Medical Research Society
ACTH. The segments are chilled to -70°C and
sectioned at 12 pm at -25°C. The sections are mounted on glass slides clipped to a holder to facilitate
simultaneous handling and they are immersed in
separate containers in which are either standard
concentrations of ACTH or suitable dilutions of the
plasma sample. After 5 min immersion the sections are
removed and plunged into the acidic ferric chloridepotassium ferrocyanide solution used for the cytochemical demonstration of reducing potency
(Alaghband-Zadeh, Daly, Tunbridge, Loveridge &
Chayen, Journal of Endocrinology, in press). The
amount of Prussian blue stain (E680-E480)in the
cells of the zona reticularis is measured with a Vickers
M85 microdensitometer fitted with red-sensitive
photomultipliers.
As was predictable, when the sections were exposed
to the hormone dissolved in T8 medium alone, there
was considerable distortion of the cells and duplicate
sections gave widely differing results, even though a
graded response was observed. However, the preservation of the cells and the reproducibility of the results
were greatly improved if a colloid stabilizer, polypeptide 5115 (a degraded collagen much used in
quantitative cytochemistry) was included (5% w/v)
in the hormone or plasma solution. With this reinforced sample medium, a negative linear relationship
has been found between reducing potency in the
ulna reticularis and the concentration (loglo) of
standard ACTH over the range 0.005-5 pg/ml.
3. THE
ANTI-ANABOLIC
EFFECT
OF
GLUCAGON ON ALBUMIN SYNTHESIS BY
THE ISOLATED PERFUSED RAT LIVER
METCALFE,
ELIZABETH
BLACKand
A. S. TAVILL,JOAN
R. HOFFENBERG
Division of Clinical Investigation, Clinical Research
Centre, Northwick Park Hospital, Harrow, Middlesex
HA1 3UJ
The effects of glucagon on albumin synthesis, albumin
degradation, ureogenesis, glucose mobilization and
polyribosome profiles have been studied in the isolated
rat liver perfused with rabbit plasma containing
rat red cells. Measurements on groups of four
perfusions made during a control period of 2 h were
compared with those observed during a further 3 h of
perfusion during which glucagon was infused at a
constant rate (0.22 nmol/min or 4.4 pmol/min) into
the portal vein. The higher rate was that shown to
produce a maximal increase in hepatic cyclic AMP in
rats of equivalent weight (Exton, Robison, Sutherland
& Park, 1971, Journal of Biological Chemistry, 246,
6166).
Glucagon (0.22 nmol/min) produced early maximal
glucose mobilization which was accompanied by a
1 3 ~
fall in albumin synthetic rates measured by immunodiffusion from 8-0+1-1 (S.E.M.) mg/h/300 g body
weight to 3.1 kO.3 mg/h/300 g, a rise in urea synthesis
from 6.1 k 0-6 mg/h/3GU g to 8.8 k 0.6 mg/h/300 g and
a reduction in aggregated polyribosomes in favour
of monomeric and dimeric species. No significant
change in the degradation of screened lZ5I-labelled
rat albumin was observed. The reduction in albumin
synthesis produced by the lower rate of glucagon
infusion was smaller but significant, and minimal
polyribosome disaggregation was now evident.
However, glucose mobilization was not accompanied
by increased ureogenesis suggesting that glycogenolysis but not gluconeogenesis had been stimulated.
Portal vein infusion of insulin (0.37 nmol/min)
lowered blood glucose, inhibited the increase in
urea synthesis but failed to prevent the inhibition of
albumin synthesis or the polyribosome disaggregation
produced by higher dose glucagon. Intraportal
amino acids promoted urea synthesis but failed to
inhibit further glucagon-induced ureogenesis or the
accompanying inhibition of albumin synthesis and
polyribosome disaggregation.
These findings suggest an anti-anabolic role for
glucagon in albumin synthesis which cannot be
prevented by increased amino acid supply or insulin.
In contrast its catabolic effects are probably confined
to intracellular proteins (Miller, 1965, Federation
Proceedings, 24, 737) and available amino acids, and
can be inhibited by a relatively low insulin :glucagon
molar ratio.
