Immunological and electrophoretical approaches to macroamylase analysis

291

Clinica Chimica Acta, 59 (1975) 291-299 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CCA 686 3

IMMUNOLOGICAL MACROAMYLASE

AND ELECTROPHORETICAL ANALYSIS

KIKUKO HARADA, TOSHIMASA TADAO SUGIMOTOa

NAKAYAMA,

MOTOSHI

Departme& of Clinical Chemistry, Toranomon Hospital and aLIepartment of Endocrinology and Metabolism, Toranomon Tokyo 107 (Japan) (Received

September

APPROACHES

KITAMURA

TO

and

Hospital,

27, 1974)

Summary The characteristics of abnormally large-sized amylase (macroamylase) of the three patients were studied. Amylase isoenzyme patterns of the three macroamylase samples on agar gel electrophoresis were different from the normal one: a post-@globulin or middle-y-globulin zone was found in macroamylase, while a fast-y, and a pre-y-globulin or both zones is found in normal amylase. With gel chromatography at pH 3.4, macroamylase dissociated to normalsized amylase. The electrophoretic patterns of the normal-sized amylase were identical with those of normal amylase. The binding proteins of the macroarnylase were identified as immunoglobulin IgG in one case and immunoglobulin IgA in the other two cases by the immunochemical criteria and immunoenzyme electropherogram, These immunoglobulins bound with serum amylase having normal molecular weight and electrophoretic mobility to form macroamylase.

Introduction In 1964 Wilding and his collaborates [l] reported a case of hyperamylasemia which resulted from the formation of an amylase-globulin complex. Berk et al. [2] in 1967 described three additional cases and introduced the designation “Macroamylasemia” as a descriptive term, because the largemolecular amylase could not be readily excreted by the kidney. About 30 cases have so far been encountered in Europe and U.S.A., and also 7 cases in Japan. No clinical syndrome consistently accompanies macroamylasemia except that some characteristics are common to several cases, such as malabsorption, alcoholism and abdominal pain. Not only the mechanism of macroamylase genesis, but also its characteris-

292

tics remain uncertain. However, at least three possible mechanisms responsible for the production of the macro-form of amylase have been suggested: (1) protein substances, most likely immunoglobulin, bind to amylase [ 3-61, (2) nonprotein substances such as polysaccharides bind to amylase [7], and (3) polymerization of amylase occurs to form a large complex [ 81. It has been shown by gel filtration that the polymerization of ol-amylase of Bacillus subtilus occurs in the presence of Zn”, but there is no experimental evidence in man to support this concept. Take et al. [7] have suggested that polysaccharideor carbohydrate-containing protein might play a role in the formation of macroamylase in vitro but no direct evidence suggesting the role of polysaccharide in vivo has been obtained. The first mechanism mentioned above is, at present, accepted as the most likely one, but only four cases supporting this mechanism have been reported so far; IgG-binding amylase in one case [ 31, and IgA-binding amylase in three cases [4-61 . This paper describes the results of the immunochemical and electrophoretical analysis on three cases of macroamylasemia which we had encountered recently. Preliminary

