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Annals of Clinical & Laboratory Science 36:461-468 (2006)
© 2006 Association of Clinical Scientists

An Immunoglobulin A1 that Inhibits Lactate Dehydrogenase Activity, with Reversal of Inhibition by Addition of NADH

Kiyotaka Fujita1, Hirohisa Sato2, Fumiko Kameko1, Fumiko Terasawa1, Nobuo Okumura1, Mitsutoshi Sugano3, Kazuyoshi Yamauchi3, Masato Maekawa4 and Ikunosuke Sakurabayashi5
1 Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, Matsumoto; 2 Department of Clinical Laboratories, Aomori Prefectural Central Hospital, Aomori; 3 Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto; 4 Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu; and 5 Department of Clinical Laboratories, Omiya Medical Center, Jichi Medical School, Saitama, Japan

Address correspondence to Kiyotaka Fujita, Ph.D., Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, Japan; tel & fax: 81 263 37 2390; e-mail address: kyfujit{at}gipac.shinshu-u.ac.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We discovered a patient with low serum lactate dehydrogenase (LD) activity and an abnormal LD isozyme pattern. We analyzed the patient’s LD inhibitor using electrophoresis, affinity chromatography, and immunochemical technologies. The LD activity of the patient’s serum was inhibited more strongly at 4°C than at 37°C. The decrease of LD activity was more marked in a mixture of the patient’s serum with purified LD5 than in that with purified LD1. The immunoglobulin responsible for LD inhibition was an IgA1-{lambda}. The LD inhibition by the patient’s IgA1 was blocked by reduction and alkylation and by NADH. Polymerization of the patient’s IgA1 might play an important role in its interaction with LD. Moreover, the possibility exists that part of the patient’s IgA1 molecule fits into a pocket of LD in instead of NADH. This is the first report of NADH reversing such LD inhibition.

Keywords: lactate dehydrogenase, LD isozyme anomaly, LD-IgA1 complexes, NAD+-binding site


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Several reports have been published on the binding of lactate dehydrogenase (LD, E.C. 1.1.1.27 [EC] ) with IgA, IgG, and IgM immunoglobulins to form complexes that produce unusual LD isozyme electrophoretic patterns [1–7]. The clinical significance of these complexes is still poorly understood. The complexes have been observed in serum of healthy individuals and patients with various apparently unrelated diseases [8]. The LD activity associated with the LD-immunoglobulin complexes has been reported to be relatively high and stable [8], but there are also reports of LD-immunoglobulin complexes in which the LD activity is low [9–17]. It is is unclear whether immunoglobulin inhibition of LD activity is the result of a specific antigen-anti-body reaction or a non-specific protein-immunoglobulin interaction.

Here we describe a patient with low serum LD activity whose LD is bound to an IgA1-{lambda} immunoglobulin. The LD inhibition by the patient’s IgA1 is blocked by the addition of NADH and/or by reduction and alkylation of the IgA1. Therefore, we have attempted to elucidate the mechanism of LD inhibition by the patient’s IgA1.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subject.  The patient whose serum contained the LD inhibitor was a 57-yr-old woman with zoster herpes. Serum enzyme activities (U/L) were: aspartate aminotransferase, 17 (reference interval 4–38); alanine aminotransferase, 15 (4–42). Total serum LD activity was 66 U/L, which was below our reference interval (245–445 U/L). Moreover, serum LD activity was inhibited more strongly at cold temperatures than at warm temperatures. The patient’s serum immunoglobulin concentrations and reference ranges (g/L) were: IgG, 11.20 (9.5–15.5); IgA, 4.57 (1.5–2.7); and IgM, 1.83 (0.75–1.75).

Measurement of LD activity.  LD activity was measured at 37 °C in a Hitachi 7350 analyzer (Hitachi Ltd., Tokyo, Japan) with use of an LD test kit (L-LDH Test; Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Separation of LD isozymes.  LD isozymes were separated on agarose gel (Titan gel; Helena Laboratory, Urawa, Japan) with the use of a barbiturate buffer (60 mmol/L, pH 8.6). To each sample well of the agarose gel, 5 µl of patient’s serum or 2 µl of normal serum was applied, after which electrophoresis was performed at a constant voltage of 90 V for 30 min. To stain LD activity, we used a LD reagent (LDH-C-II Test; Wako Pure Chemical Industries) that contains lactic acid, NAD+, and nitro blue tetrazolium chloride, incubating the agarose gel with the reagent at 37°C for 30 min.

