948
Species Specificity
Brush-border preparations from pigs, calves, rabbits
and guineapigs were used. Only strain 075 K? H5 cul-
tured on buffered glucose nutrient agar5 attached to
brush borders from only guineapigs and rabbits. This
adhesion was inhibited by 05% D-mannose. (Five to ten
fold fewer bacteria attached to the brush borders com-
pared to the adhesion of K88-positive E. coli to pig
brush borders.)
There was no attachment of 026 Kll H60 or 078
H12 to any animal brush-border preparation.
Discussion
The capacity of a microorganism to adhere to a tissue
surface may be an important factor in determining its
pathogenicity by allowing its proliferation,4and this sur-
face attachment seems to be both species and tissue spe-
cific.14
Smith and Halls3 showed that enterotoxic strains of
E. coli could only produce clinical disease if they proli-
ferated in the small intestine of experimental animals,
and Smith and Linggood15 found that enterotoxic
strains of E. coli caused severe clinical disease in pigs
only if the organisms had the surface antigen K88. Jones
and Rutted5 demonstrated in piglets that K88 antigen
was responsible for the attachment of K88-positive bac-
teria to the wall of the small intestine, and that adhesion
was essential for the virulence of these organisms in con-
ventionally reared piglets.
Strains of humanE.P.E.c. also produce enterotoxin,16
and some strains may show tissue-invasive properties.17
We have sought further parallels with the pathogenesis
of E. coli diarrhoea in animals by measuring mucosal
adherence; using strains of E. coli that are proven
human pathogens and not those known only to produce
soluble enterotoxin.
The adhesion to fetal intestine of strains 026 K60
Hll and 078 H12 was consistently much greater than
that of control organisms. There was however a wide
(10-20 fold) difference between the greatest and least
number of adhering organisms in separate experiments
with the same strain. We do not know whether this re-
flects differences in tissue maturity or other unknown
factors. It is of interest that Sellwood et al. 18 have recog-
nised genetic variation in pigs that affects the number of
K88-positive E. coli that will adhere to the intestine.
We do not think that adhesion to human fetal intes-
tine of the test strains is caused by common fimbrix.
Suspensions that were positive in the adhesion test did
not cause haemagglutination, and there was no mucosal
adhesion of the strain (075 K? H5) that caused the
strongest haemagglutination. We do not know whether
fimbrial adhesion to mucosa can be inhibited by D-man-
nose, but the results with 075 in the brush-border tests
suggest that is likely. We found that the number of
adhering organisms (026 K60 Hll or 078 H12) is un-
affected by the presence in the incubation fluid of
D-mannose, and this is further evidence that the adhe-
siveness of these bacteria is not caused by common fim-
brise.
The method used to measure mucosal adhesion is very
similar to that used in pigs by Jones and Rutter.5 This
latter method gives very similar results to the brush-
border assay that was used to test species specificity. It
is therefore likely that the adhesive property that we
have measured is species specific.
The inconsistent performance of strain 0148 K? H28
requires further study. This organism has been respon-
sible for disease in adults.19 and the test mucosa is very
immature. Not all E.P.E.C. may exert their virulence in
an identical manner, and there are strains of E. coli,
enteropathogenic to pigs, that do not have K88. How-
ever, the strain of 0148 that we used may have lost its
adhesive factor, if, like K88, it is plasmid derived.
The structural nature of the adherence of "positive"
organisms to human fetal intestine is not known. Pre-
liminary experiments suggest that it may be (lipo) poly-
saccharide. It seems likely that mucosal adherence may
be an important property of some E.p.E.c. in man, as in
animals.
We thank Prof. Charlotte Anderson for her interest and encourage-
ment, Dr H. Williams Smith and Dr P. Cauthery for help in many
ways, Dr J. M. Rutter and Dr R. Sellwood for species-specificity tests
and helpful discussions, Dr B. Rowe for the gift of bacterial strains,
and Prof. P. G. H. Gell for laboratory facilities. This work was sup-
ported by grants from the Medical Research Council, the Children’s
Research Fund, and the United Birmingham Hospitals Endowment
Research Fund.
Requests for reprints should be addressed to A. S. McN. Institute
of Child Health, Francis Road, Birmingham B16 8ET.