Results

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293

macroamylasemia. Case 1, S.N., a 60-year-old man, ventricular septal defect, pneumonia. Case 2, E.S., a 59-year-old man, mild diabetes mellitus. Case 3, F.U., a 53-year-old man, healthy. The amylase activities of the three patients were abnormally high in the serum and low in the urine throughout the long period when they were examined. We had previously studied the macroamylase present in the serum of one of the three patients by gel chromatography and proposed that his macroamylase consisted of normal-sized amylase and some kind of protein [9] . Materials and Methods 1. The specimens from the patients in the present study were serum, urine, saliva and pancreatic juice. 2. As the control, sera or urine of patients with pancreatitis or with parotitis were used. 3. Goat antisera specific to human IgG, IgA and IgM (Hyland Laboratories, Los Angeles, California) and amylose (Nikken Chemicals, Tokyo, Japan) were used for the evaluation of the immunochemical properties of macroamylase. Other reagents used were of analytical grade. 4. Total amylase activity was determined by a modification of Caraway’s amyloclastic method [lo] . 5. The isoamylase analysis was performed by means of agar gel electrophoresis as modified by Harada [ll] , so as to permit good separation between the p- and y-globulin zones utilizing the excellent effect of electroosmosis. Amylase isoenzyme was identified using the amylose-iodine reaction. In this method the amylase derived from the pancreas was seen to migrate in the fast-y-globulin zone and that derived from the parotid gland in the pre-yglobulin zone. In normal serum and urine both of these amylase fractions were found by the method used and the ratio of the two fractions varied among individuals. 6. Immunoelectrophoresis was carried out using agar gel according to the method of Scheidegger [ 121. The amylase activity in the immunoprecipitin line was visualized with the following staining method. The agar plate after immunoelectrophoresis with deproteinization was placed on another plate containing 0.1 per cent amylose buffer solution (80 mmol/l phosphate buffer pH 7.0) and the two plates were then incubated at 37°C for one hour. After the incubation, the amylose agar plate was discarded and the electrophoresis plate was further incubated at 37°C for 30 minutes under moist conditions. This plate turned purple with a few drops of Caraway’s iodine solution [lo] except in those areas having amylase activity (because of cleavage of amylose by amylase). 7. Gel filtration studies were performed using a column (2.6 cm X 100 cm) of Sephadex G-200 (Pharmacia Fine Chemicals) at room temperature equilibrated either with 0.01 mol/l Tris/HCl, 0.1 mol/l NaCl, pH 8.0 or with 0.2 mol/l glycine/HCl, pH 3.4. The fraction volume was 6 ml at flow rates of 18-24 ml/h. 8. For thin-layer gel chromatography, Excel plate gel filtration apparatus (Showei Seisaku-sho, Tokyo, Japan) and Sephadex G-200 superfine were used. The samples (the patient’s serum and the fractions from column gel chromato-

294

graphy of the serum) and the marker (bromophenol blue-containing control serum) were applied and eluted at room temperature for 3-4 h with 0.066 mol/l phosphate buffer at pH 7.2. When the color nearly reached the midpoint of the plate, the paper strips previously dipped in 0.1 percent amylose buffer solution (pH 7) were immediately placed on the plate. After being incubated for 30 minutes at 37”C, the paper strips were removed and dried in air. Then, alcoholic iodine solution (Caraway’s 0.1 mol/l iodine solution and ethylalcohol, 1 : 1, v/v) was sprayed over them. Amylase activity appeared in a decolorized clear zone against the blue background. Results 1. Column gel chromatography of the patient’s sera on Sephadex G-200 The elution patterns of amylase activity in the patient’s serum and normal serum following gel filtration on Sephadex G-200 at pH 8.0 are shown in Fig. 2. It was found that the major component of the total amylase activity was eluted from the column simultaneously with 7S globulin in the first case and eluted between 7S and 19s globulin in the other two cases. The molecular weight of the large amylase component in each case was estimated as 170 000, 250 000 and 250 000 using a calibration curve correlating the molecular weight to the elution volume. 2. Agar gel electrophoresis Serum amylase zymograms shown in Fig. 3 indicate that electrophoretic mobilities of the macroamylase in the three cases were quite different from those of normal amylase originating from the pancreas or the salivary gland. In the zymogram of the first case, the amylase activity appeared in the middle-y:-,: : : :

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Origin 1

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of serum

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obtained

with agar gel electrwhoresis.

a, control;

b, case 1; c. case 2;

globulin zone as a single peak while in the other two cases the activity migrated mainly to the post-&globulin position tailing toward the cathode up to the y-globulin. Patients’ sera showed a specific mobility as mentioned above, while their urine, saliva and pancreatic juice showed the normal mobilities; pre-yand/or fast-y-globulin, pre-y-globulin and fast-y-globulin, respectively. 3. Dissociation of macroamylase at pH 3.4 Gel filtration of the patients’ sera at pH 3.4 resulted in a complete disappearance of the macroamylases, and amylase activity was eluted at the same point as the amylase from normal control serum. The recovery of amylase activity after gel filtration was approximately 95 percent in all cases. After having neutralized the fraction containing the amylase activity by adding 0.1 mol/l NaOH, the fraction was concentrated to about one-tenth volume with Visking tubing. Following electrophoresis the concentrated fraction had the same two amylase activities originating from the pancreas and the saliva that are observed for the normal sera. The amylase activity in the first case is located predominantly in the pre-y-globulin zone on agar gel electrophoresis

296

and the other two cases equally contained fractions.

