101

Clinica Chimica Acta, 64 (1975) 101-116 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 7061

LACTATE DEHYDROGENASE (LDH)-IgG3 COMPLEXES IN HUMAN SERUM

JEIKE

BIEWENGA

IMMUNOGLOBULIN

and T.E.W. FELTKAMP

~~~artrnent of Histology, Free ~n~uersity, P-0. Box 7161,Amsterdam and ~epurtmerzt of Autoimmu~e Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (The Netherlands) (Received

April 14, 1975)

Summary Sera of fifteen patients conning complexes of LDH and IgG were analysed. The LDH-IgG complexes of fourteen of these patients were investigated for their subclass specificity. They appeared to consist of LDH and IgG3, except for one of the patients. The serum of the latter patient also contained complexes of LDH and IgG, . In this serum both types of the immunoglobulin light chains were involved in the formation of the complexes, whereas in the serum of the patients with LDH-IgG, complexes alone, they only contained one of the two immunoglobulin light chain types; it mainly concerned kappa type. The results of the present study were compared to those of a previous study on LDH-IgA complexes, in which the IgA fraction always contained light chains of kappa type. The mean LDH activity and the heat stability of the LDH activity in the sera with LDH-IgA complexes appeared to be higher than in the sera with LDH-IgG3 complexes. In contrast to complexes of LDH and IgA which occurred in all age groups LDH-IgG3 complexes were mainly found in elderly persons. Although auto-antibodies to different tissue antigens were present in the majority of the sera containing LDH-IgG3 complexes, antibodies to LDH could not be demonstrated. A relationship between the presence of the complexes in the serum and a particular disease could not be established.

Introduction Several papers have been published on the occurrence of complexes of LDH and immunoglobulins in human serum (l-61. LDH-IgA as well as LDHIgG complexes have been described, but complexes with immunoglobulins of other classes have never been reported. The formation of the complexes was thought to be due to an anomaly in the structure (either primary, secondary or

102

tertiary) of the immuno~lobulin involved, since normal LDH isoenzymes were present in the blood cells of the patients, and since the formation of complexes was also achieved with purified immunoglobulin fractions obtained from the patients and normal LDH of other subjects. Sera which contained LDH-IgA or LDH-IgG complexes showed an abnormal LDH isoenzyme pattern in the sense that one or more of the LDH fractions had abnormal electrophoretic mobility or were missing. In a previous paper [4] we reported the presence of an extra LDH band in a serum with LDH-IgG complexes. In another paper [6] dealing with LDH-IgA complexes we could demonstrate that the IgA involved was always of the kappa light chain type. It was not proved that IgA in the LDH-IgA complexes was an antibody to LDH. The purpose of the present study was to get more information on the nature of LDH-IgG complexes. We therefore investigated these complexes in the serum of fifteen patients. It will be shown that in fourteen of the cases the complexes contained IgG of the subclass IgG3, while only in one case complexes were present of LDH with immunoglobulin of the subclass specificity IgGi in addition to the LDH-IgG3 complexes. Although the clinical and serological findings of several of the patients studied were suggestive of an autoimmune disease, it could not be proved that the IgG invoived in the formation of the complexes was an antibody to LDH. Materials and methods Serum samples were obtained from patients with electrophoretically abnormal LDH patterns. Only cases in which the presence of LDH-IgG complexes was confirmed by immunoelectrophoresis with staining for LDH activity were included in the present investigation. Serum samples with normal electrophoretic mobility of the LDH isoenzymes were used as control, The antisera to the subclasses of IgG were a kind gift from Miss Marijke van der Giessen of the Netherlands Red Cross Blood Transfusion Service, Amsterdam. The techniques that were used for agar gel electrophoresis, immunoele~trophoresis, de~rmination of LDH activity, determination of the heat stability of LDH activity, staining for LDH activity, purification of LDH, gelfiltration