Vol. 30. No. 4. 1986 (301)

(Regular paper)

Serum lactate dehydrogenase isoenzyme (s) linked to immunoglobulin

G with extremely decreased activity detected in myocardial infarct

Hiroshi Sasaki*, Mitsutaka Yoshida** and Kei Furiya***

SUMMARY

LDH isoenzyme (s) linked to IgG of the lambda type found in the serum of a patienthaving had myocardial infarction is reported. This LDH-IgG complex has almost noenzymatic activity. It was found that the IgG of the patient acted as an inhibitor of LDH.LDH activity in normal human serum was inhibited and abnormal patterns of LDHisoenzyme (s) appeared when certain amounts of the patient's serum was added to acontrol serum as well as purified LDH isoenzymes. The IgG fraction in the serum wasseparated from the LDH-IgG complexes by 5'-AMP-Sepharose 4B column chromatogra-phy. Subclass analysis of IgG made it clear that the IgG in the serum was IgG3.

Key words: LDH anomaly, LDH isoenzyme, LDH linked immunoglobulin, LDH inhibitor.

INTRODUCTION

Since Ganrot1) reported an LDH-IgA complex in

human serum in 1967, many reports have been

published on the binding of LDH (EC 1.1.1.27) toIgA and IgG to form complexes that produce un-

usual LDH isoenzyme patterns2-7). Most of the cases

involved abnormalities such as; The absence of one

or more of the normal LDH fractions while addi-

tional fractions were present, the presence of one or

more additional LDH isoenzymes, and a marked

increase of activity in the LDH-3 band. We detect-

ed LDH-IgG complex (es) with extremely de-

creased enzymatic activity, and a macromolecular

inactivator which combined with LDH isoenzyme

(s) to form LDH-IgG complex (es) that showed no

activity, in the serum of a patient recovering from

myocardial infarction8). The LDH activity in the

erythrocytes showed normal patterns.

This paper describes the first example in which

the complex formation between serum LDH and

IgG of the lambda type, leading to an extremely

decreased activity, was detected in a patient's ser-

um. In addition, the results of some investigations

on abnormal immunoglobulin which inactivated A-

and B- subunits of LDH in the patient's serum are

reported and discussed.

MATERIALS AND METHODS

Subject: The subject whose serum showed an anti

心筋梗塞患者に見 られた異常免疫 グロブ リンG(λ) が結合 した不活性 血清LDHの 性質＊佐々 木博, 国家公務員等共済組合連合会立川病院 ・中央検査部生化学

＊＊吉田光孝, 東邦大学 ・理学部生理化学

＊＊＊降矢焚, 東京女子医科大学 ・生化学

Correspondence address, Hiroshi Sasaki, Federation of National Public Service and Affiliated Personnel MutualAid Association. Tachikawa Hospital, Tachikawa-shi, Tokyo 190, Japan(Received March 28 1986, Accepted June 18 1986)

-55-

(302) The Physico-Chemical Biology

-LDH activity and contained LDH isoenzyme (s)

linked to IgG with extremely decreased activity was

a 58-year-old man with cardiac insufficiency, liver

damage and ascites. He had suffered from

myocardial infarction about 6 years before. The

LDH isoenzyme anomaly was detected during

treatment of the subject after the myocardial

infarction. The results of laboratory studies

revealed elevated levels of uric acid (8.9mg/dl), γ-

globulin (21.3%), IgG (1,770mg/dl), IgA (448 mg/

dl) and IgM (221mg/dl).

Materials: Antisera against human IgG (γ), IgA

(α), IgM(μ), IgD(δ), IgE(ε), and light chains

kappa (κ) and lambda (λ) were obtained from

Dakopatts Co., Ltd. and Miles Laboratories, Inc. 5'-AMP-Sepharose 4B, Protein A -Sepharose Cl-4B

were purchased from Pharmacia Chemicals. All

other chemicals used were of analytical grade.

Enzyme activity: LDH activity was measured by

optimized standard method using a Hitachi 726

automated analyzer (Hitachi Co., Ltd., Japan) as

well as an LKB 8600 Reaction Rate Analyzer (LKB

Co., Ltd., Sweden). The activity of LDH was

expressed as IU/L.

Separation of LDH isoenzymes in serum: Isoen-

zymes were separated by electrophoresis on agarose

gels and enzyme activity was developed using

nitroblue tetrazolium with diaphorase9).