REFERENCES
1. Taylor, J.J. appl Bact. 1966, 29, 1.
2. Smith, H. W , Gyles, C. L.J med. Microbiol. 1970, 3, 403.
3. Smith, H.W, Halls, S. J. Path. Bact. 1967, 93, 499.
4. Savage, D. C. Symp. Soc. gen. Microbiol. 1972, 22, 25.
5. Jones, G. W., Rutter, J. M. Infect. Immun. 1972, 6, 918
6. Smith, H. W., Halls, S. J med. Microbiol. 1968, 1, 45.
7. Drucker, M. M., Polliack, A., Yeivin, R., Sacks, T. G. Pediatrics, 1970, 46,
855.
8. De, S. N., Chatterie, D. N. J. Path. Bact. 1953, 66, 559.
9. The Use of Fetuses and Fetal Material for Research. Report of the Advisory
Group. H.M. Stationery Office, 1972.
10. Townley, R. R. W., Bhathal, P. S., Cornell, H. J., Mitchell, J. D Lancet,
1973, 1, 1363.
11. Sellwood, R., Gibbons, R. A., Jones, G. W., Rutter, J. M J. med Microbiol
(in the press).
12. Duguid, J. P., Smith, I. W., Dempster, G., Edmunds, P. N. J. Path. Bact.
1955, 70, 335.
13. Duguid, J. P., Gillies, R. R. ibid. 1957, 74, 397.
14. Lancet, 1974, 1, 716.
15. Smith, H. W., Linggood, M. A. J. med. Microbiol. 1971, 4, 467
16. Gorbach, S. L. New Engl. J. Med. 1970, 283, 44.
17. Dupont, H. L., Formal, S. B., Hornick, R. B., Snyder, M J, Libonati,
P., Sheahan, D. G., Labrec, E. H., Kalas, J. P. ibid. 1971, 285, 1.
18. Sellwood, R., Gibbons, R. A., Jones, G. W., Rutter, J. M. Vet. Rec. 1974,
95, 574.
19. Rowe, B., Taylor, J., Bettelheim, K. A. Lancet, 1970, i, 1.
TREATMENT OF DIABETES INSIPIDUS WITH
CARBAMAZEPINE
JOHN K. WALES
Department of Medicine, University of Leeds, General
Infirmary, Leeds LS1 3EX
Summary Oral carbamazepine has been shown to
have antidiuretic activity in seven out of
nine patients with neurohypophyseal diabetes insipidus.
At the doses used side-effects were not a major problem.
In the eighth patient a carbamazepine and clofibrate
combination was effective but in the ninth carbamaze-
pine was without effect. It is suggested that carbamaze-
pine should be used initially in neurohypophyseal dia-
betes insipidus if oral therapy is indicated, but the mode
of its antidiuretic action is as yet unclear.
 949

Introduction
IN 1966 Braunhofer and Zilva’ and Arduino et al.1
reported the antidiuretic effect ef carbamazepine and
chlorpropamide, respectively, in neurohypophyseal dia-
betes insipidus. Chlorpropamide has been more widely
used in clinical practice since then, but hypoglycaemic
side-effects have limited its use. The antidiuretic activity
of carbamazepine has, therefore, been studied as an
alternative therapy in patients with diabetes insipidus of
this type.
Patients and Methods
Nine patients were investigated in the metabolic ward of the
General Infirmary at Leeds. The clinical details of these pa-
tients are shown in table J. Five patients developed diabetes in-
sipidus after hypothalamic or pituitary surgery, and four pa-
tients had the idiopathic neurohypophyseal type of the disease.
All but two patients were treated by vasopressin injections on
admission, one patient receiving chlorpropamide, the other no
therapy, despite a urine output between 7 and 9 litres/day.
On admission all patients discontinued their normal treat-
ment for diabetes insipidus but other replacement therapy was
maintained throughout the study. After 4-5 days of baseline
measurements of 24 h fluid intake, 12 h urine output, body-
weight, urine and plasma osmolality, and 24 h urinary excre-
tion of sodium, potassium, calcium, and phosphate (in four
,
patients), carbamazepine 600 mg/day in divided doses was
started and the same measurements were continued for 7 days.
