complex. However, there have been no reports on the presence of abnormal LDH isoenzymes in relation to the clinical course of diseases. We also know of no reports on production of abnormal LDH isoenzymes by tumors. In the serum of the patient described in this report, the extra band of LDH-T was found between LDH-1 and LDH-2. Gel filtration showed that this isoenzyme was a macromolecule. It was stable on heating at 56°C for 30 min. Its appearance was not due to a hereditary abnormality, since the LDH isoenzyme pattern of the patient’s erythrocytes was normal and this LDH-T disappeared from the serum after removal of the cancer by operation. Immunoelectrophoretic studies showed that LDH-T was not a complex of LDH and immunoglobulin. Moreover its appearance was probably not due to hyperthyroidism, since no abnormal extra band was found in the sera of five patients with hyperthyroidism. It is very interesting that LDH-T gradually decreased and finally disappeared after removal of the cancer tissue. Alp-O, that is often found in patients with malignant tumors [15], also decreased and disappeared after the operation. Unfortunately, we did not examine the LDH isoenzymes of the cancer tissue itself. The possibilities of the presence in the patient of serum factors which affected the mobility of normal LDH isoenzymes and of polymers of normal LDH isoenzymes were not completely excluded. However, the results described above strongly suggest that LDH-T originated from this cancer tissue. Acknowledgements The authors are grateful to Dr. E. Ichihara for help in the preparation of the manuscript in English. We wish to thank Miss T. Kawano for the secretarial assistance. References 1
Kreutzer,
2
Lundh,
3
Voigt,
4
Ganrot,
H.H.,
Jacobs,
B. (1967) A.
(1967)
P.O.
5
Kindmark,
C.O.
Biewenga.
J. and
Kitamura,
M.,
Nagamine,
M. (1972)
9
Biewenga,
11
Van
12
Kind.
13
Ogita.
Clin.
Z.,
Clin.
Klin. J. Clin.
(1970)
Hira;suka, Acta Acta
H.J.
(1962)
Clin.
Chin
(1954)
Chiba.
Chim.
Acta
11,
159-169
Y.
5, 146-147
Invest.
Chin
Chin
Acta
Y.,
Clin.
593
Lab.
Clin.
Chim.
Sakoyama.
23,
F. and Chin
E.J.
Biochem.
(Base11
Clin.
and King.
C. (1965)
16.305-309
(1965)
der Helm, P.R.N.
L.G.
Hashimoto,
J. (1972) A.L.
Chem. Scsnd.
Thijs,
7
Francke,
Acta
Experientia
(1969)
8
Babson.
Chin
Z. Klin.
(1967)
6
10
Ph. and
Clin.
A.
24,
49--53
Acta
27.293-299
(1971)
Clin.
Chim.
Acta
34.
419-423
36.139-144 40.407414 12,
210-215
Acta
J. Clin.
7.
124
Pathol.
and Masuzawa,
7, 322-326 M.
(1973)
Y.
and
Physico-Chemical
Biol.
(in Japanese)
17.
63-69 14
Sheidegger,
J.J. (1955)
15
Masuzawa,
M.,
Japanese)
17.
Kamata, 71-76
Int. T.,
Arch.
Allergy
Sakoyama,
7, 103-110 Y.,
Chiba,
Ogita,
2.
(1973)
Physico-Chemical
Biol.
(in
Clinica Chimica Acta, 68 (1976) 235-240 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
CCA 7589
ABNORMAL SERUM LACTATE DEHYDROGENASE ISOENZYME A CASE OF LARYNGEAL CARCINOMA AND THYROTOXICOSIS
IN
FUMIKO TANAKA *, NOBUYUKI AMINO, CHOZO HAYASHI, KIYOSHI MIYAI and YUICHI KUMAHARA Central Laboratory for Clinical Investigation, Osaka University Hospital, l-l-50, Fukushima, Fukushima-Ku, Osaka 553 (Japan) (Received October 6,1975)
An abnormal lactate dehydrogenase (LDH) isoenzyme was found in the serum of a patient with laryngeal carcinoma and hyperthyroidism. On electrophoresis it migrated as an additional band between LDH-1 and LDH-2. Followup studies suggested that this higher molecular weight, rather thermostable LDH isoenzyme, might have originated from the cancer tissue, though a possible relationship with the thyrotoxic state cannot be excluded.
Introduction Abnormal electrophoretic patterns of serum lactate dehydrogenase (LDH) isoenzymes have been reported by several investigators [l--9]. Recently we observed an extra band between LDH-1 and LDH-2 on electrophoresis of serum LDH of a patient with laryngeal carcinoma complicated with hyperthyroidism. This paper reports a case history of this patient and some characteristics of this abnormal LDH isoenzyme. The possibility that it originated from cancer tissue is discussed. Materials and methods LDH activities were measured spectrophotometrically by a modification (LDH kit-N, Nipponshoji Co., Ltd., Japan) of the original method of Babson [lo]. Agarose gel (Pol-E Film, Diagnostic Division, Pfizer Inc., New York, U.S.A.) was used for electrophoretic separation of the LDH isoenzymes. Electrophoresis was carried out in a cooled apparatus using barbital buffer (pH 8.6,
* To
whom
reprint
requests
should
be addressed.