on Sephadex G-200, dernonstration of antibodies to nuclear and other tissue antigens and preparation of a rabbit antiserum to human LDH have been described previously [3,6]. Rheumatoid factors were demonstrated on human 0 red cells sensitized with rabbit immunoglobuiins according to van Loghem-Langereis [7] and Feltkamp and van Rossum [S] . Immunoglobulin concentrations were determined with Tripartigen plates (Behringwerke, Marburg). IgG was isolated from serum of three out of the fifteen patients. The IgG of patient La was obtained by ionexchange chromatography on DEAE-Sephadex A-50 with 0.02 M phosphate buffer, pH 8.0. The protein fractions containing only IgG, as shown by immunoelectrophoresis were collected. Affinity chromatography was used for the purification of IgG from the serum of patients Pr and Do. The procedure was carried out as follows: Immunoelectrophoretically pure IgG from a pool of sera, obtained by ionexchange

Fig. 1. Immunoelectrophoretic pattern of purified rabbit anti-human-IgG normal human serum (NHS) and polyvalent goat anti-rabbit serum (G-a-R).

(R-a-HGG)

obtained

with

chromatography on a DEAE-Sephadex A-50 column using gradient elution with 0.02-0.3 M phosphate buffer, pH 7.0, was injected in rabbits. The rabbits were primed with a mixture of this IgG and complete Freund’s adjuvant (FCA) after which they were given booster-injections with IgG only. The antibody containing fractions of the rabbit sera were purified by affinity chromatography using human IgG coupled to CNBr-Sepharose 4B (Pharmacia, Uppsala, Sweden) according to the manufacturers prescription. The antibody preparation thus obtained precipitated in immunoelectrophoresis IgG and a minor amount of IgA from normal human serum. The protein preparation probably contained IgG which was partly polymerized, as it was not completely soluble and showed in immunoelectrophoresis with polyvalent goat antirabbit serum a precipitation line close to the well having identity with the IgG-line (Fig. 1). With this rabbit anti-human-IgG coupled to CNBr-Sepharose 4B, IgG was isolated from the serum of patients Pr (Fig. 2) and Do using columns packed with Sephadex G-25 (height 19 cm) and rabbit anti-human-IgG coupled to Sepharose 4B (height 7 cm) as a top layer. The diameter of the columns was 1.5 cm. The immunoelectrophoretic pattern of the IgG fractions of serum Pr (Fig. 3) and Do showed only one precipitation line with an antiserum to whole human serum. All IgG preparations were dialysed against demineralized water and freeze dried.

-

e,uate YOl”rm

Fig. 2. Purification of IgG from the serum of patient Pr by affinity chromatography. and II contain IgG and degradation products. respectively.

The eluate regions I

104

Fig. 3. Immunoelectrophoretic pattern of the IgG fraction purified region I, Fig. 2) obtained with an antiserum to whole human serum.

from

the serum

of patient

Pr (eluate

Fig. 4. Immunoelectrophoretic patterns of serum La with LDH-IgG complexes obtained with anti-IgG compared with a control serum (C). Upper half stained for proteins and lower half stained for LDH activity.

Since Kreutzer et al. [9] reported that anomalies in the LDH isoenzyme patterns of serum become less pronounced or even disappear after addition of NAD to the serum, the influence of NAD on the LDH isoenzyme patterns of our sera was also studied. Results In the serum of the fifteen patients with electrophoretically abnormal LDH isoenzyme patterns, LDH activity was present on the immunoprecipitation line of IgG. A representative example is given in Fig. 4. In more than 100 sera with electrophoretically normal LDH isoenzyme patterns no such activity could be observed. Abnormal electrophoretic mobility of LDH isoenzymes can be due to a genetic anomaly in the LDH isoenzymes, which is expressed both in serum and blood cells. Therefore, the LDH isoenzyme patterns of lysates of washed red cells and leucocytes of nine out of the fifteen patients (Ui, Wi, La, Sp, Vr, Pr, Ku, Do, Ed) were investigated for abnormal LDH fractions, but abnormalities were not observed. The clinical data of the patients and the results of the serum analysis are noted in Table I. LDH activity and LDH isoenzyme patterns On the basis of differences between the LDH isoenzyme patterns of the sera studied, three groups could be distinguished. The first group appeared to comprise sera in which nearly all LDH activity was present in a diffuse band in the -y-globulin region (Fig. 5). Electrophoretically normal LDH isoenzymes were not observed. Patients Ui and La pertaining to this group could be followed up; they have been described extensively in a previous paper [4]. In short, when patient Ui was admitted to the hospital he appeared to have highly increased serum LDH activity; in the LDH isoenzyme pattern electrophoretically normal LDH fractions were observed at