!f no response to the drug was noted after 7 days, the dose of
carbamazepine was increased to 1200 mg/day. All side-effects
were recorded.
Both before and after carbamazepine therapy the response
in hourly urine output and free-water clearance after intra-
venous infusions of 0.1 mU/kg and 1mU/kg aqueous vasopres-
sin (Parke Davis) was measured to determine whether there
was any change in antidiuretic response to vasopressin. During
the tests however, the patients were not catheterised but hyd-
ration was maintained by giving a volume of oral fluid hourly,
equal to the previous hour’s urine output. DAY
Plasma levels of arginine vasopressin were measured by Fig. 1-Mean response in urine output, fluid intake, plasma and urine
radioimmunoassay (by Dr J. J. Morton, M.R.C. Blood Pres- osmolality, and body-weight in six patients with diabetes insipidus
sure Unit, Western Infirmary, Glasgow) on two occasions treated with carbamazepine 600 mg/day
before and after carbamazepine therapy in two patients in Shaded area in the bottom graph indicates osmolar clearance and
whom a satisfactory clinical antidiuretic response was stippled area free-water clearance expressed/12 h period.
observed.
Urinary electrolytes and plasma and urinary osmolalities
were measured by standard laboratory methods and free-water
clearance was calculated3. Statistical analysis of results was In the seventh patient continued for
treatment was not
performed by paired t test at a significance level of 5%. 7 days because social problems necessitated her early
All patients gave informed consent to the trial of carbamaze-
pine therapy. discharge. Plasma osmolality fell significantly after 7
days and there was a significant increase in urinary
Results osmolality and reduction in free-water clearance. A
Seven patients treated with carbamazepine showed a small increase in body-weight was noted but this was not
satisfactory response in urine output and fluid intake. significant. From a clinical point of view all these pa-
The results obtained in six patients are shown in fig. 1. tients were able to sleep through the night after 2-3
TABS E I—CI INICAI DFTAII S OF PATIENTS STUDIE
D
950
days of carbamazepine therapy without drinking or
passing urine. The two patients who did not respond are
described in detail below.
Table n shows plasma-arginine-vasopressin levels
before and during carbamazepine therapy. Both patients
showed good clinical response but no change in plasma-
vasopressin levels.
There is little evidence that carbamazepine therapy
greatly increases the antidiuretic response to injected
vasopressin either at a low (0.1mU/kg) or high
(ImU/kg) dose. Fig. 2 shows the response in free-water
clearance after both injections before and after carba-
mazepine in five patients who had a satisfactory clinical
response to the drug. When the % fall in free-water
clearance in the hour immediately after the vasopressin
injection is compared before and after 7 days carbama-
zepine therapy the result shows that after 0.1mU/kg
vasopressin dose there is a 63% fall before and 87% after
carbamazepine and after the ImU/kg dose, 197% before
Fig. 2-Mean changes in free-water clearances, in five patients (B) befor
and (A) after treatment with carbamazepine.
The value of 0.0ml/min is equivalent to the mean osmolal
clearance.
and 206% after carbamazepine. There was no signifi-
cant change in response at either dose comparing the
results before and after carbamazepine. However, it is
clear that due to the greatly reduced free-water
clearance in patients receiving carbamazepine therapy
only a small change in urine output in the hour was
required to alter the calculated response in free-water
clearance after either vasopressin dose.
There were no significant changes in the urinary excre-
tion of sodium or potassium due to the drug; urinary
calcium rose and urinary phosphate fell but neither
change was significant.
The first two patients treated with carbamazepine
had a return of their symptoms when the carbamazepine
was replaced by placebo.
Carbamazepine has continued to be an effective anti-
diuretic therapy in those patients who responded for
over 6 months but symptoms returned whenever the
drug was discontinued.
