Immunichemical determination of cathodal elastase in human duodenal juice

215

CIinica Chimica Acta, 96 (1979) 215-223 @ Elsevier/North-Holland Biomedical Press

CCA 1079

IMMUNICHEMICAL DETERMINATION HUMAN DUODENAL JUICE

S. BORULF

*, T. LINDBERG

February

20th,

ELASTASE

IN

and M. MANSSON

Departments of Paediatrics and Experimental General Hospital, Malmij (Sweden) (Received

OF CATHODAL

Research,

University of Lund, MalmG

1979)

Summary

In human duodenal juice enzymes hydrolysing the elastase substrate succinyl-trialanine-p-nitroanilide have both an anodal and cathodal mobility in agarose gel electrophoresis. The cathodal enzyme, also having an elastinolytic activity, was purified. An application of electroimmunoassay for separate determination of this cathodal elastase is presented. Parallel estimations of esterolytic, elastinolytic and immunochemical activities in duodenal juice from a group of children revealed discrepancies suggesting both variations in the distribution of the two forms of “elastases”, and presence of inactive forms.

Introduction

One anionic enzyme with elastase-like properties and one cationic elastase have been isolated from human pancreatic tissue [1,2]. In human duodenal juice, Fric et al. [3] demonstrated one enzyme with anodal and one with cathodal mobility in gel electrophoresis; both had activity against the elastase substrate succinyl-trialanine-p-nitroanilide (Suc(Ala)3NA). The cathodal enzyme also had an elastinolytic activity. Knowledge about these enzymes in human duodenal juice in physiological and pathological conditions is scanty and incomplete. This is perhaps because the methods for assaying elastinolytic activity 14-61 have a relatively low sensitivity or are too laborious to be used in routine work. Moreover, the synthetic substrates for elastase assays, introduced in recent years, are split by both enzymes [1,2]. These methods, therefore, provide no information about the amount of the separate enzymes in duodenal juice. Immunochemical methods, however, could quantify the elastases separately. * Correspondence Hospital, S-21401

should be addressed MalmB, Sweden.

to:

Stefan

Borulf.

Department

of Paediatrics.

Malmii

General

216

In this study we have purified the human cathodal elastinolytic elast,asr from duodenal juice and evolved a method for immunochemical determination of this enzyme. Material Duodenal juice was obtained from two healthy adults for the purification and elaboration of the immunochemical method. Juices for the application of the method were obtained from a group of 9 children aged 8 months to 7 years. Seven of the children had normal pancreatic exocrine function. Two had signs of pancreatic insufficiency (trypsin and amylase activity, electrophoretic pattern of duodenal juice [ 71). The juices were collected into tubes in crushed ice, as described earlier [ 71, from the distal duodenum in the fasting state and after intake of water 100300 ml depending on age. The juices were kept frozen at -20” C until analysed. Chemicals SP Sephadex C-50, Sepharose 4-B, CNBr-activated, and Blue Starch Polymer (BSP), i.e. PHADEBAS Amylase Test, from Pharmacia, Uppsala, Sweden. Bovine elastin from Worthington Biochem. Corp., Freehold, N.J. USA. Porcine elastase (EC 3.4.21.11) E0127, bovine albumin, n-benzoyl-DL-arginin-p-nitroanilide (BAPNA), n-benzoyl-tyrosin-ethylester (BTEE), n-carbo-/3-naphtoxy-Lphenylalanine (CNPA), and N-tert. -butyloxycarbonyl-L-alanine-p-nitrophenylester (Boc-Ala-NP) from Sigma Chemicals, St. Louis, U.S.A.: Succinyl-trialanine-p-nitroanilide (Suc(Ala),NA) from Peptide Institute Inc., Protein Research Foundation, Osaka, Japan. Agarose (medium-electroendoosmosis) from Marine Colloids Inc.-Miles Laboratories Ltd., Stoke Poges, U.K. Freund’s complete adjuvant from Difco Lab., Detroit, U.S.A. Rabbit antisera against human anodal and cathodal trypsins and chymotrypsin were available in our laboratory [ 81. All chemicals were of analytical grade. Methods Enzyme assays Elastase esterolytic actirrity was measured according to Fric et al. [ 31 with Suc(Ala),NA as substrate with the purified human elastase and porcine elastase as standards. The substrate was included in the blank sample. The reaction was stopped with acetic acid. A unit of activity was defined as an absorbance change of one absorbance unit/min at 410 nm. Specific activity was calculated as units per mg protein. For detection of elastase esterolytic activity in chromatographic fractions, Boc-Ala-NP [9] was used parallel to Suc(Ala)3NA. Suc(Ala),NA was used for detection in gels [ 31. Elastase elastinolytic activity on bovine elastin was assayed with radial diffusion in elastin-agarose [6] and with the spectrophotometric method of Ardelt et al. as modified by Gertler [4]. The same standards as above were used. Fractions with a salt molarity exceeding 0.02 were dialyzed against the reaction