Electrophoretic analysis of serum proteins: Ser-

um proteins were electrophoretically separated by

the method described by Ogawa et al.10) with slight

modifications, using an automated electrophoresis

system Model AES (Olympus Co., Ltd.).

Immunoelectrophoresis: Immunoelectrophoresis

was carried out according to the technique de-

scribed by Graber et al.11)

Purification of LDH isoenzymes from human eryth-

rocytes: Each isoenzyme was highly purified

from human erythrocytes by employing the tech-

niques of general ligand affinity chromatography

using 5'-AMP-Sepharose 4B according to the

methods of Bachman et al.12) and Igarashi et al.13)

with slight modifications.

Separation of free immunoglobulin fraction that

acts as an inhibitor of LDH in the serum: The

free immunoglobulin fraction in the patient's serum

was separated from LDH as well as LDH-im-

munoglobulin complex by 5'-AMP-Sepharose 4B

column chromatography. The free immunoglobulin

fraction was obtained and its subclass analysis was

performed by Protein A-Sepharose Cl-4B column

chromatography 14).

Identification of free immunoglobulin in the ser-

um was performed by the method of Tozawa et al.15)

RESULTS

Electrophoretic analysis of LDH isoenzymes:

The LDH isoenzyme patterns of the patient's serum

are given in Fig. 1. The patient's serum showed a

marked decrease of a slow-moving band near LDH-4 (A3B) and complete loss of LDH -1 (B4), -2

(AB3), -3 (A2B2) and -5 (A4). The LDH isoen-

Fig. 1 Electrophoretic patterns of LDH isoen-zymes.

(a) patient's serum; (b) mixture of the patient's serumand patient's hemolysate with the ratio of 1:1 byvolume; (c) patient's hemolysate; (d) mixture of the

patient's serum and "control" serum with the ratio of1:1 by volume; (e) "control" serum

-56-

Vol. 30. No. 4. 1986 (303)

zyme patterns of the lysates of the patient's red

blood cells showed neither additional LDH isoen-

zyme fractions nor irregularities in electrophoretic

mobility (Fig. 1).

In agreement with the above observations the

total activity of LDH was almost zero in the

patient's serum when either pyruvate or lactate was

used as a substrate, though the value of this

patient's serum had been normal till 5 years before.

Effects of pH or some reagents on the LDH-isoen-

zyme-IgG complex (es) on to the enzyme activity

was as follows: Total activity of LDH in the

patient's serum was determined under various con-

ditions of pH ranging from 2.1 to 8.1; however, any

LDH activity was not restored at this pH range.

Treatment of the patient's serum with 3 M urea, 100

mM β-mercaptoethanol, 100mM dithiothreitol or

20mM glutathion did not cause any restoration of

LDH activity.

LDH-inactivating activity in the serum: To

investigate the inactivating effect of the patient's

serum on LDH activity, equal volumes of the

patient's serum and control serum were mixed and

the mixture was assayed after being kept at 4℃

overnight. As shown in Fig. 1, all bands detected in

the control serum disappeared and a slow-moving

band near LDH-4 appeared instead. These results

are summarized in Table 1. Effects of addition of

the patient's serum to the hemolysates were

examined in the same way as above. As in the case

of the serum, a slow-moving band near LDH-4

appeared and an additional faint band near LDH-3

was observed (Fig. 1).

Further evidence of the inactivating effect of the

patient's serum on LDH isoenzymes was provided in

more detail by the following experiments. Equal

volumes of the patient's serum, diluted to various

degree, were mixed with the control serum and the

mixtures, after kept at 4℃ overnight, were subject-

ed to electrophoretic analysis and the activity bands

were visualized as mentioned above. As shown in

Fig. 2, LDH-inactivating activity was observed

even in the patient's serum diluted 128-fold. The

electrophoretic patterns of LDH-3, -4 and -5 isoen-

zymes began to change by incubation with the

patient's serum of 128×dilution. Slight changes of

LDH-3, -4 and -5 fractions were observed and a few

broad bands appeared between LDH-3 and -5 frac-

tions. Contrary to the decreases of these fractions,

LDH-1 and -2 showed little change on agarose gel

electrophoresis. Complete disappearance of the

original bands and appearance of a broad band

corresponding to LDH-4 isoenzyme by incubation

of the normal serum with the patient's serum of 16

×dilution were seen (Fig. 2). To investigate

which type of the subunits (A or B) of LDH tended

Table 1. Inhibition of enzymatic activity of LDH isoenzymes by the patient's serum

*: Tested serum was mixed with an equal volume of the patient's serum

and allowed to stand overnight at 4℃.