TABLE I

Sl

Groua III

II

I

-

f m

,f

f m

m

f

f f f m m

m m f

SEX

81 12

67 38 67 91 52

68 61 69 64 70

51 68 82

Age

~-----

Heart failure and goitre Oesophagus carcinoma

Colon diverticulosis Duodenal ulcer Hypertensione Carcinoma of the prostate Hyper~poprote~em~ with hypertension

Cerebra vascular accident Rheumatoid arthrltisd Heart failure, rheumatoid arthritisd Cholelitbiasis, thyrotoxic adenomae Severe lung function disorder, rheumatic complaints

SLE Chronic asthmatic bronchitis Active chronic hepatitis

-

Diagnosis

+, slightly increased; ++, mWerateIy increased and +++, highly increased. Values of about 70% were found in serum of healthy individuals. Analysed at the period of greatest aberration. Definite rheumatoid arthritis according to the ARA criteria Ill]. e Treated with or-methyl DOPA.

a b c d

Le Ed

tH Er R KU Do

SP Vk‘

Grow

Group

Hg HI!

Wi La

Ui

Patient

CLINICAL DATA AND RESULTS OF SERUM ANALYSIS 0

-

f +++

normal +* + +

+

normal + + + +++

+ +++C

+++c

LDH activitya

--

67 48

nd. 67 65 73 80

n.d. 41 70 64 68

45c 45 15-2OC

___

Heat stability of LDH activity in %b

__---_~-.-

4.7 3.2

1.9 2.9 1.0 3.6 3.2

3.2 1.9 2.3 3.6 5.5

2.0 2.9 8.6

IgA

1.6 1.2

2.2 1.2 1.6 1.5

1.4

0.8 1.4 2.6 1.5 2.5

1.1 1.4 2.0

IgM

Concentrations of the ~rnu~o~obul~ __~--

15.9 13.2

13.9 12.4 IO.8 13.6 12.5

12.3 33.8 13.2 10.6 24.8

24.0c 14.2 25.0e

I&

Fig. 5. Protein and LDH isoenzyme patterns proteins; b, stain_ed for LDH activity.

of the serum

of patients

Ui and La (Group

I). a. stained

for

that time. Two weeks later the LDH activity was still highly increased but the LDH isoenzyme pattern showed a very diffuse band in the +y-globulin region (Fig. 5). About one month after admission the serum LDH activity decreased and also the pattern of the LDH isoenzymes became normal within about three months. Patient La also showed on admission to the hospital highly increased serum LDH activity that decreased slowly to slightly elevated levels until about one year after admission, during which period the LDH isoenzyme pattern did not change. In the following half year separate LDH isoenzyme fractions, with still abnormal electrophoretic mobility, became visible and the LDH activity further decreased. The patient finally died from an acute infection of the respiratory tract. The LDH isoenzyme patterns of the sera of group I did not change on addition of NAD. The heat stability of the LDH activity of the sera was decreased (Table I).

c SP .> ”

Vr C Fig. 6. LDH isoenzyme serum (C).

patterns

of the serum

of patients

SP

and VI (Group

II), compared

with a control

107

In the second group the LDH activity was found in the fl-r-globulin region (Fig. 6). The LDH activity was normal to slightly increased except for patient Vr whose serum showed a highly increased LDH activity. Four out of the five sera belonging to this group showed also a sharp band with weak LDH activity in the region of a normal LDH-5 fraction. In the fifth serum (Vr) such a band situated between LDH-4 and LDH-5 was also present, but the LDH activity was that low, that it was only discovered on gelfiltration of the serum followed by electrophoresis of the eluates. The LDH isoenzyme patterns of the serum of the patients Hr and Vr remained unchanged for at least one month. Two years before the abnormal LDH isoenzyme pattern was observed, the serum of patient Vr had shown decreased LDH activity and normal r-globulin concentration. Follow up of the LDH isoenzyme patterns of the other patients was not possible. When prior to electrophoresis NAD was added to the sera of group II a remarkable change occurred: the LDH isoenzyme patterns showed sharp LDH-1 and LDH-2 bands, except for patient Vr. In the latter serum LDH