21-i

buffer before analysis. tion method [lo].

pepsin

In gels elastinolysis

and chymotrypsin

was detected

nativities were assayed

with the elastin digeswith BAPNA

ill]

and

BTEE [ 121, respectively.

Carboxypeptidase

A and amylase activities were identified

as described

earlier [8] with CNPA and BSP, respectively.

Other methods ~otein concentration

was measured

according

to Lowry

et al. [ 131 with

bovine albumin as standard.

Polyacrylamide electrophoresis was carried out at pH 4.3 according

to Reis-

feld et al. [14].

Agarose gel ekctrophoresis

was done with 0.075 mol/l sodiumbarbital taining 0.002 mol/l calcium lactate (pH 8.6) [ 151.

con-

Isolation of cuthodaE elastase All procedures

were done at 4°C unless otherwise

stated.

Step I. Ethanol precipitation as described earlier [ 81. Step II. Ion exchange chromatography on SP Sephadex C-50 in 0.05 mol/l Na acetate,

0.02 mol/l CaCI, (pH 4.3) with a linear salt gradient

Step III. Affinity

IS].

chromatography on ~Z~stin-Sepharose 4-B. Soluble elastin

prepared from bovine elastin according to Keller and Mandl [16] was coupled to CNBr-activated Sepharose 4-B according to the manufacturer’s instructions and a 2 X 18 cm column of this material was equilibrated with 0.02 mol/l TrisHCI, pH 8.8. The elastinolytic fractions from step II were pooled and dialyzed for 2 h against the same buffer and applied to the column. After passage of unretarded proteins, as read at Az8,,, the column was eluted with 1 mol/l sodium acetate 0.05 mol/l CaC12 (pH 4.0). Resulting peak fractions were immediately analysed for protein concentration, Suc(Ala),NA activity, and elastinolytic activity. Aliquots were kept at -20°C to be used as standards for assays. Immunization procedure. The elastase-containing fractions from step III were mixed with equal amounts of Freund’s complete adjuvant and injected intracutaneously and subscapularly into rabbits. Booster shots were given after 3 weeks, and serum was harvested and IgG fractions were isolated [ 171. Immunoelectrophoresis according to Scheidegger [18] was run in 0.8% agarose in the electrophoresis buffer. Crossed immunoelectrophoresis [19] and electroimmunoussuy [20] were done in the electrophoresis buffer with 3% polyethyleneglycol in the anti-body containing gels. Results

Isolation of cathodal e~~stase Table I summarizes

the procedure.

Step II. SP Sephadex chromatography As Fig. 1 shows, elastinolytic part of the fractions containing

activity is recovered in a peak that is only a Suc(Ala),NA activity. Suc(Ala&NA activity

218 TABLE

I

PURIFICATION

OF HUMAN

step

CATHODAL Volume (ml)

0. I. II. III.

Duodenal juice Ethanol precipitation SP-Sephadex C-50 El&in Sepharose 4-B

528 232 51 12

ELASTASE Total protein

Total Suc(Afaf~NA

(mg)

UJ)

2217.6 507.5 14.8 6.2

22.7 16.6 4.03 0.57

ReCOVeXy W)

