Medical Research Society
THE EFFECT OF FASTING AND FEEDING ON
ENZYMES CONTROLLING POLYAMINE METABOLISM
IN SMALL BOWEL EPITHELIAL CELLS
118
33
P
120 HIBbl%BIBIT OF PBOLIFRRATIVE pBsp(asB M ASTICHI
BY IITBPLKUKII-2: SIlIILllp BPPKTS I1 SdpcDIWSIS
PATIBBTS AIJJ COITBOJS
T BAMBA, S VAJA, GM MURPHY AND RH DOWLING
Gastroenterolo y
London SE1, 9 R f
IJnit, Guy's
Campus,
UMDS,
Fasting and feeding have orofound p f f p r t s o n
small -bowel mucosar growth-but-the -mechanisms
whereb food stimulates villus tip enterocytes
to infyuence crypt cell production is unknown
One possible factor is the growth-associateh
polyamines, and the enzymes controllin
their
O h , nnd
synthesis (ornithine decarboxvlnsp:
degradation (diamine o x i d a s e r ' -DAOj. To-'st;dy
DAO
this, we measured the activities of ODC
and a marker of mature enterocytes
alkAliine
phosphatase, in epithelial cells ioiated bv the
mid villi;
Weiser technique from villus tips
lower
villi
and crypts from' the
ileum
non-fasted controls ( n
6 ) and rats fasted for
1 8 - 2 4 h before study ( n = 4 ) .
The reliability
of the cell isolation technique was confirmed
histology and alk phos activity. RESULTS:
::stin
causes reduced crypt cell pro uc ion
and vifius hypoplasia but, paradoxicall:,
tDAO
activity
mU/g protein
increased from 9 . 6
f SEM 0 . h
in control villus tips to 1 2 . 3
f 1.5 after fastin (NS)' from 7 . 6 f 0.4 to
1 3 . 9 f 3 . 0 in mid vifli I D ' <
0.01):
5 . 7 f 1.0
to 10 7 f 1 4 in lower villi (
( 6 . 0 1 ) and 5I4
f
0:9
t o ' 1 2 . 8 f 1 . 5 in crypfs ( p <
0.001).
Thus
fasting abolished the villus ti
DAO 'gradient - presumably because t!H
became populated by functionally hypermature
cells since alk phos activity showed a similar
pattern of results.
In contrast
fastinf
rofoundly lowered ODC activity ( m o i mg protf-1
from 3 1 9 f 8 2 in control viylus tips to
1 6 . 4 f 3.0 after fasting from 2 9 7 f 5 9 to 1 0 . 7
2 8 4 f 4 5 to 6 . 3 f . 2 . 8 in
3 . 6 in mid villi
in t . h n
cower villi and 150'f 3 1 to 5 . 8 + 3 . 3
crypts.
Indeed, fasting reduced ODC activits
to a greater extent than did treatment of fed
rats with the competitive inhibitor of ODC
dif luormethyl
ornithine which reduced
ODt
57 f 5
44 f 4 and 4 9 f 7
activity to 7 2 f 7
res ectively.
CbNCLUSIONQ:
These
results
congirm previous studies of ODC induction by
food (Amer J Physiol 1 9 8 6 2 5 1 '
12370) and
suggest
that not oniy does 06C
activity
increase to initiate intestinal mucosal growth.
but also that reduced ODC,may be responsible
f o r the hypoplasia of fasting.
CffP:
119
PROTEASE INHIBITION BY BISMUTH SUBCITRATE (DE-NOL):
D J Lyons, D N Nitchell, E B Nitchell, and G L
Asherson
CLINICAL RESEARCH CENTRE VATFORD ROAD HIDDLESEX
Sarcoidosis is characterised by granuloma formation
and by impaired cell-mediated immunity. Production of
interleukin-2 (IL-2) in response to stimulation is
impaired. Ve therefore studied the effect of exogenous
recombinant IL-2 on the proliferative response to
antigen in vitro. Peripheral blood mononuclear cells
were separated on Ficoll-Hypaque and resuspended in
RPHI culture medium at a concentration of lO"/ml.