activity was also observed in the (Y1-, p-r- and r-globulin region but the bands were diffuse. The heat stability of the LDH activity of these sera was normal or only slightly decreased (Table I). The third group is characterized by the presence of an extra LDH fraction between LDH-4 and LDH-5 (Fig. 7). In four out of the five patients the electrophoretic mobility of the other LDH fractions was similar or differed only slightly as compared to the corresponding LDH isoenzymes in normal serum. Although in the serum of the fifth patient (Do) the LDH-2 band was missing and the LDH-3 band was diffuse we included this patient in the third group, since these abnormalities can be fully ascribed to complexes of LDH and IgA which were also present. The LDH activity was normal to moderately increased in all five sera. Addition of NAD to the sera had no influence on the LDH isoenzyme patterns. The LDH isoenzyme patterns of the serum of patients Ku and Do

c DO

Fig. 7. LDH isoenzyme patterns of the serum of patients Ku and Do (Group III) compared with control serum (C). The serum of patient Do contained LDH-IgA complexes next to LDH-IgG complexes.

108

Le C

I, i

Ed c

Fig. 8. LDfi isoenzyme

patterns of the serum of patients

Le and Ed compared

with control

serum (Ct.

remained unchanged for at least two weeks. No change was noted in the serum of patient Pr during 2% years. The other patients were not followed up. The heat stability of the LDH activity was found normal in the serum of patients Er, Pr and Ku, but it was slightly increased in the serum of patient Do, which may be due to the presence of LDH-IgA complexes in the latter serum 131. Two cases (Le, Ed) could not be classified because their serum LDH isoenzyme patterns were different from those of the patients fitting in the groups (Fig. 8). The LDH isoenzyme pattern of serum Le was much more diffuse than that of control serum. After addition of NAD to this serum sharp LDH-1 and LDH-2 bands could be observed. The LDH activity of serum Ed was highly increased. This serum showed five LDH bands, the LDH-1, LDH-2 and LDH-3 bands were more diffuse than in control serum and had increased LDH activity. No change was noted after addition of NAD to serum Ed.

The results of immunoelectrophoretic analysis with staining for LDH activity are presented in Table II. Gelfiltration of five out of the fifteen sera (La, Vr, Pr, Ku, Do) was performed on Sephadex G-200. The abnormal LDH fractions of these sera were eluted more quickly than fractions containing the normal LDH isoenzymes. It is evident from Table II that with an antiserum to whole human serum not all sera with LDH-IgG complexes can be detected. With anti-IgG, these complexes could be demonstrated in all sera, although in some instances the reactions were weakly positive. In the majority of the sera the LDH activity coincided with the IgG precipitation line. In the serum of patients Ui, La, Hr and Ku the LDH activity was found to run parallel to, but at a somewhat greater distance from the trough containing the antiserum than the IgG line. LDH activity could not be demonstrated on the precipitation lines of IgA

II

____-~

-

Le Ed

tH EI Pr KU DO

Hg Hr Sl SP Vr

Ui Wi La

Group III

Group II

Group I

Patient

-

POS

POs

Pas

weakly pos

Pas

weakly pm

PO5

__I_~__-

Pas POS PO5 POs Pas

POS POs POs POs

neg

POs

Pas

POS

weakly pas weakly pos weakly pas

neg neg neg neg

WeaMY

POS

neg

anti-&G

WITH

STAINING

-

--._-

-.__

FOR LDH ACTIVITY,

-~~-

OF THE SERA

___-.

----__~-~

neiz weakly pos

nag neg weakly pos

Pas

neg

neg

neg

neg neg neg

neg

neg n-a

neg neg

n.d.

anti-IgG 1

I_ .__ ____ __-_-_.

neg weakly pos

neg

n.d.

neg neg neg neg

n.d.

neg POS neg POS

pos

n.d.

nag POS POS

n.d. weakly

n.d. neg

neg

n.d. weakly pas

..-~II

lambda

anti-BeneeJones

neg

kappa

anti-Bence-Jones

lines in ~munoeleetrophoresis with various antisera ___.-_ ____._.--_ _---

--____~..~--.--.--~

ANALYSIS

on ~munoprecipitation

net2

human

anti-whole

LDH activity

OF IMMUNOELECTROPHORETIC

n.d.. not done.

RESULTS

TABLE

--_

-

nefi neg

nefi neg

neg neg

neg

nw

n.d.

---~

anti-&G2

--.-

~. --

_~___

L._-_._

POS POS

POS POS POS POS POS

POS POS POS POS

POS

n.d.

anti-&G-j

_

-

neg

neg

neg

neg

neg

neg

neg neg neg neg

n.d. neg neg