both pancreatic

and salivary amylase

4. Formation of macroamylase in vitro The proteins obtained by gel filtration of the patient’s serum at pH 3.4 were fractionated into four proteins as shown in the right side of Fig. 4. Each region was adjusted to pH 7.0 and concentrated to one tenth, and an equal volume of control serum was then added and allowed to stand at room temperature for 3 h. The result of thin-layer gel chromatography of these mixtures is shown in the left side of the figure. The proteins of regions II, III, and IV in case 1, and that of region II in cases 2 and 3 converted the amylase present in the control serum to larger molecular forms similar to that of the patient’s serum. The isolated patient’s amylase (region V) did not bind to protein present in normal serum. 5. Precipitation of macroamylase with anti-immunoglobulin serum To determine that the large amylase is an amylase-immunoglobulin complex, quantitative immunochemical precipitation was performed with goat antiserum against IgA, IgG and IgM. Increasing volumes of the patient’s serum were added to 0.1 ml of the goat antiserum as shown in Fig. 5 and incubated at room temperature for 24 h. The samples were centrifuged at 3000 rpm and the amylase activity in the supernatant was determined. After washing with saline

t

t

A

G

t

M

Fig. 4. (A) Thin-layer gel chromatogram of amylase activity on Sephadex G-200 Superfine. Right figure (B) ilk&rates column chromatographic fractions as in Fig. 2. Patterns as follows: a, serum case 1; b, region II protein ease 1 + control serum; c, region III protein + contrcd serum; d, region IV protein c control serum; e, serum case 2; f, region II protein case 2 + control serum; g, control serum. The lowest pattern is serum protein used as reference: A, aibumin; G, immunoglobulin G; M, macroglobulin

297 Case

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1 Anti-IgG

1 Potlent

3 *erwn

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Case

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5

10

25

( p )

of amylase

2

Anti-IgA

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with anti-~rnun~~ob~in

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100

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amylase activity:

3 times, the precipitate was completely dissolved in 2.4 ml of 0.05 mol/l gIycine/HCl buffer solution (pH 3.4). The protein concentration was determined by absorbance at 280 nm. As the zone of equivalence was reached, the quantity of protein precipitate increased whereas the soluble amylase reciprocally decreased. At the maximal IgG precipi~tion in the first case and at the maximal IgA precipitation in the second and the third cases the maximal removal of amylase from the supernatant was observed. No precipitation of amylase occurred by adding various volumes of macroamylase serum to antiserum specific for IgA or IgM in the first case and to antiserum specific for IgG or IgM in the other two cases. 6. Development of enzyme activity on the ~~munu~~ecipitin line Enzymatic activity detection at the immunoprecipitin line was further attempted to prove the postulated binding of amylase to in~munoglobulin. Fig. 6 shows the enzyme activity of the serum of case 2 developed by the iodine-amylose reaction after immunoelectrophoresis, demonstrating the amylase activity as a decolorized line; the specific amylase activity was observed in

Fig. 6. Amylese activity on the precipitin line of immunoelectrophoresis caee 2; b, control serum,

using anti-IgA serum. a, Serum of

298

the zone to the anode side half of the IgA immunoprecipitin line. The lower side showed a control serum. No amylase activity lines were observed in this sample. Discussion As in the previous reports, no anomaly in the function of either pancreas or kidney was observed in the three cases of macroamylasemia reported here. Our cases were diagnosed as macroamylasemia because of extremely low values of amylase clearance (Fig. 1). The ratio creatinine clearance/amylase clearance, an index to detect macro~ylase [ 131, was also extremely low. Our enzymatic and kinetic attempts to distin~ish ma~ro~yl~e from amylase, including tests for affinity to substrates and for the inhibition by EDTA or wheat cx-amylase inhibitor were unsuccessful, as in Berk’s experiments