Cells were cultured in a MZincubator for seven days.
Proliferative response was assessed by the
incorporation of =[HI thymidine. Thirty eight patients
and fourteen control subjects were studied. The
proliferative response of patients to PPD was 7816cpm
f 798'7 (counts per minute; mean f sd), and was
significantly reduced compared to that of controls
16654cpm f10755 (P<O.OOl Wann-Vhitney). However in the
presence of IL-2 the response of the patient group to
PPD was slightly, though not significantly, greater
than that of controls 25311cpm f 16'73'7 and 24171cpn
i13864 respectivley. This enhancement could not be
explained by a larger response to IL-2 alone as this
was similar in the two groups. Enhancement occurred
through a synergistic effect between PPD And IL-2.
This effect was seen with similar frequency in
patients 422, and controls 402, although its magnitude
was greater in patients. Similar enhancemnt of
cellular responses to antigen and nitogen have been
reported in other diseases; lepromatous leprosy;
rheumatoid arthritis; primary and acquired
immunodeficiency states. Our data shows that
enhancement also occurs in sarcoidosis and healthy
subjects and m y represent a normal phenopanon,
perhaps expressed to a greater extent in certain
disease states.
121 MACROPHAGE (AM)
AND NEUPROPHIL (F'MNL) CHEMILIMINJ3SCENCE
A POSSIBLE THERAPEUTIC MECHANISM ?
(a)IN ERONCHOALVEOLAR LAVAGE (BAL)
N.M. BIRD, H.T. GREEN AND JONATHAN M. RHODES
WARDC, KELLY CA,
and WALTERSEH
University Dept. of Medicine and Dept. of Microbiology
Walton Hospital, Liverpool
Bismuth subcitrate (De-Nol)has proved a very efEective ulcer healing agent but its mode of action is poorly
understood. Studies have been performed to assess its
effect on protease activity since bacterial, leucocyte
and pancreatic proteases may all have a pathogenic role
in intestinal mucosal ulceration in both the upper and
lower intestine.
Azocasein 2.5 m g / m l in 0.05M Tris HC1 pH 8 . 5 was used
as substrate and a bacteria free filtrate of a normal
faecal homogenate used as protease source with trypsin
0-0.08 mg/ml as positive control. Incubation for 60
minutes at 37OC was performed in the presence of bismuth
subcitrate at concentrations 0, 0.4, 0.8. 2.0, 4 . 0 , 6 . 0 ,
8.0 mg/ml. Enzyme activity was stopped by the addition
of 5% (w/v) trichloroacetic acid and 1 unit of enzyme
activity defined as a change in O.D. of 0.001 at 440nm.
Mean protease activity in the absexce of inhibitor was
87.5 u/ml (equivalent to bovine pancreatic trypsin 4.58
BAEE units). Marked protease inhibition was demonstrated
in the presence of bismuth subcitrate: 50% inhibition
at 1 . 4 mg/ml and 83% inhibition at 4.0 mg/ml. These
concentrations are comparable to those likely to occur
therapeutically at the mucosal surface particularly in
the presence of mucosal ulceration.
The potent anti-protease effect of bismuth subcitrate
may well explain its mucosal protective effect and
deserves further investigation.
STENIONSC,
FLUID
DUDDRIDGEM, HENDRICKDJ
The Chest Unit, Newcastle General Hospital, University of
Newastle upon Tyne
It has previously been shown that there is a linear
relationship between luminol amplified cL and PMNL count in
BAL fluid and we have confirmed this in 20 subjects
undergoing routine diagnostic bronchoscopy (r=O.83,p(0.001).
This suggests that luminol CL may not be an appropriate
marker for AM activity in mixed cell populations. We have
further defined the relationship between cell count and CL
for a wide range of PMNL and AM concentrations by adding
PMNLs from peripheral blood to cells obtained at BAL.