~~~

anti-IgGq

.-,__---

-

110

and IgM except for patient Do, whose serum showed also LDH activity on the IgA line. In eight sera the LDH activity was found on the precipitation line obtained with anti light chain sera. Kappa light chains were involved in six cases, lambda light chains in one (Ku) and kappa and lambda light chains in the remaining case (Ed). When antisera to the four subclasses of IgG were used, LDH activity was found on the precipitation line of IgG3 exclusively, except for patient Ed; in this case LDH activity was present on the IgG, as well as on the IgG, line. Investigation for complex formation with LDH of various species Binding of LDH of homologous or heterologous specificity was investigated on mixtures of the serum of the patients with either a human control serum or with serum from different animal species (Table III). In some cases binding of LDH could not be demonstrated due to a low LDH binding capacity of the serum (Sl, Le) or to the fact that the LDH isoenzyme patterns became too complicated (group III) to decide on binding of LDH. The serum of patient Pr must have bound LDH from horse and hog serum because the LDH activity of the LDH-3 bands of the mixtures was relatively low. As the LDH-2 band was missing in the serum binding of LDH could also be demonstrated for serum Do. With purified IgG of the patients Do, La and Pr it was shown that LDH from various species could be bound to the IgG fractions. In a control experiment we found that a rabbit antiserum to human LDH could bind human, bovine and hog LDH, but not horse and rabbit LDH, this in contrast to the serum of the patients. Investigation for autoantibodies By agar gel electrophoresis of mixtures of rabbit anti-LDH with human control serum it was found that LDH-anti-LDH complexes were retained at the application slit. These immune complexes were insoluble over a wide range of LDH/anti-LDH ratios. The antibody activity of the rabbit anti-LDH was localized in the IgG fraction as demonstrated by immunoelectrophoresis. The LDHIgG complexes of the patients were always soluble, even in mixtures of serum of the patients or purified IgG fractions thereof with varying amounts of human serum containing all LDH isoenzymes or with animal sera. The sera were further tested for the presence of antinuclear and rheumatoid factors and for antibodies to thyroid colloid and cytoplasm, gastric parietal cells, adrenocortex and heart cells and to smooth and skeletal muscle. The results are shown in Table IV. A high incidence of antinuclear antibodies (ANA) was found in the patients belonging to groups I and II, antibodies to other tissue antigens were present as well. Several of these patients showed clinical signs of an autoimmune disease (Table I). Such signs were not observed in patients pertaining to group III. In an alternative experiment the sera of patients with confirmed SLE and of patients with definite rheumatoid arthritis (RA) [lo] were tested for LDHimmunoglobulin complexes. No LDH-IgG complexes were detected in hundred

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aau

sod sod

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Sod Sod Sod Sod

‘pm

=?

‘P‘U sod sod u

‘P’”