**: The arithmetic mean of activities of both sera.

***: Activated of tested mixture.

-57-

(304) The Physico-Chemical Biology

to be bound and inactivated by the patient's serum,

we continued a similar electrophoretic analyses

using each isoenzyme fraction obtained from human

red blood cells, instead of normal serum. Fig. 3

demonstrates that by incubation of each isoenzyme

with the patient's serum each isoenzyme is made to

move further toward the cathode, each original

band disappears, and a slow-moving band which

corresponds to LDH-4 appears. Although dialysis

of the patient's serum against physiological saline at

4℃ for 24hrs did not affect the inactivating activity,

this activity decreased gradually by incubation of

the serum in physiological saline at 56℃ for up to 60

min.

Inhibitory effects of the patient's serum on activ-

ities of several enzymes other than LDH: In

addition to LDH, the activity of α-hydroxybutyrate

dehydrogenase (α-HBDH) also markedly de-

creased after equal volumes of the patient's serum

and control serum were mixed and kept at 4℃

overnight. Contrarily, cholinesterase, amylase and

isocitrate dehydrogenase activities were little

inhibited by the serum (Table 2).

Identification of inactivating factor: The

patient's serum was treated with specific antisera, e.

g. anti-IgG, -IgA, -IgM, -IgD, -IgE, -lambda chain

and -kappa chain. The resultant precipitates were

added to control serum having adequate LDH

activity (Fig. 4). There was no significant

decrease of LDH activity when the immuno-

globulins other than IgG in the patient's serum

precipitated. On the other hand, precipitation of

IgG or lambda chain resulted in an almost complete

loss of LDH activity of the normal serum. The

binding, inactivating substance for LDH thus

proved to be IgG (λ) (Table 3).

Fig. 2 LDH isoenzyme patterns of "control" ser-um combined with various amounts of the

patient's serum (a-e)

The patient's serum was diluted 128, 64, 32, 16 and 8times for a, b, c, d and e respectively, and mixed with"control" serum in ratio 1:1 by volume; (f) "con-

trol" serum

Fig. 3 Changes in electrophoretic patterns of pur-ified LDH isoenzymes combined with the

patient's serum.

Each isoenzyme fraction was mixed with an equal

volume of the patient's serum.

-58-

Vol. 30. No. 4. 1986 (305)

Subclass determination of the patient's serum

IgG: The patient's serum was diluted two times by

10mM phosphate buffer, pH 7.0, and 0.4ml of the

diluted serum was applied onto a 5'-AMP-Sephar-

ose 4B column, 0.5cm by 1.0cm, which was eluted

with 1mM NADH2. The eluate containing free IgG

was applied onto Protein A-Sepharose Cl-4B col-

umn (0.5cm×l.0cm) and was eluted with 10mM

phosphate buffer, pH7.2. This eluate containing

IgG3 exhibited inactivating activity for LDH. The

effective subclass of patient's serum IgG was there-

fore identified as IgG3.

Table 2. Inhibitory effects on several enzymeactivities by the patient's serum

*: Tested serum was mixed with an equal volume of the patient's serum

and allowed to stand overnight at 4℃.

LDH, Lactate dehydrogenase; AST, Aspartate aminotransferase; ALT, Alanineaminosransferase; ALP, Alkaline phosphatase; ChE, Cholin esterase; γ-GTP, γ-

Glutamyl transferase; AMY, Amylase; CK, Creatine kinase; ICD, Isocitrate dehy-drogenase; GDH, Glutamate dehydrogenase; α-HBDH, α-Hydroxybutyrate dehy-

drogenase; SDH, Sorbitol dehydrogenase

Table 3. Identification of the immunoglobulin

in the patient's serum

*: E570×103

-59-

(306) The, Physico-Chemical Biology

DISCUSSION

The presence of macromolecular LDH's in serum

has previously been reported1-7). However, the

clinical significance of these high molecular weight

complexes is still poorly defined. The complexes

have been observed in the serum of healthy individ-

uals and patients of certain diseases, e.g. cardiac

asthma, myocardial infarction or ulcerative colitis. In

the present case, however, investigation of the

poorly understood, decreased LDH activity led to

the discovery of circulating LDH complexes.