BAL fluid frcm 5 subjects undergoing diagnostic bronchoscopy
w a s centrifuged at 4'C and the cell pellet resuspended in
Medium 199 to a final concentration of 0.5x106cells/ml. m e
differential cell counts (Wright Giemsa) were 97-98% AMS,
0-1% PMNLs and 0-2% lymphocytes. Aliquots of 0.5ml were
stimulated with 5% latex and luminol CL was measured (Luuac
2010) at 37'~. In 7 aliquots a proportion of the AMS were
replaced by PMNLs separated from peripheral blood of the
same subjects by Ficol centrifugation (>95% pure).
Final
PMM. concentrations were <2%, lo%, 20%, 40%, 502, 70% and
>95% with AMS constituting almost all of the remainder of the
cells in each case. Luminol amplified CL increased linearly
from 2.1 -10.9 x10'counts
per sec (cps) at < 2 % PMNLS to
ii.ix10~cpsat >95% PMNLS (r-0.996, P<O.WI) with
47.3 2
the y intercept approximately at 0. zhus ldnol amplified CL
appeared to reflect exclusively Pb@& activity.
Assay of lucigenin amp1ified.n using the same technique
34 P
Medical Research Society
showed a f a l l from 35 22.3 xlO%ps in aliquots with (2% PMNLs
(>97%AM)to 20 f2.3 xlO+cps a t )95% PMNL8 (OXAH) indicating
that both c e l l types c o n t r i b u t e t o lucigenin CL with AMs
contributing approximately twice a s much per c e l l as PMNLs.
Allowing for the contribution of PMNLs to t o t a l CL, lucigenin
amplified CL increased l i n e a r l y with increasing numbers of
AUs ( ~ 0 . 9 8 3 , pa.001). Lucigenin CL thus appears to be an
appropriate technique for measuring AM activation.
122
REGIONAL RED BLOOD CELL TRANSIT TIMES I N HUMAN LUNGS
W.MACNEE,
B.A.
M A R T I N AND J . C .
HOGG
UBC Pulmonary Research Laboratory, S t Paul's Hospital,
Vancouver, B.C., Canada.
The t r a n s i t time ( T T ) of red blood c e l l s ( R B C ) through the
pulmonary c i r c u l a t i o n , which is proportional t o t h e blood
v e l o c i t y , is a c r i t i c a l determinant of gas exchange, and
has been shown t o vary regionally i n animal lungs, w i t h
longer TT i n the upper lung zones (Hogg e t a l , JAP 1985;
59:1266). We have measured t h e frequency d i s t r i b u t i o n and
mean pulmonary TT i n man by two independent techniques.
Pulmonary TT of an i . v . bolus of 9gmTc l a b e l l e d RBCs were
measured i n 16 healthy s u b j e c t s , s t u d i e d u p r i g h t , using a
gamma camera linked t o a computer. TT was c a l c u l a t e d by
deconvolution using the t i m e / a c t i v i t y curves from regions
of i n t e r e s t drawn around the r i g h t and l e f t v e n t r i c l e s and
the l e f t lung o r i t ' s regions. Mean TT between the r i g h t
v e n t r i c l e and t h e l e f t lung was 4.8+0.8s(n=16). TT was
longer i n t h e upper (4.8+1.0s) compared w i t h t h e mid
( 3 . 9 ~ 0 . 8 s )or lower lung zones (4.0+0.8s,p<0.05). T h i s
regional v a r i a t i o n i n pulmonary t r a n s i t times was reversed
i n 5 s u b j e c t s studied supine, associated w i t h a f a l l i n
c a r d i a c index and an increase i n c a p i l l a r y blood volume
(p<0.05). I n 8 a d d i t i o n a l s u b j e c t s undergoing lung
r e s e c t i o n f o r peripheral bronchial carcinoma, RBC TT were
measured i n t r a - o p e r a t i v e l y by i n j e c t i n g a flow ( 9gmTc
macro-aggregated albumen) and a volume marker (5'Cr
l a b e l l e d R B C s ) i n t o the c e n t r a l c i r c u l a t i o n w i t h l a t e r
l e s from the
counting of these markers i n multiple s
i n these
resected 1ung.TT was then measured a s
samples of resected lung. Mean TT ( n = p % 4.6+1.9s,
and was longer i n t h e upper ( 6 . 1 ~ 1 . 1 s )than i n the lower
I n 5 of these s u b j e c t s prezones (3.7+0.6,p<0.05).