Jo 9% Pa!J!J’“d

oa

Sod Sod Sod sod

‘pm

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u u u u u

p3 a? ~.~ ___-

sod sod sod sod sod

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JO Hal

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XH

‘P’U sod sod sod sod

~~_

SflOf)O?Ot’IOH

ds

Sod sod Sod sod sod

HUM

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‘P’U sod

‘p-U

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sod Sod sod Sod sod

112 TABLE

1V

RESULTS n.d.,

not

OF

SERUM

ANALYSIS

FOR

THE

PRESENCE

OF

AUTO-ANTIBODIES

done. _.._-_.~_~~__~~_~.__~~._~_

Patient

Anti-nuclear antibodies

Group

_~ Rheumatoid

(ANA)

~~~_ ~~.._

Antibodies

POs

n.d.

Red

neg

ne!z

NlXle

POS

POS

Smooth

Hg

POS

net2

None

Hr

POS

neg

NOIX?

Sl

neg

ad.

Smooth

SP

Pm

neg

None

POS

neg

None

neg neg

nez

NOIll?

EX

neg

NOIE

Pr

neg-pm*

neg

Red

KU

neg

neg

Heart

DO

neg

neg

Gastric

Le

POs

neg

Smooth

Ed

weakly

neg

Gastric

La

cells

(direct

Coombs

test

positive)

Coombs

test

positive)

.--

___~_.

muscles

II

Vr

muscles

III

tH

* This

antigens

I

Wi

Group

autologous

factors

Ui

Group

~~~~~ ~~~~__~~.~

to other

serum

became

pm

positive

during

this

cells

(direct

muscle parietal

cells

muscles par&al

cells

study.

RA sera, 60 of which gave a positive reaction in the Waaler-Rose test, and in 19 SLE sera. LDH-IgA complexes were demonstrated in one of the RA sera. Discussion Among a number of fifteen patients with serum LDH-IgG complexes three groups could be distinguished on the basis of differences in the LDH isoenzyme patterns of their sera. The first group was characterized by an anomalous LDH fraction in the y-globulin region. The second group had an anomalous LDH fraction with /3-r-mobility and the third group showed an extra LDH band between LDH4 and LDH-5. Two patients could not be classified in any of these groups. Enlargement of the material may lead to the detection of more groups. Next to the differences in the LDH isoenzyme patterns other differences were found. In group I the heat stability of the LDH activity was lower than in the other groups. In group II changes occurred in the LDH isoenzyme patterns after addition of NAD. These differences suggest that, although in all cases the formation of the complexes must be attributed to an anomaly in the structure of IgG, this structural variation in IgG is not the same in the three groups. The presence of an IgG fraction that binds LDH appears not to be connected with elevated serum IgG concentrations. The serum LDH activity was increased in the majority of the patients. In some patients this can, at least partially, be due to their clinical condition, but

113

TABLE V LDH ACTIVITY

OF THE SERA WITH LDH-IMMUNOGLOBULIN _~-~-

__ _..___.-

----

--~

COMPLEXES _ __..__

-

_.__~_ _____--

LDH activity*

-.-.-

Normal _~_

.-___.._...-l_-

LDH-&A**: Group I Group II Patient Vo

_

2

f -._.-__---..

++ ____._

3 2

4 1

+++

++++

_ ._ 4 1

Total LDH-IgG: Group I Group II Group III Patients Le and Ed Total __-

2

5

1 1

1 3 3 1

2 ---.--_--_

5

4

1

2 1 1 1

8

1

4 -___. -~

____

_.- .__.

* The LDH activity is expressed as in Table I. ** These patients were described previously [61.

in the other cases such a relation is less obvious. Therefore, we assume that a relation exists between the presence of the LDH-IgG complexes in the sera and the increased LDH activities. Increased LDH activities were also observed in sera with LDH-IgA complexes [6] in which the mean LDH activity was higher than in sera with LDH-IgG complexes (Table V). Reduced catabolism of LDH when it is complexed to IgG or IgA is supposed to have caused the increased serum LDH activities. The LDH activity in the sera with LDH-IgA complexes was more resistant to heating than in sera with LDH-IgG complexes (Table VI). Although the significance of this observation is unknown, it may be related to a diminished stability of IgG3 compared to the other subclasses, under various conditions [ll-131. Clinical improvement in two out of the patients was accompanied by a decrease in serum LDH activity, whereas the anomalies in the LDH isoenzyme patterns became less pronounced in one or even disappeared in the other. These observations indicate that, at least in some patients, a relationship exists between disease and the presence of the LDH-IgG complexes. Such relationship was not observed for patients with LDH-IgA complexes [6]. TABLE VI HEAT STABILITY

OF THE LDH ACTIVITY O-30% -.-_

LDH-IgA* LDH-IgG

1

OF SERA WITH LDH-IMMUNOGLOBULIN

31-50% ~--

51-10% --.

