Molecular forms of acetylcholinesterase in Hirschsprung's disease

297

Clinica Chimiw Acta, 145 (1985) 297-305 Ekevier

CCA 03093

Molecular forms of acetylcholinesterase Hirschsprung’s disease

in

James R. Bonham a**,Gordon Dale ‘, David Scott b and John Wagget’ *~~purr~~n~ o!Clinical ~~~~ernist~ and ’ department of H~sto~~t~o~o~,~~~cast~e General ~os~jtu~ and ’ Fleming Memorial Hospital for Sick Children, Newcastle upon Tyne (UK] (Received July 16th, 1984; revision October 9th, 1984)

Summary

We describe changes in the levels of different molecular forms of acetylcholinesterase in four cases of Hirschsprung’s disease linked to the transition from aganglionic to normal bowet. In addition changes in a control case with histologically normal bowel is reported. In all patients with Hirschsprung’s disease there is a marked increase in the level of the tetrameric form of the enzyme in the aganglionic region. The changing level of this form of the enzyme correlates well with the hist~hemical appearance suggesting that quantitative measurement of this molecular species might form the basis of an improved diagnostic test for the disease.

Historically the diagnosis of Hirschsprung’s disease has rested upon the confirmation of aganglionosis occurring in Auerbach’s plexus from the terminal portion of the large intestine. More recently, a simpler technique requiring only suction biopsy and relying upon the histochemical demonstration of hypertrophied nerve trunks in the lamina propria and muscularis mucosa has proven very useful [1,23. In this procedure nerve fibres are visualised by an increase in associated cholinergic staining. It has also been known for some time that this increased cholinesterase activity observed histochemically could be measured quantitatively in suction biopsy homogenates [3]. This has formed the basis of a useful diagnostic test showing increased

* To whom correspondence should be addressed. 0009-8981/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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acetylcholinesterase (AChE) activity in Hirschsprung’s disease when compared with other causes of constipation in childhood [4,5]. AChE is known to occur in a variety of differing molecular forms [6], some of which predominate at particular sites [7,8] and may be used as markers for structures such as the neuromuscular junction [8]. While elevated AChE levels are known to occur in Hirschsprung’s disease, the particular molecular forms involved in this increase have not been properly identified. Electrophoresis has shown the presence of an abnormally migrating form of AChE present in suction biopsy tissue in Hirschsprung’s disease [9] but the use of sucrose density gradient centrifugation has not been hitherto reported. We report the change in molecular forms of AChE along strips of resected bowel which include an aganglionic region and the transition into proximal. histochemitally normal, gut. These results are compared with the changing histochemical appearance in each case. Materials and methods Tissue sampks Resected distal colon was obtained from four cases of Hirschsprung’s disease treated by the Soave operative procedure (cases A, B, C and D). Case A. Recta1 sleeve measuring 10 cm, comprising mucosal layer only, from a male infant aged 10 mth. Case B. A 16-cm length of bowel including upper rectum and sigmoid colon and involving both muscle and mucosal layers obtained from a male infant aged 11 mth. Case C. A 24-cm length of bowel including distal colon and rectum, comprising both muscle and mucosal layers from a male infant aged 11 mth. Case D. Recta1 sleeve measuring 14 cm, comprising mucosal layer only, from a male infant aged 1 Y’. Control tissue consisted of a 30-cm segment of redundant bowel extending from upper rectum into sigmoid colon, containing both muscle and mucosal layers. excised from a lCyr-old girl with no evidence of Hirschsprung’s disease. Tissue preparation In each case the segment of bowel was divided longitudinally into three strips for histological, histochemical and biochemical studies. These strips were then divided transversely into between 5 and 9 sections depending upon strip length. Sections were stored in liquid nitrogen for biochemistry, fixed in 10% formalin for histology and 8 pm cryostat sections cut immediately for histochemical investigation. Histological technique The formalin fixed material was processed routinely and embedded in paraffin wax. Sections were stained with haemotoxylin and eosin and examined for the presence or absence of ganglion cells. Cryostat sections were stained by the method of Karnovsky and Roots [lo] for acetylcholinesterase. For the purpose of this paper a positive result indicates a