236
ionic strength 0.05). The isoenzymes were coloured by the method of Van der Helm [ll]. Serum alkaline phosphatase activity was determined by the method of Kind and King [l2] with slight modifications. Alkaline phosphatase isoenzymes were separated by the method of Ogita et al. 1131 using polyacryl~ide gel electrophoresis. Gel filtration was performed on a Sephadex G-200 column (2.4 cm X 65 cm) in phosphate buffer (pH 7.4, ionic strength 0.17) at a flow rate of S-9 ml/h. Immunoelectrophoresis was carried out as described by Sheidegger [ 14 1. Case report A 55year old male electrical engineer was admitted to this hospital on March 13th, 1975 suffering from inspiratory dyspnea. Except for an attack of malaria at the age of 24, his own previous history and his family history were not remarkable. Tracheotomy was carried out as a first aid operation and 3 weeks later, on April 4th, 1975, after diagnosis of cancer of the larynx, total laryngectomy was performed. The cancer was identified histologically as squamous cell carcinoma. No metastases to the regional lymphnodes were observed. After the operation, he continued to lose weight and complained of palpitation. Although he had no obvious goiter or abnorm~ity of the eyes, he was diagnosed as having hyperthyroidism with high levels of serum thyroxine and triiodothyronine and low cholesterol. Therapy with an anti-thyroid drug was started on April 30th, 1975. The abnormal pattern of LDH isoenzymes was first observed on examination of serum obtained on April 23rd, when the LDH activity was 255 units. The serum alkaline phosphatase level was 11.0 King-Armstrong units and on electrophoresis a small fraction (Alp-O) was seen which did not move from the origin. The erythroeyte sedimentation rate was 25 mm after 1 h. Mild anemia was detected but otherwise routine blood and urine analysis gave normal results. The levels of serum glutamic oxalacetic transaminase and glutamic pyruvic transaminase were normal. The electrophoretic pattern of serum protein and the levels of serum immunoglobulins IgG, IgA and IgM were normal. Anti-thyroglobulin antibody was not detected but anti-thyroid microsomal antibody with a titer of 1 : lo3 was found. Physical and radiological examination showed no evidence of distant met‘astasis. Results The electrophoretogram of LDH isoenzymes of the patient’s serum showed an extra band between LDH-1 and LDH-2 (Fig. 1). The gel filtration pattern of the patient’s serum on Sephadex G-200 is shown in Fig. 2, Two abnormal peaks of LDH activity were detected in front of the main peak, which had the same mobility as normal LDH. The material in these abnormal peaks was found to give the extra band on electrophoresis. No staining for LDH activity was demonstrated after immunoelectrophoresis of the serum against antisera to immunoglobulin IgG, IgA and IgM. The LDH isoenzyme pattern of a lysate of the patient’s erythrocytes did not show either
231
before
after
88 days
7- l-1975 _--._
assayed
after
days
43
5-17-1975
sera were
after
days
4-30-197
* Sample
after
19 days
26
4-23-1975
after
*
*
OF
AND
25.5 25.4 0 0
76.5
69.7
71.0
70.0
at -20°C.
---.--____-
65.4 47.5
50.4
73.7
LDH-T
(units)
LDH
LDH-I
activities
storage
260
260
270
255
270
300
days
11
LDH
ACTIVITIES
Total
after
5
SERUM
relative
ON
operation
to
Time
UP STUDY
7 days
I
4-11-1975
3-241975
Date
FOLLOW
TABLE
-__
16.2
75.1
66.7
62.2
67.8
65.1
LDH-2
--__~
ALKALINE
11.0 11.5
7.3 11.2 15.7 19.2 15.6
11.9 17.3 22.9 24.2 25.0
61.8 62.2 69.7 70.5 71.2 ----___
15.0
20.1
16.2
82.8
13.6
12.5
16.6
LDH-5 Total
Alkaline LDH-4
-___
ISOENZYMES
LDH-3
PHOSPHATASE
0
6.2 6.0
0
6.2
4.6
0
0 3.2
2.3
0
0
Alp-3
(units)
5.8
6.3
2.8
2.9
9.2 8.6
Alp-2
activities
Alp-1
phosphatase
1.4
0
0
1.6 1.7
1.6 0.9
2.1
3.5
Alp-0
0.8
0.9
1.0
Alp-4
239
Fractcm
No
Fig. 2. Gel filtration of the patient’s .seum on a Sephadex G-200 column. 3.0 ml of serum were applied to a 2.4 cm X 65 cm column. Elution was performed at a flow rate of 6.6 ml/h 4’C. l -r. protein: l - - - - - -*, LDH activity.