4. PLASMA SECRETIN ASSAY I N MAN
K. D. BUCHANAN,
J. D. TEALE,G. HARPER,J. R.
HAYESand E. R. TRIMBLE
Department of Medicine, The Queen's University,
Berfast
We have encountered two major difficulties in the
establishment of a plasma secretin assay. Firstly, the
rarity of pure hormone preparations has limited the
experimental possibilities, and secondly the absence
of a tyrosine residue in the molecule has prevented
the preparation of 1251secretin by conventional
methods. We have been successful in raising high
titre, high affinity antibodies, using only very small
immunizing doses (5-25 pg) of pure secretin (Buchanan, Teale & Harper, 1972, Hormone and Metabolic
Research,4,507). Secretinwas iodinated with 12510dine
and specific activities of at least 40-70 pCi/pg were
achieved by using either pH modifications of the
chloramine-T method (Chisholm, Young & Lazarus,
1969, Journal of Clinical Investigation, 48,1453) or by
an enzymatic method (Holohan, Murphy, Buchanan
& Elmore, in press). Trasylol only partially prevented
secretin degradation in plasma. However, plasma
14~
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extraction (Heding, 1971, Diabetologia, 7 , 10)
successfully protected the hormone from plasma
enzymes. Sensitivities in the range of 30-100 p g / d
have been achieved for plasma secretin assay. The
assay has been found to be specific in that no crossreaction has been detected with other gut and islet
hormones. Immunoreactive secretin was present in
extracts of human duodenum and upper jejunum, and
these extracts diluted identically with pork secretin
standards. Plasma secretin levels in nine human
subjects declined after 50 g oral glucose from a fasting
level of 87+45 (MkSEM) to 43+13 at 60 min
although this suppression did not achieve significance
(P<0.15). On the other hand, the secretin levels after
25 g oral protein in seven subjects were significantly
elevated at 15, 30, 45 and 60 min after the meal
(P<O.Ol, < 0.025, < 0.05 and <0.025 respectively).
Secretin therefore does not appear to have a role in
the entero-insular axis for glucose, although a role for
protein is possible.
surgery patients. The gastrin response to protein
feeding in three patients with recurrent ulceration was
similar to that in asymptomatic patients. In a fourth
patient studied the increase in gastrin output was
320% which was more than two standard deviations
greater than the increase in asymptomatic patients.
This abnormal response was abolished by antrectomy
and gastroenterostomy.
Thus in at least some patients an excessive gastrin
response to feeding may be of importance in the
aetiology of duodenal ulceration. Identification of
these patients may lead to more rational treatment.
6. A COMPARISON OF STOOL FLUID AND
IN VIVO STOOL DIALYSATE
HAZELTHOMand MICHAEL
J. TARLOW
Department of Child Health, University of Aberdeen
In vivo dialysis of stool has been used for well over 10
years in the investigation of stool composition
(Wrong, Metcalfe-Gibson, Morrison, Ng & Howard,
5. GASTRIN AND DUODENAL ULCERATION
1965, Clinical Science, 28, 357). Sealed bags, preJ. R. HAYES,J. ARDILL,T. L. KENNEDY
and K. D. pared from dialysis tubing, and containing a small
BUCHANAN
amount of an oncotic agent, are swallowed and later
Departments of Medicine and Surgery, The Queen's recovered from the stool. It has hitherto been assumed
that the fluid within the bags represents the extraUniversity of Belfast
cellular component of faecal water.
Two healthy adult volunteers swallowed dialysis
The finding of elevated circulating gastrin levels in
patients with the Zollinger-Ellison syndrome suggested bags on nine separate occasions; these bags were
that the antral hormone might be implicated in the later recovered from the faeces. The stool in which the
pathogenesis of simple duodenal ulceration. However, bags were passed was homogenized, centrifuged, and
in most respects, basal gastrin levels do not differ the resulting supernatant analysed at the same time as
significantly from normal. It is possible, however, that the dialysis fluid. The dialysate had a higher pH
the gastrin response to feeding in duodenal ulcer (6.3 ? 0.7), and lower osmolality (373f37 mOsm/kg),
patients might be different from that in controls. sugar concentration(240f 130mg/100 ml) and sodium
We have therefore studied plasma gastrin levels concentration (24k 16 mEq/l), than the stool water
following oral ingestion of a standard protein meal. In measured directly (pH 5.7k0.2, osmolality 519f 31
fifteen patients with uncomplicated duodenal ulcer- mOsm/kg, organic anion concentration 213 ? 19
ation and in thirteen control subjects the absolute mEq/l, sugar concentration 8 7 0 5 610 rng/100 ml,
gastrin levels and the pattern of gastrin release were and sodium concentration 37 f22 mEq/l). Similarly,
similar. The increase in gastrin output following the dialysate had an appreciably higher bicarbonate
protein was 96+ 19% (mean? SEM) in the duodenal and lower ammonia concentration than the stool
ulcer patients and 63+13% (mean ?SEM) in the fluid itself.
These differences might be accounted for by the
control group. This difference was not significant
(0.1 >P > 0.05). However, as a significant negative continued bacterial fermentation of the stool in the
correlation was found between the increase in gastrin distal colon and rectum. This would produce more
output and stimulated acid secretion, the gastrin organic anions, in a more acid medium, and might be
response in the duodenal ulcer patients might have associated with the breakdown of complex polybeen expected to be less than in the controls. A saccharides to glucose. In the distal colon and rectum
different pattern of response was seen in five patients the stool is already semi-solid, and diffusion from
with pyloric stenosis. At no time during the test were this material into dialysis bags would be impaired to a
gastrin levels significantly different from the fasting greater or lesser extent; thus the dialysis bags would
level. The basal gastrin levels in eight patients with no longer be at equilibrium with the stool surrounding
recurrent duodenal ulceration were not significantly them. Therefore in vivo dialysate of stool may not
different from those in asymptomatic post-gastric represent the extracellular component of faecal water.