299

marked increase in the number of AChE positive nerve fibres in the muscularis mucosae. Biochemistry Samples were carefully thawed and where appropriate, (cases B, C and control), muscle and mucosal layers were separated by dissection under a binocular microscope. Buffer systems throughout comprised disodium hydrogen and potassium dihydrogen phosphate in appropriate proportions to give the desired PH. In cases A, B, C and control, 5% w/v homogenates were made in 100 mmol/l phosphate buffer, pH 8.0, using a Potter Elvehjem homogeniser. These were then centrifuged at 15000 x g to remove debris and the clear supernatants subjected to sucrose density gradient centrifugation. In case D, in order to distinguish the easily soluble from membrane bound cholinesterase forms, samples were very mildly homogenised by 20 passes in a Potter Elvehjem homogeniser on ice in 100 mmol/l phosphate buffer, pH 7.3. The homogenate was then spun at 36000 x g (average) for 30 min to ensure that only soluble species (< 100s) remained in the supernatant. The pellet was then resuspended by vortexing for 1 min in 10 mmol/l phosphate buffer, pH 7.2, containing 1 mol/l NaCl, 1 mmol/l EDTA and 0.5% w/v Triton. This suspension was also spun at 36000 X g (average) for 30 min. In cases B and C, AChE activity was also determined by the routinely used quantitative AChE assay 141. Density gradient centrifugation In case A, 0.2 ml samples of pre-centrifuged homogenate were layered together with sedimentation markers &galactosidase (S2,,, W; 16.OS), alcohol dehydrogenase (Sz,,, W; 4.8s) and catalase (so, W; 11.35) onto 5 ml linear sucrose gradients (5 to 20% w/v with a 0.5 ml 50% w/v sucrose cushion) made up in 10 mmol/l phosphate buffer pH 7.2, containing 1 mol/l NaCl and 0.5% w/v Triton. Centrifugation was carried out with a Beckman L2-75B or Spinco L centrifuge in an SW 65 swing out rotor at 102000 x g (average) at 4°C for 17 h. Approximately 35 fractions were collected drop-wise following puncture of the gradient tube into pre-weighed fraction tubes. Improved resolution of the lower molecular mass forms was obtained by increasing the viscosity of the sucrose gradient to 10-40s w/w and appropriately increasing the centrifuge speed to 147000 X g (average) for 24 h in an SW 50.1 rotor or to 216 000 X g (average) for 17 h in an SW 65 rotor. This modification was adopted for cases B, C, D and the control specimen. AChE activity was measured by a previously described method 1111adapted for use on a centrifugal fast analyser (Cobas Bio, Roche Diagnostic Ltd., Welwyn Gdn., UK) without change in final reagent concentration. Within-batch precision measurement gave a coefficient of variation (CV) value of 3.9% at a level of 2.4 U/l. Following collection, the fraction tubes were reweighed and the percentage sucrose in each fraction calculated from refractive index measurement, enabling the volume of each fraction to be determined. Enzyme activity was plotted directly against cumulatively increasing volume change on the abscissa. This activity was

300

corrected for interference in cholinesterase assay produced by the increasing concentration; this was effectively a reagent blank correction.

sucrose

Results The sedimentation pattern of AChE activity from the extreme ends of case B (a 16-cm section of bowel including upper rectum and sigmoid colon) is illustated in Fig. 1. There is a marked increase in AChE activity sedimenting at 9.2s f 0.8 (SD), n = 52, (G4) in the aganglionic (distal) region compared with the normal (proximal) region. 3.5s + 0.5 (SD), n = 47, (G,) AChE is also increased in the aganglionic zone but this is much less pronounced. 5.0s + 0.6 (SD), n = 47, (G,) AChE activity remains largely unaltered as does the form sedimenting at 16.7s k 0.8 (SD), n = 41, (A,,). This A,, species comprises such a small percentage of the total AChE activity (5% in the aganglionic region) that experimentally determined changes are probably not significant. Changes in G, and G, forms seen in Fig. 1 are qualitatively similar in both muscle and mucosal layers; however their magnitude is greater in the muscle layer. Marked changes in the levels of G, AChE activity were found in all cases (A-D). Changes in activity are shown for cases A, B and C in Figs. 2-4. Variation in the levels of these molecular forms closely reflect histological and histochemical changes and the level of G, AChE rises sharply as the aganglionic zone is reached. Changes

CASE

AChE

220

activity (U I”)

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B: MUSCLE

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B.