Patients Who Did Not Respond to Carbamazepine Alone
Patient 8 (table I) developed diabetes insipidus at age
4 years after an attack of mumps, and was well con-
trolled on vasopressin tannate in oil injections. As he
passed into adolescence he became increasingly anta-
gonistic and resentful about his injections, and this
caused a great deal of family strife. In July, 1972, chlor-
propamide was tried but, despite increasing the dose to
500 mg per day, no antidiuresis occurred. However, he
had a severe hypoglycxmic episode on this dose and was
unconscious for 24 h. Vasopressin injections were then
started again. In September, 1974, he was readmitted
for a trial of carbamazepine, and without his vasopressin
injections his urine output was 10-11 litres/day. Carba-
mazepine at a dose of 600-1200 mg/day resulted in
some fall in urine output to around 6 litres/day, but on
the higher dose he complained of ataxia and pains in his
arms and legs. On the addition of clofibrate and reduc-
tion of the carbamazepine dose to 800 mg/day, as sug-
gested by Bonnici,4 his urine output fell to 3 litres/dav
and he was discharged on this regimen. Since then he
has made a good clinical response to intranasal desmo-
pressin (’DDAVP’). At all times anterior pituitary func-
tion has been normal.
Patient 9, presented in 1968 with facial pain thought
at first to be trigeminal neuralgia. In 1973, however,
further investigations revealed a suprasellar tumour. At
craniotomy (Mr D. J. Price) the pituitary fossa was
found to be normal, but a cystic craniopharyngioma was
located entirely within the hypothalamus. This tumour
was totally removed. Moderate diabetes insipidus fol-
lowed, and regular vasopressin injections were required.
When these injections were discontinued, urine output
increased to around 5 litres/day. Carbamazepine in
doses up to 1200 mg/day made little impression on this
level of urine output. Her vasopressin injections were
resumed with complete control of her symptoms and
urine output. She would not use intranasal desmopres-
sin.
Discussion
This report has confirmed the efficacy of oral carba-
mazepine alone in controlling the symptoms of neurohv
pophyseal diabetes insipidus in seven out of nine
patients. In the eighth patient the addition of clofibrate

tocarbamazepine was effective. The average dose used
was 600 mg/day, and at this dose level no side-effects of
note were recorded.
The first line of therapy in neurohypophyseal diabetes
insipidus is intranasal desmopressin.5 However, patients
object to this form of therapy. The principal alternative
oral therapy is chlorpropamide, but this drug can cause
hypoglycæmia.6 7Carbamazepine should be used in pre-
ference to chlorpropamide in cases where oral therapy is
required. Neither vasopressin, chlorpropamide, or car-
bamazepine8 are effective in nephrogenic diabetes insi-
pidus.
The mode of action of carbamazepine in diabetes insi-
pidus is unclear. The activity of this drug in epilepsy and
trigeminal neuralgia points to a central hypothalamic
effect increasing the secretion and/or synthesis of vaso-
pressin. Indeed, the experiments of Frahm et al.,9
Cabezas-Cerrato et al.,10 and Kimura et al.11 have
shown an increase in plasma antidiuretic activity, as
measured by bioassay, after carbamazepine therapy.
However, Hanefeld et al.12 could find no evidence of an
effect of carbamazepine on the area of vasopressin pro-
duction in the rat hypothalamus using enzymatic and
histochemical techniques. Meinders et al.13 were unable
to measure any increase in radioimmunoassayable
arginine-vasopressin in the plasma of patients with dia-
betes insipidus treated by carbamazepine, and my
results in two patients accord with this finding.
It has also been suggested 14 that carbamazepine in-
hibits an enzyme, vasopressinase, which destroys vaso-
pressin in the plasma and thus prolongs its action. One
might also expect this type of activity to increase the
plasma levels of vasopressin. The clinical experimental
results do not suggest a prolongation of the action of
vasopressin by carbamazepine either at a low or high
dose. However, after carbamazepine free-water
clearance was so reduced that reliable changes in urine
flow measurement in uncatheterised patients were
doubtful and an effect could have been masked.
The experiments of Uhlich et al. 15 using the toad
bladder do not support the concept of a direct, or poten-
tiating, effect of vasopressin on the kidney. This is in
contrast to chlorpropamide which does potentiate the
action of vasopressin on water transport in this experi-
mental model. 16
There is no suggestion in these patients that carbama-
zepine has any antidipsic action. Further work is
required to elucidate the mode of action of carbamaze-
pine in neurohypophyseal diabetes insipidus.