the additional LDH isoenzyme fraction or irregularity in electrophoretic mobility. On electrophoresis of a mixture of equal amounts of normal serum and the patient’s serum, the distribution patterns of activities in the two were superimposed. When the patient’s serum was incubated at 56°C for 30 min, the bands of LDH-4 and LDH-5 disappeared, but the abnormal extra band still appeared. During this heat inactivation treatment the total LDH activities of normal serum and the patient’s serum decreased to 67% and 77%, respectively. Table I shows the results of follow up studies on this patient. The activity of the abnormal LDH isoenzyme, which was tentatively named LDH-T, decreased after removal of the cancer tissue and had disappeared on May 17,1975. When serum obtained at this time was fractionated by gel filtration on Sephadex G200, no abnormal peaks of LDH activity were observed. Another specimen of serum, obtained 11 days before the operation was analysed by electrophoresis after storage at -20°C for 56 days and showed high LDH-T activity (21.8% of the total activity). The appearance of LDH-T was not due to freezing, because a sample of serum from a normal subject did not show any abnormal LDH isoenzyme or an irregular electrophoretic pattern after storage at -20°C for 2 months. Discussion Extra bands of a sixth isoenzyme of serum LDH have been reported by several investigators [2,3,8]. Lundh [2] and Voigt [3] detected an abnormal band between LDH-3 and LDH4 on electrophoresis of sera from a patient with leukemia and a healthy woman, respectively. Nagamine [8] reported an extra
240
band between LDH-2 and LDH-3, attributed to formation of an LDH-immunoglobulin A complex. However, there have been no reports on the presence of abnormal LDH isoenzymes in relation to the clinical course of diseases. We also know of no reports on production of abnormal LDH isoenzymes by tumors. In the serum of the patient described in this report, the extra band of LDH-T was found between LDH-1 and LDH-2. Gel filtration showed that this isoenzyme was a macromolecule. It was stable on heating at 56°C for 30 min. Its appearance was not due to a hereditary abnormality, since the LDH isoenzyme pattern of the patient’s erythrocytes was normal and this LDH-T disappeared from the serum after removal of the cancer by operation. Immunoelectrophoretic studies showed that LDH-T was not a complex of LDH and immunoglobulin. Moreover its appearance was probably not due to hyperthyroidism, since no abnormal extra band was found in the sera of five patients with hyperthyroidism. It is very interesting that LDH-T gradually decreased and finally disappeared after removal of the cancer tissue. Alp-O, that is often found in patients with malignant tumors [15], also decreased and disappeared after the operation. Unfortunately, we did not examine the LDH isoenzymes of the cancer tissue itself. The possibilities of the presence in the patient of serum factors which affected the mobility of normal LDH isoenzymes and of polymers of normal LDH isoenzymes were not completely excluded. However, the results described above strongly suggest that LDH-T originated from this cancer tissue. Acknowledgements The authors are grateful to Dr. E. Ichihara for help in the preparation of the manuscript in English. We wish to thank Miss T. Kawano for the secretarial assistance. References 1
Kreutzer,
2
Lundh,
3
Voigt,
4
Ganrot,
H.H.,
Jacobs,
B. (1967) A.
(1967)
P.O.
5
Kindmark,
C.O.
Biewenga.
J. and
Kitamura,
M.,
Nagamine,
M. (1972)
9
Biewenga,
11
Van
12
Kind.
13
Ogita.
Clin.
Z.,
Clin.
Klin. J. Clin.
(1970)
Hira;suka, Acta Acta
H.J.
(1962)
Clin.
Chin
(1954)
Chiba.
Chim.
Acta
11,
159-169
Y.
5, 146-147
Invest.
Chin
Chin
Acta
Y.,
Clin.
593
Lab.
Clin.
Chim.
Sakoyama.
23,
F. and Chin
E.J.
Biochem.
(Base11
Clin.
and King.
C. (1965)
16.305-309
(1965)
der Helm, P.R.N.
L.G.
Hashimoto,
J. (1972) A.L.
Chem. Scsnd.
Thijs,
7
Francke,
Acta
Experientia
(1969)
8
Babson.
Chin
Z. Klin.
(1967)
6
10
Ph. and
Clin.
A.
24,
49--53
Acta
27.293-299
(1971)
Clin.
Chim.
Acta
34.
419-423
36.139-144 40.407414 12,
210-215
Acta
J. Clin.
7.
124
Pathol.
and Masuzawa,
7, 322-326 M.
(1973)
Y.
and
Physico-Chemical
Biol.
(in Japanese)
17.
63-69 14
Sheidegger,
J.J. (1955)
15
Masuzawa,
M.,
Japanese)
17.
Kamata, 71-76
Int. T.,
Arch.
Allergy
Sakoyama,
7, 103-110 Y.,
Chiba,
Ogita,
2.
(1973)
Physico-Chemical
Biol.
(in