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activity 2oo (U I-’ ) t

--

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180 160

Agangl~onlc .

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160 140 -

G4

120 100 -

2

80 -

0

10

20

30

40

50

60

70

80

90

100

Percentage fraction

0

10

20

30

40

50

80

70

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100

volume

Fig. 1. Sedimentation patterns of AChE activity from extreme ends; normal (proximal) and aganglionic (distal) of resected bowel case B for both muscle and mucosal layers. Enzyme activity is expressed as U/l per ml of 5% w/v homogenate applied to the gradient and the fraction volume as a percentage of the total volume (approximately 5 ml). The positions of the marker enzyme, Escherichia co/i fl-galactosidase (B-gal.; 16.OS), bovine heart catalase (cat.; 11.3s) and equine liver alcohol dehydrogenase (a.d.h.; 4%) are labelled. The positions of 16.7s (A,,), 9.2s (G4), 5.0s (G,) and 3% (G,) molecular forms are shown.

301 CASE A 80-

G, + G:, C-4

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% A, 2

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10 ,____-.-- c -.---- -*...__

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I

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1

4

,

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6

I

I

8

10 distal

cm

HISTOLOGY HISTOCHEMISTAY

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,

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Fig. 2. The variation in the levels of AChE molecular forms (G,, G,, G4 and A,,) shown at five intervals along the 10 cm strip of rectal sleeve of Case A. The histological and histochemical appearance is also shown for comparison. Enzyme activities are expressed as mu/ml of 5% w/v homogenate originally applied to the gradient. CASE 6: MUCOSAL

AChE activity (mu ml” homogenate)

LAYER

40 30 20 -

0

0

I 2

I 4

.r ___.--L-w-_ I , ____1_-_ , 8 10 12 14 16

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HISTOLOGY HISTOCHEMISTRY

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4o CASE B: MUCOSAL

I

LAYER

AChE activity (&log-1 )

10

2SD

normal range

0 0 2 proximal

4

6

8 cm

10

12

14 16 distal

Fig. 3. The variation in the levels of AChE molecular forms (G,, G,, G4 and A,,) are shown at seven intervals along the 16 cm length of resected bowel from the mucosal layer of Case B. The histological and histochemical appearance is also shown for comparison. Enzyme activities are expressed as mu/ml of 5% w/v homogenate originally applied to the gradient. Total AChE levels as determined by the routinely used quantitative assay are shown as they vary along the same 16 cm section of bowel. Enzyme activity is expressed as U. lo-“g-l wet weight tissue. The diagnostically used normal range is shown for comparison.

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CASE C: MUCOSAL G,

50 AChE activity (mu ml-’ homogenate)

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I

6

9

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18

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24

HISTOLOGY

I

2SD normal range

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0 0

3

proximal

6

9

12 cm

15

18

21

24

distal

Fig. 4. The variation in the levels of AChE molecular forms (G,, G,, G, and A,, ) are shown at 8 intervals along the 24 cm length of resected bowel from the mucosal layer of Case C. The histological and histochemical appearance is shown for comparison. Enzyme activities are expressed as mu/ml of 5% w/v homogenate originally applied to the gradient. Total AChE levels as determined by the routinely used quantitative assay are shown as they vary along the same 24 cm section of bowel. Enzyme activity is expressed as U.lO~‘~g-’ wet weight tissue. The diagnostically used normal range is shown for comparison.