Purification of LD.  LD was purified from pooled serum that showed no LD isozyme anomaly electrophoretically, and a human liver that was obtained from a medicolegal autopsy case. Liver tissue was homogenized in 0.1 mol/L sodium phosphate buffer (pH 7.2) containing 150 mmol/L of NaCl and then centrifuged (2750 x G for 5 min). The supernatant was further centrifuged at 3000 x G for 20 min. The liver supernate was mixed with two volumes of pooled serum and applied to a 5'-AMP-Sepharose-4B column (15 x 0.9 cm I.D., Amersham Pharmacia Biotech, Buckinghamshire, England). Non-binding components were eluted with the same buffer at a flow-rate of 0.2 ml/min. The bound LD was eluted with the same buffer containing 1 mmol/L NADH and dialyzed against 0.1 mol/L Tris-HCl buffer (pH 8.0).

Purification of LD1 and LD5.  LD1 was purified from human erythrocytes by 5'-AMP-Sepharose-4B affinity chromatography, followed by DEAE-Sephacel (Amersham Pharmacia Biotech) chromatography, as previously described [18]. LD5 was prepared by the same methods from human liver that was obtained from a medicolegal autopsy case.

Detection of LD inhibitor in patient’s serum.  We separately mixed 250 µl of antiserums to human IgG ({gamma} chain specific), IgA ({alpha} chain), IgM (µ chain), kappa light chain, and lambda light chain (DAKO, Copenhagen, Denmark) with 50 µl of the patient’s serum and incubated the mixtures at 37°C for 60 min. After centrifuging to remove precipitates, the supernates were concentrated by ultrafiltration with Minicon-B15 concentrators (Amicon Corp., Beverly, MA). The supernates were mixed with normal pooled serum, incubated at 4°C for 20 hr, and assayed for LD activity.

Purification of IgA1 from patient’s serum.  Two ml of serum was applied to a 15 x 0.9 cm I.D. column packed with Jacalin-Agarose (Funakoshi Corp., Tokyo, Japan). Jacalin is a lectin from jackfruit (Artocarpus integrifolia) that binds IgA1 [19]. The column was washed with 0.1 mol/L sodium phosphate buffer (pH 7.2) and the bound IgA1 eluted with 0.8 mol/L of galactose in sodium phosphate buffer. The IgA1 fraction was immediately dialyzed against 0.2 mol/L Tris-HCl buffer (pH 8.0) and concentrated by ultrafiltration with an Amicon Minicon-B15 concentrator. After concentration, the IgA1 was loaded onto a DEAE-Sephacel ion-exchange chromatography column (55 x 1.6 cm I.D.) and eluted at a flow-rate of 0.3 ml/min with a linear gradient of 0 to 0.5 mol/L NaCl in the dialysis buffer.

LD inhibition assay by NADH.  To investigate the possibility that the LD inhibition disappeared in the presence of NADH, we incubated a mixture of purified patient’s IgA1 and purified LD with NADH at concentrations of 1, 3, 5, or 10 mmol/L at 4°C for 2 hr. After incubation, LD activities and LD isozyme electrophoretic patterns were examined.

LD inhibition assay by 5'-AMP-Sepharose-4B affinity chromatography.  To see whether LD coupled to 5'-AMP ligand was inactivated by the patient’s IgA1, affinity chromatography was carried out as follows: 3 ml of the purified LD was applied to the 5'-AMP-Sepharose-4B column. The LD coupled to 5'-AMP column was washed with 0.1 mol/L sodium phosphate buffer (pH 7.2), and 1 ml portions of 3.5 g/L solutions of the purified IgA1 were applied to the column. Unbound fractions were eluted with more of the same buffer, until the absorbance at 280 nm was <0.02 at a flow-rate of 0.2 ml/min. Bound fractions were eluted with the same buffer containing 1 mmol/L NADH and dialyzed against 0.1 mol/L Tris-HCl buffer (pH 8.0). After concentration with an Amicon Minicon-B15 concentrator, LD activity was assayed and the LD isozyme electrophoretic pattern was determined.

Immunofixation electrophoresis.  Serum prorein electrophoresis was performed at room temperature on agarose gels (Universal gel; Helena Laboratory, Urawa, Japan) in barbiturate buffer (60 mmol/L, pH 8.6). To each sample well of the agarose gel, 1 µl of the patient’s IgA1 was applied and electrophoresis was performed at a constant voltage of 90 V for 35 min. Immunofixation was performed with rabbit polyclonal antiserums specific for human IgG ({gamma} chain specific), IgA ({alpha} chain), IgM (µ chain), kappa light chain, and lambda light chain (DAKO), as previously described [20].