A mixture of the patient's serum and a normal

serum gave the same abnormal electrophoretic

pattern as the patient's serum alone, e.g. all LDH

isoenzymes in the control serum disappeared.

Furthermore, the marked decrease of LDH activity

was observed when the normal serum was incubated

with the patient's serum. Similar results were

obtained for the purified LDH isoenzymes from

human erythrocytes.

Kitamura et al.16) reported a case of complete

deficiency of B-subunit (s) of LDH isoenzymes. In

that case, specimens of the serum, saliva and

hemolysate did not contain any of LDH-1, -2, -3

and -4. In the case presented here, however, defi-

ciency of A- or B-subunit could be ruled out

because the hemolysate gave normal isoenzyme

patterns. Furthermore, the serum of this patient

had shown a normal LDH activity and a normal

electrophoretic isoenzyme pattern until several

years ago.

Nagamine17) presented an abnormal LDH isoen-

zyme pattern of LDH-2, -3, -4 and -5 with de-

Fig. 4. Method of identification of the immunoglobulin in

the patient's serum

-60-

Vol. 30. No. 4. 1986 (307)

creased activity. A heat labile protein component

which inactivated A-subunit (s) of LDH in the

serum of the patient was reported at the same time.

Gray et al.18) reported the first example of IgG

which complexed with LDH only at lower tempera-

tures and resulting in irreversible loss of activity of

the LDH isoenzymes having A-subunits (i.e., all but

LDH-1). The present case is entirely different

from these reports. It is presumed that abnormal

IgG (λ) of the patient bind to normal LDH mole-

cules to form LDH-IgG complex (es) with marked-

ly decreased enzyme activity. The binding within

the LDH-IgG complex (es) was particularly tight

due to the specific nature of the immunoglobulin,

and it is unable to separate the complex by treating

it with buffers of various pH ranging from 2.1

to 8.1 or with 3M urea, 100mM β-mercaptoethanol

or dithiothreitol. It was previously reported that an

LDH-linked inhibitory factor was exclusively

bound to the A-subunit of LDH isoenzymes since

each of the isoenzymes inactivated had A-subunit

(s) in common. In the present case, the inactivatingeffect of the patient's serum was observed for both

A- and B-subunits of LDH isoenzymes.

The reconstitution experiments, using normal

serum or purified isoenzymes from human eryth-

rocytes, revealed that A-subunits were more-

strongly affected then B-subunits by the inhibitory

factor. This case was unique since the activities of

both the A- and B-subunits were simultaneously

suppressed. Although identification of the inhibi-

tory factor in the LDH-IgG complex (es) failed due

to markedly decreased enzyme activity of the com-

lex (es), at least it was proven from the results

shown in Table 3 that IgG (λ) was concerned,

Nakagawa et al.19) reported that IgG, which bound

to both placental and intestinal ALP's was present

in the circulation. In addition, this immunoglobulin

had an inhibitory effect on the placental ALP. This

supports the hypothesis that the enzyme-linked

immunoglobulin is an oligoantigenic antibody. The

mechanism of interaction of IgG with LDH subunits

may be different between the A- and B-subunits.

LDH-immunoglobulin complexes have been repor-

ted in serum from, healthy subjects2,5-7) as well as

subunits with various diseases2-4,7,20-23). In our sub-

ject the anti-LDH IgG activity appeared to have

been present for a few years and constant in quan-

tity for at least seven months.

The inhibitory effects of the patient's serum on

some other enzymes were studied and it was obser-

ved that the degree of inhibitory effect on α-HBDH

was the same as that of LDH. Further studies on

this LDH-IgG complex. (es) are in progress to reach

a definite conclusion.

ACKNOWLEDGEMENT

The authors appreciate Mr. Itsuo Kuroda, in

Division of Biochemistry of Central Laboratory,

Mutual Aid Tachikawa Hospital, for his excellent

technical assistance and Dr. Kousuke Mori, a direc-

tor of Central Laboratory of the Hospital, for his

encouragement.