in total AChE activity demonstrated by the quantitative assay already in use as a diagnostic service [4] (normal range shown) are included in Figs. 3 and 4 for cases B and C. The magnitude of increase is greater in the G, species alone than in total AChE activity which includes all AChE species. In the control case (Fig. 5) while levels of G,, G, and G, forms fluctuate in both muscle and mucosal layers along the length of resected bowel, there is no consistent trend. The A,, species again comprises a very small percentage of the total activity. In case D two extraction methods are used, a mild aqueous homogenisation medium more nearly equivalent to that in cases A, B and C and also a medium containing the detergent Triton capable of releasing membrane-bound forms of the enzyme. As in cases A, B and C there is an increase in the easily soluble form of G, in the aganglionic zone when compared with the ganglionic region which acts as an in-built control (Fig. 6). The increase in Triton releasable G, in the aganglionic region is even more noticeable (Fig. 6). Less marked but consistent changes are also seen in G,. The combination of high salt and Triton would be expected to release a large proportion of any A,, form of the enzyme present; however this species showed a low level throughout.

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CONTROL:

MUSCLE

CONTROL:

LAYER

MUCOSAL

LAYER

--_ i

G

1

_.-.

G2

-

G4

.----_

A,2

t

0

10

, 30 0

20

proximal

cm

distal

10

proximal

20 cm

30 distal

Fig. 5. The variation in the levels of AChE molecular forms (G,, G,, G, and A,,) are shown at seven intervals along the 30 cm section of resected bowel for both muscle and mucosal layers of the Control (no evidence of Hirschsprung’s disease) case. Enzyme activities are expressed as mU/ml of 5% w/v homogenate originally applied to the gradient. 150

CASE D: TRITON

RELEASABLE

AChE activity (mu mlM1 homogenate)

0 0

AChE activity (mu ml“

;;

2

4

6

10

12

14

CASE D: EASILY SOLUBLE

proximal HISTOLOGY HISTOCHEMISTRY

8

GANGLIONIC NEGATIVE

cm

1 I

distal

AGANGLIONIC POSITIVE

Fig. 6. The variation in the levels of AChE molecular forms (G,, G,, G4 and A,,) are shown at seven intervals along the 14 cm section of resected bowel employing two different homogenisation media: low salt without Triton (easily soluble), pH 7.3, 100 mmol/l phosphate buffer and high salt with Triton (Triton releasable), pH 7.2, 10 mmol/l phosphate buffer containing 1 mol/l NaCl and 0.5% w/v Triton. The results are expressed in mU of activity of each form per ml of 5% w/v homogenate applied to the gradient. The histological and histochemical appearance is shown for comparison.

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Discussion It is welt estabhshed that there is both histochemical [Z] and direct quantitative [s] evidence of increased a~etyIcholinesterase activity within the aganglioni~ region of Hirschsprung’s disease. However, in neither situation have the particular molecular species of AChE involved been identified. AChE occurs in several distinct molecular forms. Studies of tissue from a variety of sources demonstrate a monomer (G,) with a sedimentation coefficient of 3.5-4.7s. dimer (G,) of 6.0-6.7s and a tetramer (G,) of approximately 10.0s ]12,13]. In addition to these globular forms. collagen containing asymmetric species which are predominantly membrane-bound, also exist; the most frequently reported of these is a 16.05 (A,*) form believed to comprise three tetramers linked to a collagen tail [13]. We describe the presence of G, (3.5S), G, (S.OS), G, (9.2s) and A,, (16.7s) in normal and aganglionic bowel. In all cases levels of G, are markedly increased in the aganglionic region. G, also shows an increase in the muscle layer of case B; however this is not seen in the mucosa of the remaining three cases (Figs. 2-4). Both G, and G, have been described as major forms of the enzyme in nerve trunks [7] and may be secretory products [13]. It would appear, therefore, that the nerve trunk hypertrophy seen in Hirschsprung’s disease is responsible for the increased levels of G,. It is possible that the increase in the level of G, in case B (Fig. 1) is due to some spontaneous disaggregation of the G, molecule. Transection of sciatic nerve produces marked increase in G, in the proximal nerve stump [15] and it may be that the changes which we observe in aganglionically related nerve trunk hypertrophy have some related cause. It should be mentioned that transected nerves also accumulate A,, [15] and although the levels of A,? in gut appear low we did not observe any consistent increase in the aganglionic region. Triton~containing buffer used in homogenisation discloses an even more marked increase in the level of G, activity in the aganglionic region (Fig. 6). This suggests that there is a large increase in the membrane-bound as well as soluble form of this species. These findings are in accord with the histochemically observed distribution of the enzyme showing increased AChE activity outlining hypertrophied nerve trunks in the aganglionic zone. The value of histochemistry and the quantitative measurement of AChE activity in the diagnosis of Hirschsprung’s disease is now well established [2,5]. The identification of a specific form of AChE (G,+) in increased activity in regions where there is nerve trunk hypertrophy gives a clearer understanding of the basis of these tests. The difference in levels of this form between normal and aganglionic areas is more marked than the change in total AChE activity (the sum of all forms present). This creates the potential to design a more sensitive and specific assay by direct measurement of this AChE species alone. Acknowledgement We should like to record our thanks to Dr. R.J.T. Bennington, Department, NGH for the use of laboratory facilities.