Reduction and alkylation.  To determine whether or not the LD activity inhibition of the patient’s IgA1 disappeared after reduction and alkylation, we reduced 1 ml portions of 3.5 g/L solutions of the purified IgA1 with a 0.1 mol/L solution of 2-mercaptoethanol (2-ME), and after 1 hr at room temperature alkylated the IgA1 with iodoacetamide (final concentration 0.02 mol/L). After dialysis against 0.1 mol/L Tris-HCl buffer (pH 8.0), the LD activity and the LD isozyme pattern were compared with the untreated IgA1.

Western blotting analysis.  The patient’s IgA1 was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretically transferred to polyvinyl-idene difluoride (PVDF) membrane (Nihon Millipore Kogyo, Yonezawa, Japan), as described by Towbin et al [21]. Immuno-staining was performed with rabbit polyclonal antiserums specific for human IgG ({gamma} chain specific), IgA ({alpha} chain), IgM (µ chain), kappa light chain, and lambda light chain (DAKO) as primary antibodies, and peroxidase-conjugated goat anti-rabbit IgG antibody (DAKO) as a secondary antibody. After the membranes were washed with 0.05% Tween 20- phosphate-buffered saline (pH 7.4, PBS), the bound antibodies were detected with 3,3'-diaminobenzidine tetrahydrochloride.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LD isozyme pattern and time-course of LD activity.  The LD isozyme pattern of the patient’s serum showed only a faint band at the origin (Fig. 1Go). A mixture of the patient’s serum with normal serum displayed the same abnormal pattern as the patient’s serum alone. On the other hand, the patient’s hemolysate from erythrocytes had a normal LD isozyme pattern. The time-course of the LD activity of the patient’s serum when kept at 37°C and at 4°C is illustrated in Fig. 2Go. LD activity was lost more rapidly at 4 °C.


Figure 1
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  		Fig. 1. Electrophoretic pattern of LD isozymes. A, normal serum; B, patient’s serum stored for 48 hr at 4°C; C, equivolume mixture of patient’s serum and normal serum (same as A) after incubation for 20 hr at 4°C. Anode is to the right in Figs. 1, 5, and 6.

 

Figure 2
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  		Fig. 2. Time-course of the LD activity of the patient’s serum kept at 37°C and 4°C. NS, normal serum; PS, patient’s serum.

 
Capacity of patient’s serum to inhibit LD1 or LD5.  To investigate which LD isozyme was inactivated by the patient’s serum, we separately mixed purified LD1 and LD5 with the patient’s serum at a ratio of 1:1 (v:v) and LD activity was compared to each mixture after 2 hr and 20 hr at 4°C. The residual LD activities were measured after incubation. As shown in Fig. 3Go, the capacity of the patient’s serum to inhibit LD activity was more marked in the mixture with purified LD5 than in that with purified LD1.


Figure 3
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  		Fig. 3. Loss of LD activity by incubation with patient’s serum. DW + LD1, equi-volume mixture of distilled water and purified LD1; PS + LD1, equivolume mixture of patient’s serum and purified LD1; DW + LD5, equivolume mixture of distilled water and purified LD5; PS + LD5, equivolume mixture of patient’s serum and purified LD5. LD activity of each mixture was assayed after incubation for 2 hr and 20 hr at 4°C, respectively.

 
Identification of the LD inhibitor.  To identify the LD inhibitor, we mixed samples of normal pooled serum and of the patient’s serum with antibodies specific for IgG, IgA, IgM, kappa light chain, or lambda light chain. The residual LD activity was 24%, 88%, 26%, 28%, and 93%, respectively, of the original activity. Therefore, the serum LD inhibitor was removed only when IgA or lambda-specific antibodies were used, which suggests that the inhibitor is an IgA-{lambda} immunoglobulin.

Identification of IgA subclass with LD inhibition.  The patient’s IgA1 was purified by Jacalin-Agarose affinity column and DEAE-Sephacel ion-exchange chromatography. To remove IgG and IgM from the Jacalin-unbound fraction (containing IgA2), the unbound fraction was filtered through a column that contained anti-IgM (µ chain) coupled to Protein G and the IgA2 rich fraction (unbound fraction) was immediately dialyzed against 0.2 mol/L Tris-HCl buffer (pH 8.0). To confirm the LD inhibition by IgA1 or IgA2, we separately mixed the IgA1-rich and IgA2-rich fractions with purified LD at a ratio of 3:1 (v:v) and LD activity was compared to each fraction after 30 min and 60 min at 4°C. Fig. 4Go shows the LD inhibitory activity of the IgA1-rich and IgA2-rich fractions from the patient’s serum when mixed with purified LD. The LD inhibition of the patient’s IgA was more marked in the IgA1 fraction than in the IgA2 fraction.