71--75%

COMPLEXES >75%

._ ~._

2 4 _

_1 6 ._-

* See ref. 6. ** This serum contained LDH-IgA next to LDH-IgG complexes.

1 _____-.. -__

11 ** 1

114

TABLE

VII

DISTRIBUTION

OF

PATIENTS

WITH

LDH-IMMUNOGLOBULIN

COMPLEXES

OVER

VARIOUS

AGE

GROUPS

Number

Author(s)

O-20

LDH-IgA Biewenga, Ganrot.

1967

Markel, Total

Total

1

years

41 m-60 years

61m-70

years

>70

4

4

5

years

3

1

[21

1

1 1

1

4

7

4

5

1

2

8

4

1

2

8

complexes

paper

group

[61

[51

group

Kindmark.

21-40

[141

1972 1974

LDH-IgG This

years

in age groups

complexes Feltkamp

Nagamine,

of patients

1969

1

[ll

5

The LDH-IgG complexes were observed more frequently in elderly patients than in the other age groups (Table VII). LDH-IgA complexes occurred in all age groups. Lubrano et al. [15] suggested a relationship between diseases of the liver and abnormal LDH isoenzyme pattern as they found an extra LDH fraction (called “LDH-T”), similar to that we found in our group III, in the serum of several patients with highly increased serum bilirubin concentration. Whether there was a correlation of “LDH-T” with LDH-IgG complexes has never been investigated. In constrast to the patients described by Lubrano et al., the patients of group III had neither increased serum bilirubin concentrations nor were they suffering from a hepatic disease. The clinical data of this group rather suggest that there is no relationship between disease and presence of the extra LDH fraction. Immunoelectrophoretic analysis of the sera led to some remarkable results. Firstly, the formation of complexes with LDH was found to be restricted to the IgG3 subclass. This finding explains why the LDH activity in immunoelectrophoresis with anti-IgG was often found to run parallel to, but at a somewhat greater distance from the trough containing the antiserum than the IgG line. Precipitation of IgG3 at that position was one of the properties of the antiserum. Secondly, only one of the two types of light chains was found in the complexes, except for one case (Ed). The kappa type was mainly involved in LDH-IgG complexes; in LDH-IgA complexes light chains of this type only were found [6]. The presence of the LDH-IgG complexes might be suggestive for an autoimmune disease with antibodies to LDH. Antibodies of restricted immunoglobulin class occur. Karpatkin et al. [16], for instance, demonstrated that the antibodies to platelets in patients with autoimmune thrombocytopenic purpura were of the IgG3 subclass. Kappa and lambda light chains were found in these

115

anti-platelet antibodies whereas in the IgG3 forming complexes with LDH only one type of light chain is present. With regard to the light chains the LDH-IgG3 complexes are more comparable to the anti-red cell antibodies in patients with chronic cold agglutinin disease. The latter antibodies are nearly always of the IgM class with kappa light chains [17]. Although the presence of antibodies can thus be restricted to a single IgG subclass or to one type of the light chains, the results of our analysis gave no answer to the question whether the IgG3 is an antibody to LDH or not. It was not possible to get a precipitate of LDH-IgG3 complexes in mixtures of serum of the patients and human control serum. This may point to a monovalent binding between LDH and IgG3. Furthermore, the binding of LDH was not species specific. The rabbit antiserum to human LDH, however, had some species specificity. Further studies, i.e. enzymatic cleavage of the IgG fraction and testing of the fragments for binding of LDH, will be necessary to proof that we are not dealing with autoantibodies to LDH. The finding of autoimmune phenomena in patients belonging to groups I and II suggests that the presence of LDH-IgG, complexes is related to an autoimmune disease. However, this hypothesis, is not supported by the results of our investigation of the serum of patients with SLE and rheumatoid arthritis. The patients of group III resemble in some respects the patients with LDH-IgA complexes [6]. Firstly, in addition to the anomalous band(s) normal LDH isoenzymes were present. Secondly, the presence of the complexes was not related to a particular illness. Thirdly, the LDH isoenzyme patterns of one patient (Pr) studied over a long period did not change. It is surprising that in the serum of one of the patients of group III (Do) both LDH-IgA and LDH-IgG complexes occurred. These findings suggest that the anomalies in the structure of the IgA and IgG3 fractions leading to binding of LDH are of the same origin. Similarities in the structure of the IgA and IgG3 immunoglobulins, if existing, might be comparable to those described by Wolfenstein-Todel et al. [18] for IgGzh and IgG, h myeloma proteins from a single patient. Both myeloma proteins had identical light chains and identical variable regions of the heavy chains. In only one case (Ed) two subclasses of IgG and both types of light chains were involved in the formation of the complexes. As it could not be proved that the IgG3 fraction is an antibody to LDH the complexes were called immunoglobulin complexes to distinguish them from the classical immune complexes which are antigen-antibody complexes. A relationship between the occurrence of the complexes and a particular disease could not be established. In view of the broad variation in medical treatment of the patients without any common denominator, it is very unlikely that the formation of the LDH-IgG3 complexes was induced by drugs. IgG3 has a higher molecular weight than the other subclasses due to an extended hinge region. It also has more disulfide bridges in the hinge region and a higher number of proline residues in this region, as compared to the other subclasses [19]. Whether or not a relationship exists between these properties of IgG, and the formation of the complexes with LDH is unknown. However, it might be possible that its special structure of the hinge region is an important factor in the formation of the complexes.