Specific activity

Purification

w/mg protein) 100 73 18 2.5

0.01 0.03 0.27 0.09

1 3 27 9

appeared cathodally as well as anodally in electrophoresis of the elastinolytic fractions. The elastinolysis was confined to the cathodal band. In non-elastinolytic fractions, Su~(Ala)~NA activity only appeared in an anodal band. Fig. 1 also gives the localizations of anodal and cathodal trypsins, chymotrypsins, and carboxypeptidase A. Boc-Ala NP-splitting activity was found not only in the fractions with Suc(Ala),NA activity but also in fractions containing anodal and cathodal trypsins and ~hymotrypsins. Assays with purified human cathodal trypsin and chymotrypsin showed that both enzymes had a Boc-Ala-NP-splitting activity (AA/min for 1 Erg of cathodal trypsin and chymotrypsin was 0.58 and 1.33, respectively).

0.15

E

c 0

x 6

z

0.10

s D $ 8 0.05

50

60

70

80

90

100 c

110 TrAN

120

130

140

150

---I

ChTr

160 I

,

170

TrCAT ChTr

1110 190

200

, 4

CXP

Fract ton number Fig. 1. Chromatography of ethanol-precipitated human duodenal juice on SP-Sephadex C-50. Fraction volume 5 ml. On top: Localizations of Suc(Ala)~NA-splitting activity in agarose gel after electrophoresis. x Zone of lysis in elastin-agarose after electrophoresis. l ----0, absorbance at 280 nm: +-+++, elastinoIytic activity [61: A-, Suc(Ala)3NA-splitting activity [31, y AA/30 min; o-0, Boc-Ala-NPsplitting activity [91, y AA110 min. Tr, trypsin; CbTr, chymotrypsin; CXP, carboxypeptidase A.

219

Step III. Elastin-Sepharose chromatography In the chromatogram (Fig. Z), one protein peak, appearing as a single band in polyacrylamide electrophoresis (Fig. 3), was eluted; it had cathodal mobility in agarose electrophoresis and elastinolytic activity and also Sucf Ala)3NA activity. This Suc(Ala),NA activity was reduced to a third of that recovered from step II (see Table I). The remaining two thirds of the Suc(Ala)3NA activity (having anodal mobility) was found in the unretarded protein fraction that was almost devoid of elastinolytic activity. Assays of the peak fractions for trypsin, chymotrypsin, c~boxypeptidase, and amylase were negative.

Stability of the enzyme When incubated at 37”C, the adult’s duodenal juice retained its immunoactivity and elastinolytic activity virtually unchanged after 4 h (Fig. 4), during which time, the SuefAla),NA activity in the juice decreased to 90%. The juice

Fractton

number

Fig. 2. Chromatography of elastinolytic fractions from SP-Sephadex C-60 on Elastin-Sepharose 4-B. FracSuc(Ala)gNAsplitting activity 131, Y AA/ tion volume 5 ml. -, absorbance at 280 nm; A-, 30 min; 0-0, elastinolytic activity [41, Y AA/30 min. Fig. 3. Polyacrylamide disc electrophoresis in 8% gels at pH 4.5 of peak fraction from Step III. Running direction from anode (top) to cathode (bottom).

i_ 1

2 T

4h

/ME

Fig. 4. Immunoactivity. elastinolytic activity and Suc(Ala)3NA-splitting activity in human duodenal juice elastinolytic activactivity against cathodal elastase antibodies: .\-F\, incubated at 37’C. 13----0, Suc(Ala)3NA-splitting ity; @e, Suc(Ala)3NA-splitting activity in juice from an adult; l --------0, activity in juice from a B-month-old child.

from a 6 months old child, which had no detectable elastinolytic activity, decreased to 62% in Suc(Ala)3NA activity, whereas the immunoactivity was unchanged. At 4°C and -2O”C, the immunoactivity, the elastinolytic, and Suc(Ala)3NA activities in duodenal juice remained unchanged for at least 1 months. In the purified state, at both pH 4.0 and 8.6, the enzyme retained its activities unchanged for months at 4” C and -20” C. Immunochemical