Figure 4
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  		Fig. 4. LD inhibition of patient’s IgA1 and IgA2 when mixed with purified LD. LD activity in the mixture of patient’s IgA1 or IgA2 fraction and purified LD was measured after incubation for 30 min and 60 min at 4°C, respectively.

 
Effect of NADH on inhibition of LD by the patient’s IgA1.  As shown in Fig. 5Go, the electrophoretic pattern for a mixture of the patient’s IgA1 and purified LD with NADH showed a normal LD isozyme pattern with NADH concentration as low as 1 mmol/L. Moreover, the LD inhibition by the patient’s IgA1 disappeared in the presence of NADH concentrations as low as 1 mmol/L.


Figure 5
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  		Fig. 5. LD isozyme pattern for the mixture of patient’s IgA1 and purified LD with NADH. A, the mixture of patient’s IgA1 and purified LD with NADH in concentration of 1 mmol/L; B, the mixture of patient’s IgA1 and purified LD without NADH; C, purified LD.

 
LD inhibition of the patient’s IgA1 for LD coupled to 5'-AMP-Sepharose 4B.  To determine whether LD coupled to 5'-AMP ligand is inactivated by the patient’s IgA1, we applied the purified IgA1 to the column and eluted the bound fraction (LD) with NADH. The electrophoretic LD isozyme pattern for the concentrated bound fraction was normal, and no LD inhibition was demonstrated. The unbound fraction was devoid of LD activity. Therefore, we mixed the purified LD with the unbound fraction at a ratio of 1:3 (v:v) and the LD activity was measured after incubation for 1 hr and 24 hr at 4 °C. The results showed that the LD activity of the mixture was inhibited 32% at 1 hr and 65% at 24 hr. Moreover, the LD isozyme electrophoretic pattern of the unbound fraction and the purified LD were the same as the patient’s serum alone. These results demonstrate that the patient’s IgA1 cannot bind to LD when the LD is coupled to 5'-AMP ligand.

Characterization of the patient’s IgA1.  Fig. 6Go shows the immunofixation electrophoretic pattern of the patient’s IgA1. Although inhibition of LD by the patient’s IgA1-{lambda} was demonstrated, an increase in monoclonal IgA1 was not confirmed.


Figure 6
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  		Fig. 6. Electrophoretic pattern (a) of the patient’s IgA1 and immunofixation pattern, and (b) with antiserums to human IgG (4), IgA (5), IgM (6), kappa light chain (7) and lambda light chain (8). 1, normal serum; 2, patient’s serum; 3, patient’s IgA1.

 
To determine whether or not the LD inhibitory activity disappeared after the patient’s IgA1 was reduced and alkylated, we mixed the purified LD and the patient’s IgA1 after reduction and alkylation (mixture ratio = 1:3, v:v). The LD activity and LD isozyme electrophoreic pattern were compared with the patient’s untreated IgA1 after incubation for 1 hr and 2 hr at 4°C. Inhibitory activity for LD was only 0.3% at 1 hr and 0.5% at 2 hr, compared to 26% and 34% inhibition of LD by the patient’s untreated IgA1 at 1 and 2 hr, respectively. Thus, LD inhibition was not produced by the patient’s IgA following reduction and alkylation. The LD isozymes of the mixture of this treated IgA1 and purified LD showed a normal pattern.

The patient’s IgA1 was found by Western blotting to be composed of two {alpha} chains of about 61 kDa and two {lambda} chains of 28 kDa.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of LD-immunoglobulin complexes in serum has been reported [1–7]. The increased half-life of these complexes often results in an apparently unexplained increase in serum LD activity. In contrast, we observed a patient with decreased LD activity associated with circulating LD-immunoglobulin complexes. There are two categories of causes of low LD activity in serum. In one, the LD activity is low, not only in serum but also in all cells or tissues, and is attributed to subunit variants [22], subunit deficiency [23,24], and the like; that is, to genetic variants, In the other category, LD activity is low in serum only and this is attributed to the presence of so-called LD inhibitors. In our case, we eliminated the possibility of an H- or M-subunit deficiency by finding a normal LD isoenzyme pattern in the patient’s erythrocytes, and found that the patient’s IgA1-{lambda} was the immunoglobulin responsible for LD inhibition. The LD activity of the patient’s serum was inhibited more strongly at 4°C than at 37°C, as in earlier reports [10,11,15,16], suggesting cryo-type temperature dependence.