REFERENCES

1) Ganrot, P.O.: Experientia, 23: 593, 1967.2) Nagamine, M.: Clin. Chim. Acta, 36: 139,

1972.

3) Biewenga, J.: Clin. Chim. Acta, 41: 139,1973.

4) Biewenga, J.: Clin. Chim. Acta, 16: 149,

1977.

5) Trocha, P.J.: Clin. Chem., 23: 1780, 1977.6) Weijers, R.N.M. et al.: Clin. Chem., 29:

272, 1983.

7) Biewenga, J. and Feltkamp, T.E.W.: Clin.Chim. Acta, 58: 239, 1975.

8) Sasaki, H. et al.: Physico-Chem. Biol.

(Seibutsubutsurikagaku), 28: 143, 1984.9) Kuroda, I. et al.: Physico-Chem. Biol.

(Seibutsubutsurikagaku), 29: 403, 1985.10) Ogawa, T. et al.: Physico-Chem. Biol.

(Seibutsubutsurikagaku), 11: 351, 1966.11) Graber, P. et al.: Biochim. Biophys. Acta,

10: 193, 1953.

12) Bachman, B.K, and Chi-Yu, L.: Anal.Biochem., 72: 153, 1976.

13) Igarashi, F. et al.: Jap. J. Clin. Chem., 11:115, 1982.

14) Bird, P. et al.: J. Immunol. Methods, 71:

-61-

(308) The Physico-Chemical Biology

97, 1984.

15) Tozawa, T. et al.: Physico-Chem. Biol.

(Seibutsubutsurikagaku), 26: 243, 1982.16) Kitamura, M. et al.: Clin. Chim. Acta,

34: 419, 1971.

17) Nagamine, M.: Clin. Chim. Acta, 50: 173,1974.

18) Gray, G.W. and Smith, M.J.: Clin. Chem.,30: 11, 1984.

19) Nakagawa, H. et al.: Clin. Chem., 29: 375,1983.

20) Biewenga, J. and Feltkamp, T.E.W.: Clin.Chim. Acta, 64: 101, 1975.

21) Gorus, F. et al.: Clin. Chem., 28: 236, 1982.22) Pudek, M.R. and Jacobson, B.E.: Clin.

Chem., 28: 2434, 1982.

23) Markel, S.F. and Janich, S.L.: Am. J. Clin.Pathol., 61: 328, 1974.

要 旨

患者血 清 中に乳 酸脱 水素 酵素 (LDH; EC1.1.1.27)

の活 性阻害 因子が 存在 す る こ と, また その阻害 因子 は

免疫 グロブ リンで あ り, 主 に A-subunit に対 す る阻 害

がみ られ た例 が報告 され て いる. 我々 は, 血 清LDH活

性 はほ とん ど検 出で きなか ったが赤 血球LDH活 性 が

正 常 に認 め られた心 筋梗塞 の既往 歴 の あ る症例 に遭遇

した.

この症 例 の血清 中のLDH阻 害 因子 につ い て生化 学

的 ・免疫化 学的性 質 に関 す る解析 を行 った. その結果,

この 阻害 因子 は トリス緩衝 液(pH7,4) に 対 して透

析 されず, 56℃, 60分 間の熱処 理 ・クエ ン酸緩 衝液 に

よるpH解 離 実験 ・3M尿 素 お よび各SH試 薬処 理 な

ど に よる変 化 はみ られ なか った. この阻 害 因子 を保 存

す る血清 を, ヒ ト赤血 球か ら精 製 したLDHア イ ソザ

イ ムに添加 す ると各ア イ ソザ イム に95～99%の 酵素 活

性 阻 害 が認 め られ た. さ らにLDH結 合 免疫 グロ ブ リ

ンの検 索 に よ り従 来の報告 にみ られ な い異常免 疫 グ ロ

ブ リンG, λ (IgGλ) 結合 型 で あり, LDH-Aお よび

-B subunit の両 者 に対す る活 性阻害 が観察 された. 5'

-AMP Sepharose 4Bお よび Protein A-Sepharose

Cl-4Bな ど を用 いて 分 離 ・精製 さ れ た患 者 血 清 中の

IgGは, 他 の血清LDHに 対 して阻害 効果 を示 し, さ ら

に この 異常IgGの サ ブ クラス のIgG3も 同様 の 阻害 効

果 を示 した.

-62-