Although most investigators have not reported any
antidiuretic activity of carbamazepine in normal sub-
jects, the occasional patient on this drug for other rea-
sons might be expected to show inappropriate antiduire-
tic activity and there is a clinical report of such a pa-
tient.17
I thank Sister R. Dyer and the metabolic ward staff for their help
in these studies and my colleagues for referring these patients to me.
REFERENCES
1. Braunhöfer, J., Zilva, L. Med. Welt. 1966, 36, 1875.
2. Arduino, F., Ferraz, F. P. J., Rodriquez, J.J. clin. Endocr. 1966, 26, 1325.
3. Smith, H. W. Bull. N.Y. Acad. Med. 1959, 35, 293.
4 Bonnici, F. Clin Endocr., 1973, 2, 265.
5 Edwards, C. R. W., Kitau, M. J., Chard, T., Besser, G. M. Br. med. J. 1973,
ii, 375.
6. Wales, J. K., Fraser, T. R. Acta endocr. 1971, 68, 725.
951
Preliminary Communications
DIAGNOSIS OF HIRSCHSPRUNG’S DISEASE
BY QUANTITATIVE BIOCHEMICAL ASSAY OF
ACETYLCHOLINESTERASE IN RECTAL
TISSUE
V. E. BOSTON G. DALE
K. W. A. RILEY
Departments of Child Health and Clinical Biochemistry,
Newcastle University Hospitals, Newcastle upon Tyne
Summary Acetylcholinesterase activity was meas-
ured in rectal-biopsy specimens
obtained from nineteen children. Seven children in
whom the diagnosis of Hirschsprung’s disease was estab-
lished were found to have a significantly higher enzyme
activity than the twelve in whom this diagnosis was
excluded. This estimation may prove to be of value in
the diagnosis of Hirschsprung’s disease.
INTRODUCTION
HIRSCHSPRUNG’S disease is not easy to diagnose. The
histological examination of rectal-biopsy specimens is
subjective and requires considerable experience. Age-de-
pendent morphological differences in ganglion cells can
lead to confusion, and neural-plexus proliferation, an
important diagnostic feature, may be difficult to demon-
strated1 The initial histological diagnosis is often
wrong.23 Although anorectal manometry, when used in
the neonatal period, has been shown to be reliable in
about 90% of cases on first examination,the patients
who are most at risk may be impossible to diagnose. Fur-
thermore, in older children, manometry has been shown
to be less reliable.2
Semiquantitative histochemical methods have demon-
strated increased acetylcholinesterase activity in the sub-
mucosal and myenteric plexus of the aganglionic bowel
in Hirschsprung’s disease.s6 We describe here a quanti-
tative biochemical assay which is relatively simple to
perform and avoids subjective interpretation.
PATIENTS AND METHODS
Nineteen children, aged between 3 days and 12 years, with
a provisional diagnosis of Hirschsprung’s disease were investi-
gated. Five had constipation, and fourteen presented with neo-
natal intestinal obstruction.
Immediately before biopsy, manometry was performed to
determine the presence or absence of the anorectal reflex.24
7. Erlich, R. M., Kooh, S. W. Pediatrics, 1970, 45, 236.
8. Tietze, H. U., Finkerwirth, H. Mschr. Kinderheilk. 1970, 118, 237.
9. Frahm, H., Smejkal, E., Kratzenstein, R. Acta endocr. 1969, suppl, 138, p.
240.
10. Cabezas-Cerrato, J., Jiménez de Diego, L., Fernández-Cruz, A. Revta clin.
esp. 1970, 119, 11.
11. Kimura, T., Matsui, K., Sato, K., Yoshimaga, K. J. clin. Endocr. 1974, 38,
356.
12. Hanefeld, F., Lavsen, I., Stefan, H. Z. ges. exp. Med. 1970, 153, 95.
13. Meinders, A. E., Cejka, V., Robertson, G. L. Clin. Sci. mol. Med. 1974, 47,
289.
14. Smejkalova, E., Smejkal, J., Frahm, H. Rev. Roum. Endocr. 1970, 7, 339.
15. Uhlich, F., Loeschke, K., Eigler, J. Acta endocr. 1972, 69, suppl. 159, p. 51.
16. Wales, J.K.J. Endocr. 1971, 49. 551.
17. Radø, J. P. Br. med. J. 1973, ii, 479.