LD inhibitors have been reported to be immunoglobulins: IgG [13–16], IgA [11], or IgM [12]. The presence of an LD inhibitor may be suspected from the property of cold-dependence, as in the present case. However, the mechanism that is responsible for formation of immunoglobulins with LD inhibition is unclear.

In an LD-IgM complex case with loss of LD activity [12], it was suggested that LD inhibition was due to physical steric hindrance of IgM, rather than the fact that it was acting as an autoantibody to the active site of the enzyme, because the LD activity increased approximately 2-fold when the IgM was treated with 2-ME. Gershbein et al [25] reported that the LD activity of patients’ serum treated with 2-ME and other sulfhydryl compounds did not change. Serum IgA is present in several polymeric forms, ranging from monomers to pentamers. In normal human serum the monomeric form usually predominates, but in patients’ serums with various diseases the relative amount of each polymeric form varies.

In the present case, it appears that the patient’s LD inhibitor is an polymeric form of IgA1 because no LD inhibitory activity of the patient’s IgA1 was demonstrated after reduction and alkylation of the IgA1. However, the reduction and alkylation data may be not strong enough to support this inference, since a possibility exists that the patient’s IgA1 was denatured by reduction and alkylation.

We previously reported a case with IgG1-{lambda} type M-proteinemia whose LD-IgG complexes (no LD inhibition) were dissociated either by adding NADH or by passing the patient’s serum through a column containing 5'-AMP-Sepharose-4B, which can be considered as a fragment of NAD+ [18]. Gorus et al [26] succeeded in dissociating LD-IgG complexes (no LD inhibition) by adding NAD+, which presumably bound to LD and altered its antigenicity. Moreover, we found a multiple myeloma patient with LD-Bence Jones protein (BJP) complexes (no LD inhibition), which bound LD2, LD3, LD4, and LD5 [27]. When the purified LD was mixed with NADH and eluted through a column of CNBr-Sepharose-4B coupled to the patient’s BJP, no binding was demonstrated. In regard to the mechanism of complex formation, it is interesting that LD inhibitory activity of the patient’s IgA1 was not demonstrated after adding NADH, and that the patient’s IgA1 showed non-binding for the LD coupled to 5'-AMP ligand.

5'-AMP has been used as a general ligand for NAD+-binding enzymes such as LD. The binding constant for the enzyme-"pseudo"-ligand complex is usually much weaker than the binding constant of the corresponding enzyme-cofactor complex [28,29]. From these facts, it can be considered that the affinity of the LD molecule for immunoglobulins is very weak as compared to its affinity for NADH or 5'-AMP. Although LD-IgG complexes with LD inhibition that are not dissociated by added NADH have been reported [13], the finding that the LD-IgG complexes, not dissociated by added NADH, were easily dissociated by passing the patient’s serum through a column containing 5'-AMP-Sepharose-4B [30,31] supports the possibility that these LD-IgG complexes also become dissociated on the column.

The dinucleotide-fold region in LD, which is responsible for the NAD+ binding site, has been identified [29,32]. This region is composed of two similar conformations: one binds to the adenosine phosphate portion of the coenzyme and the other one binds to the nicotinamide mononucleotide portion. It appears that the adenosine of NAD+ binds in a pocket of LD that is not specific for the ligand, but also binds other aromatics [33]. Therefore, the region may recognize molecules that can mimic the conformation of NAD+. In the LD-BJP complexes [27], the BJP had an aromatic amino acid (ie, tyrosine) at the first portion of the N-terminus, and abnormal 3-dimensional structure (ie, longer ß-sheet structure of N-terminus).

In the present study, the possibility exists that part of the patient’s IgA1 gets into a pocket of LD in the absence of NADH, and that LD recognizes the patient’s IgA1 at the NAD+-binding site of LD, since that part of the patient’s IgA1 may appear similar in structure to NAD+. It is not clear whether the mechanism of action of the patient’s IgA1 is via occupying an NAD+-binding site or via both occupying and steric hindrance to substrate. Why was LD1 not involved in binding to the patient’s IgA1, despite the fact that LD1 also has an NAD+ binding site? It is conceivable that the amino acid residues of LD1 molecule in the NAD+-binding site may be different from that of LD5 [33]. Therefore we speculate that LD1 is uninfluenced by the IgA1 molecule because the affinity of LD1 for the patient’s IgA1 is very weak.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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