[141Agar gel electrophoresis of macroamylase, however, showed a peculiar pattern (Fig, 3). Such a pattern was first encountered by us in about 900 cases of hyperamylasemia. Small scale gel chromatography [ 151 or amylase clearance have so far been applied as screening methods to detect macroamylase, but the former is a troublesome method for laboratory personnel and the latter also for patients. Diversified results have been reported regarding the electrophoresis of macroamylase; clearly distin~ishable [ 6,161, slightly distin~ishable [ 121 and indistin~ishable [3,17,18]. Our agar gel electrophoresis proved to achieve the best separation of macroamylase under specified conditions, and it may be a practical method of routine testing for macroamylasemia. Cellulose acetate electrophoresis was also found to give a good separation 1211. The results of the immunoprecipitation studies suggest that IgG in the first case and IgA in both the second and the third cases may bind with amylase. In the second case, this was proved successfully for the first time by The formation of a specific antibody to enzymatic immunoelectrophoresis. amylase is suggested by the finding that the amylase activity was detected on the anode side half of the immunoprecipitin line. This is also indicated by the fact that macrotype amylase was not formed between the patient’s amylase and the control serum but between some protein component of the patient’s serum and normal amylase. Amylase, dissociated from macroamylase in gel chromatography at pH 3.4, was eluted at the same fraction number as for normal amylase (Fig. 4) and its mobility in electrophoresis was identical with that of normal amylase. In the first case, the pre-y fraction was detected in greater quantities than the fast-y fraction, while nearly equal quantities of both fractions were observed in the second and the third cases. This fact suggests that the antigenicity of pancreatic amylase is quite similar to that of salivary amylase, and confirmed a report [19] of difficulty in distinguishing immunologically pancreatic amylase from salivary amylase of man. It was demonstrated in the present study that the amylase showed the same properties as those of normal amylase and that electrophoresis of the patient’s urine, saliva and pancreatic juice gave the normal mobilities. Considering these findings, it is strongly indicated that the amylase fraction of the

299

patient’s serum has the same molecular weight and properties as normal amylase. The discussion leads to the conclusion that normal amylase binds with immunoglobulin to form macroamylase by some autoimmune reaction. Agar gel electrophoresis has proved to be an effective technique for screening macroamylase. Since immunoglobulins specific to enzyme have been found in the cases of LDH [20] and transaminase (lida, K., personal communication) similar phenomena might exist in relation to other released enzymes. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Wilding, P., Cooke. W.T. and Nicholson, G.I. (1964) Ann. Intern. Med. 60, 1053 Berk, J.E., Rizu, H. and Wilding, P. (1967) New Eng. J. Med. 27’7, 941 Hansen, H.R., Kley, H.V. and Knight, W.A. (1972) Am. J. Med. 52. 712 Levitt, M.D. and Cooperband, S.R. (1968) New Eng. J. Med. 278,474 Levitt, M.D., Gietzl, E. and Cooperband. S.R. (1968) Lancet i, 957 Koami, E., Yakata, M., Niwayama, M. and Ito, G. (1972) Jap. J. CIin. Chem. 1, 192 Take, S., Fridhandler, L. and Berk, J.E. (1970) Clin. Chim. Acta 27, 369 Kakiuchi, K., Kate. S., Imanishi, A. and Isemura. T. (1964) J. Biochem. Tokyo 55, 107 Honda, E., Ichiyasu, H., Kuzuya, T. and Yoshida. S. (1972) J. Jap. Sot. Intern. Med. 61, 1534 Caraway, W.T. (1959) Am. J. Clin. Path& 32, 97 Harada, K. and Kitamura, M. (19713 Medicine and Biology 82, 155 Seheidegger. J.J. (1971) Intern. Arch. Allergy 7, 103 Levitt, M.D., Rapoport, M. and Cooperband, S.R. (1969) Ann. Intern. Med. 71, 919 Berk, J.E., Kizu, H., Geller, E. and Fridhandler, L. (1969) Proc. Sot. Exp. Biol. Med. 131, 154 Fridhandler. L., Berk, J. and Ueda, M. (1971) Clin. Chem. 17, 423 Hedger, R.W. and Hard&on. W.G.M. (1971) Gastroenterology 60, 903 Davis. J.. Berk. J.E., Take, S. and Fridhandler, 1~. (1971) Clin. Chim. Acta 35. 305 Joseph, R.R. (1968) New Eng. J. Ivied. 278, 508 McGeachin, R.L., Reynolds. J.M. and Huddle&on, J.1. (1961) Arch. Biochem. Biophys. 93, 387 Biewenga, J. and Thijs, L.G. (1970) Clin. Chim. Acta 27. 293 Harada, K,, Nakayama, T., Kitamura, M., Sugimoto, T. and Yoshida, A. (1974) Igaku no Ayumi (Japan),

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