studies

Rabbit antisera raised against the elastase preparation gave only one single precipitate when tested against duodenal juice by immunoelectrophoresis (Fig. 5). No cross-reaction was observed against the anodal Suc(Ala)3NA-splitting band, nor was any cross-reaction observed against the two trypsins or chymotrypsin. The purified elastase preparations gave no precipitate when tested against human anodal and cathodal trypsin and chymotrypsin antisera in the same manner. No precipitate occurred with porcine elastase. Fig. 6 shows antigen-antibody crossed immunoelectrophoresis of human duodenal juice against rabbit anticathodal elastase antiserum. The precipitate pattern indicated mono-specificity. The duodenal juice tested in the same system against a mixture of this antiserum (5%) and of antiserum against human cathodal trypsin (2%) gave a pattern of non-identity. Immunochemical

determination

of cathodal

elastase

Fig. 7 shows the rocket pattern for anti-cathodal elastase antibody-containing gels tested against standard solutions and human duodenal juice from 9 children. As in crossed immunoelectrophoresis, the precipitates occurred ano-

Fig. 5. Immunoelectrophoresis of human duodenal elastase run for 70 min at 18 V/cm at pH 8.6.

juice with rabbit

antibodies

against

human

cathodal

Fig. 6. Crossed imnrunoelectrophoresis of human duodenal juice with 5% rabbit antibodies against human cathodal elastase. Running times: eleetrophoresis 90 min. immunoelectrophoresis 10 h, 15 V/cm, PH 8.6. Fig. 7. Electroimmunoassay of 9 samples (10 ~1) of human duodenal juice in agarose gei with 10% antibodies against human cathodal elastase run for 12 h at 11 V/cm. Dilution of samples: Pat. No. 1-2. 1 : 1; 3-4.1 : 4: 5,1 : 3; 6,1 : 10; 7-8.1 : 4; 9.1 : 10. Dilution of standard (0.52 g/i purified human cathodal elastase) from left to right: 1 : 60; 1 : 45; 1 : 30; 1 : 25; 1 : 22.5. (Standard: left part of Fig.)

dally despite cathodal mobility in this system for the antigen per se. The rockets were distinct and easily measured. Table II summarizes the results of elastase determinations with immunochemical and enzymatic (Suc(Ala)3NA and elastinolysis) assays of the duodenal juices from the nine children. Of the seven children with normal pancreatic function (Nos. 3-9), two had no detectable elastinolytic activity, but had immunoactive elastase and Su~(Ala)~NA activity. One child (No. 1) with pancreatic insufficiency with no detectable enzyme activity had a small amount of immunoactive elastase. There is no evident correlation between Suc(Ala)3NA activity, elastinolytic TABLE II Suc(Ala)3NA-SP~ITTIN~ ACTIVITY, ELASTINOLYTIC DAL ELASTASE IN HUMAN DUODENAL JUICE

ACTIVITY

Patient No.

Suc(Ala)g NA activity

Diagnosis

Age

AND I~~UNOACTIVE

ElastinImmunoolytic activity activity (mgP) Standard:

Standard:

1

2 3 4

7y 2y ly 5Y

Om lm 8m 11 m

Oy

9m 10m 6m

OY ly Oy ly

8m

4m

Shwachman’s syndrome Pancreatic insufficiency Unclassified malabsorption Hypoglycemia + subtoW pancreatectomy ;c Coeliac disease Unclassified malabsorption Cow’s milk protein intolerance Unclassified malabsorption Unclassified malabsorption

CATHO-

Porcine elastase (mg/I)

Human elastase (mg P)

0 0.25 2.25 2.92

0

0

65.3 592.0 795.6

0 59

0 64.9 42.8

1.57 8.85 11.55 2.34 3.25

419.1 2408.0 3142.6 631.8 882.2

0 70 88 143 130

19.9 144.9 54.6 52.1 159.6

Human elastase (mgn)

0

3.99

activity, and immunoactivity. stronger Suc(Ala),NA-splitting 270 times).