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Acknowledgements The authors are indebted to the clinical chemists and clinicians who supplied the serum samples and clinical data, especially to H.A. Bonte, C.H. Dickhout, H.B.A. Hellendoorn, Ph. Jacobs, H.H. Kreutzer, A.H.J. Maas, J. Mulder, H. Verleur, J.T. Coflet, J.L. de Graaff, L.A. Kater, A.C. Taverne, A.M. Valkhoff, G. v.d. Waa and M. Zeeman. We are grateful to Peter Kreuger for her excellent technical assistance, Dr. Erna van Loghem and Dr. Marijke van der Giessen for their critical support, and to Drs. J. Mulder for the determination of the immunoglobulin concentrations. Professor Dr. H.L. Langevoort and Anneke Knappers were helpful in preparing the manuscript. References l_Kindmark, C.O. (1969) Stand. J. Clin. Invest. 24. 49-53 2 Nagamine, M. (1972) Clin. Chim. Acta 36, 139-144 3 Biewenga, J. (1972) Clin. Chim. Acta 40, 407-414 4 Biewenga, J. (1973) Clin. Chim. Acta 47, 139-147 5 Markel, SF. and Jan&h, S.L. (1974) Am. J. Clin. Pathol. 61, 328-332 6 Biewenga, J. and Feltkamp, T.E.W. (1975) Clin. Chim. Acta 58. 239-249 7 van Loghem-Langereis, P.E. (1952) Bull. Centr. Lab. Bloedtransf. Dienst Ned. Roode Kruis 2, 230-235 8 Feltkamp, T.E.W. and van Rossum, A.L. (1968) Clin. Exp. Immunol. 3, l-16 9 Kreutzer. H.H.. Jacobs, Ph. and Francke. C. (1965) Clin. Chim. Acta 11, 159-169 10 Ropes, M.W. et al. (1958) Bull. Rheum. Dis. 9,17&-175 11 Jefferis, R., Weston. P.D., Stanworth. D.R. and Clamp, J.R. (1968) Nature 219, 646449 12 Turner, M.W., Bennich, H.H. and Natvig, J.B. (1970) Clin. Exp. Immunol. 7, 603-625 13 Virella, G. (1971) Experientia 27, 94-96 14 Ganrot. P.O. (1967) Experientia 23. 593-593 15 Lubrano. T., Dieta, A.A. and Rubinstein, H.M. (1971) Clin. Chem. 17, 882-885 16 Karpatkin, S., Schur, P.H., Strick, N. and S&kind, G.W. (1973) Clin. Immunopathoi. 2, 1-8 17 Pirofsky, B. (1969) in Autoimmunization and the hemoiytic anemias, pp. 438-439, Williams and Wilkins, Baltimore 18 Wolfenstein-Todel, C.. Franklin. E.C. and Rudders, R.A. (1974) J. Immunol. 112, 871-876 19 Michaelsen, T.E. and Natvig, J.B. (1974) J. Biol. Chem. 249, 2778-2785