Neurochemistry

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References I Meier-Ruge W, Lutterbuck activity in suction biopsies

PM, Herzog of the rectum

B, Morger R, Moser R, Scharli in the diagnosis of Hirschsprung’s

A. Acetylcholinesterase disease. J Pediatr Surg

1972; 7: 11. 2 Lake BD, Nixon HR. Hirschsprung’s Disease: an appraisal of histochemically demonstrated acetylcbolinesterase activity in suction rectal biopsy specimens as an aid to diagnosis. Arch Pathol Lab Med 1978; 102; 244247. 3 Boston VE, Dale G, Riley KWA. Diagnosis of Hirschsprung’s disease by quantitative biochemical assay of acetylcholinesterase in rectal tissue. Lancet 1975; ii: 951-953. 4 Dale G, Bonham JR. Riley KWA, Wagget J. An improved method for the determination of acetylcholinesterase activity in rectal biopsy tissue from patients with Hirschsprung’s disease. Clin Chim Acta 1977; 77: 407-413. 5 Dale G, Bonham JR, Lowden P, Wagget J, Rangecroft L, Scott DJ. Diagnostic value of rectal mucosal acetylcholinesterase levels in Hirschsprung’s disease. Lancet 1979; i: 347-349. 6 Chubb IW, Smith AD. lsoenzymes of soluble and membrane-bound acetylcholinesterase in bovine splanchnie nerve and adrenal medulla. Proc R. Sot London B 1975; 191: 245-261. 7 Skau KA, Brimijoin A. Multiple molecular forms of a~tylcholinesterase in rat vagus nerve, smooth muscle and heart. J Neurochem 1980; 35: 1151-1154. 8 Vigny M, Koenig J, Reiger F. The motor endplate specific form of acetyicholinesterase: appearance during embryogenesis and re-innervation of rat muscle. J Neurochem 1976; 27: 1347-1353. 9 Bajgar J, Hak J. Acetylcholinesterase activity and its molecular forms in rectal tissue in the diagnosis of Hirschsprung’s disease. Clin Chim Acta 1979; 93: 93-95. 10 Karnovsky MJ, Roots L. A ‘direct coloring’ thiocholine method for cholinesterases. J Histochem Cytochem 1964; 12: 219-221. 11 Bonham JR. Gowenlock AH, Timothy JAD. Acetylcholinesterase and butyrylchoiinesterase measurement in the prenatal detection of neural tube defects and other fetal malformations. Clin Chim Acta 1981; 115: 163-170. 12 Carson S, Bon S, Vigny M, Massoulie J, Fardeau M. Distribution of acetylcholinesterase molecular forms in neural and non-neural sections of human muscle. FEBS Lett 1979; 97: 348-352. 13 Massoulie J. The ~I~o~hism of cholinesterase and its physiological significance. TIBS 1980; 5: 160-164. 14 Brimijoin S. Axonal transport and subcellular distribution of molecular forms of acetylcholinesterase in rabbit sciatic nerve. Mol Pharmacol 1979; 15: 641-648. 15 Di Giamberardino L, Couraud JY. Rapid accumulation of high molecular weight acetylcholinesterase in transected sciatic nerve. Nature (London) 1978; 271: 170-172.