The table shows that porcine activity than human cathodal

elastase elastase

had a (about

Discussion In 1970 [Zl], trialanine compounds were reported to be specific substrates for porcine elastase. Since then, some studies have been carried out to detect enzymes with trialanine esterolytic activity in human pancreatic [1,2,22,23] and duodenal [3] juice. Different authors (1,2,3] have reported at least two substances with this activity: one enzyme with cathodal and one with anodal mobility in gel electrophoresis. Conflicting results concerning the activity of the anodal variant on the natural elastin substrates have led to confusing differences in nomenclature: “non-elastolytic Protease E” [ 221 and “Elastase 1” [l]. Fric et al. after studies of duodenal juice proposed that “anodal elastase” is a precursor of “cathodal elastase” [ 33. In this study, a major portion with Suc(Ala),NA activity was found in duodenal juice; a minor fraction of this had elastinolytic activity. This activity, confined to the part with cathodal mobility, could be isolated further with elastin as an affinity ligand. The resulting purified enzyme so far resembles elastase ‘2 of Largman et al. [Z], the elastase purified by Ohlsson et al. [23], and “the cathodal elastase fraction” of Fric et al. [ 31. The remaining Suc(Ala),NA activity, lacking elastinolytic properties, corresponds to the Protease E of Mallory and Travis [ 221 and the anodal fraction of Fric et al. [ 31. The lack of cross-reactivity, also reported by Largman et al. [ 21, between the rabbit antibodies raised against the purified cathodal elastase and the anodal nonelastolytic fraction sustains the presumption of two separate enzymes. This study reveals a remarkable difference in substrate specificity between the two substrates Boc-Ala-NP and Suc(Ala),NA illustrated in the SP-Sephadex chromatogram. High activities for Boc-Ala-NP were found in the chymotrypsin/ trypsin regions. The major part of this activity was confined to ~hymotrypsin. This renders Boc-Ala-NP unsuitable for efastase determinations in human duodenal fluid. Our results suggest that quantitation of elastase in duodenal juice by the elastin radial diffusion method described above is seemingly insensitive and unreliable. Perhaps the great influence of salt molarity on the adsorption of porcine elastase to elastin reported by Gertler 1241 is the explanation. We found, as did other authors [ 2,231, that human cathodal elastase had a very low Suc(Ala)3NA activity and elastinolytic activity compared with porcine elastase. For the former substrate, the ratio was 1 : 270; for elastinolysis, the ratio was 1 : 25. The cathodal elastase in purified state and also in duodenal juice was fairly stable at low temperatures, as judged from enzymatic and immunochemical determinations. Similar results for elastinolytic activity were reported by Largman et al. [2] for Elastase 1 and 2. At 37”C, we found a difference in stability for different juices: see Fig. 4. The proportionately greater loss of Suc(Ala),NA activity in the infant’s juice could possibly be explained by (age-dependent?) differences in the proportions

223

of the two components of the total Suc(Ala)3NA activity. The non-elastinolytic anodal fraction might be more unstable and perhaps accounts for a greater part of the infant’s Suc(Ala)3NA activity. This evidence for differences in composition of the Suc(Ala),NA activity strongly suggests that separate determinations of the two components are necessary. This might be possible with the aid of immunochemical methods or with more specific enzymatic methods. This study points to the possibility and value of electroimmunoassay for the cathodal elastase. Studies of the possible occurrence of inactive forms of the enzyme must await an enzymatic assay specific for the cathodal elastase. Our patient group revealed several discrepancies between the results of the three different methods (Suc(Ala),NA activity, elastinolysis, and immunoactivity). In one of the two patients with pancreatic insufficiency (No. 2) the discrepancy might indicate the presence of anodal “elastase” only. In the other patient (No. l), with zero values for enzymatic activities, the measurable immunoactivity could represent an inactive form of cathodal elastase. Two patients (Nos. 3 and 5) with unclassified malabsorption and coeliac disease, respectively, also had results suggesting the presence of inactive cathodal elastase. Further studies in many more patients are necessary to decide how important these discrepancies are, in order to throw more light on normal and pathological variations. Acknowledgements We are very much indebted to Dr. K. Ohlsson for valuable methodological advice. This work was supported by the Swedish Medical Research Council (Project Nos. 5143 and 5364), The Swedish Baby Food Industry Fund for Nutritional Research, The Swedish Nutrition Research Foundation, Semper Nutrition Research Foundation and Albert Pihlsson Fund. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

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