operative TT measured by t h e gamma camera/computer system
(TT=4.6+0.9s) were s i m i l a r t o i n t r a - o p e r a t i v e measurements
( T T = 3 . 7 ~ 1 . 7 , n s ) . T h u s , both techniques measure s i m i l a r
mean and frequency d i s t r i b u t i o n s of RBC TT and t h e r e s u l t s
i n d i c a t e t h a t t r a n s i t times a r e longer i n the upper lung
zones i n man, confirming animal experiments.
123
lIEILsDaEs OF S I * ) H . G RIBUVIOW M D SMOKE IJFTAKE
AUD WIdlIB
IE-CPS
A.J.T. K I R K H A M ,
and G CUMMIRG
A.R.
GUYATT, D.C.
H A R I N E R , A.G.
BALDRY
p = C0.05,
Mean v a l u e s , + SD, NS = non s i g n i f i c a n t ,
** = p<O.Ol, iFi* = p<O.OOl 10 cm H20 = 1 kPa.
.
Women
2.3
0.7
2.0
0.6
2.04
Puff volume
(ml)
56.7
15.2
45.0
12.6
4.40
Maximum flow
(ml/s)
45.1
11.2
40.4
11.2
2.24
Puff d u r a t i o n
(s)
Sig.
level
t value
Men
Although men had a s i g n i f i c a n t l y g r e a t e r smoke i n t a k e ,
t h i s d i f f e r e n c e was l o s t when puff volume was normalised
f o r body s i z e ( p u f f v o l u m e / h e i g h t cubed, t = -0.15,
p>0.05), suggesting t h a t smoke uptake i n r e l a t i o n t o body
s i z e was n o t s i g n i f i c a n t l y d i f f e r e n t between m a l e s and
females.
124
PHRENIC NERVE TERMINAL MOTOR LATENCY I N HYPERTHYROIDISM
A.
MIER, C . LAROCHE, C. BROPHY, J. WASS AND MALCOLM GREEN
Brompton Hospital, London SW3 6Hp and S t . Bartholomew's
Hospital, London EC1 7BE
No abnormalities of p e r i p h e r a l nerve terminal motor l a -
tency have been reported i n hyperthyroidism. However,
s i n c e t h i s i s a hyperactive condition w e wondered
whether phrenic nerve conduction v e l o c i t y might a l s o be
increased. Terminal motor l a t e n c y w a s s t u d i e d of the
phrenic nerve i n 6 p a t i e n t s w i t h hyperthyroidism (mean
T4 = 228 nmol/l: normal 58-174 nmol/l) who were i n i t i a l l y
b r e a t h l e s s on e x e r t i o n , b e f o r e and a f t e r treatment w i t h
carbimazole. Diaphragm electromyqrams were recorded
w i t h s u r f a c e e l e c t r o d e s i n t h e 7 t h and 8th i n t e r c o s t a l
spaces. The phrenic nerves w e r e stimulated w i t h S U P
face e l e c t r o d e s a t t h e p o s t e r i o r border of t h e SternOmastoid muscle, using square wave impulses 0.1 m s i n
duration a t 1 H z and supramaximal voltage. Phrenic nerve
terminal motor l a t e n c y was measured as t h e time between
t h e stimulus a r t e f a c t and t h e o n s e t of t h e diaphragm
muscle a c t i o n p o t e n t i a l . Median phrenic nerve terminal
range = 5.5 - 7.5
motor l a t e n c y was i n i t i a l l y 6.5 m s ;
m s ; (normal 5.5-9.2 m s )
S t u d i e s were repeated 3
months l a t e r when a l l p a t i e n t s were euthyroid(mean T4 =
9 5 nmol/l)and t h e i r b r e a t h l e s s n e s s had resolved. Phrenic
nerve terminal motor l a t e n c y had increased i n a l l patients: median = 7.0 m s ;
range = 6.0 - 8.0 m s j ( p < 0 . 0 5 ) .
W e conclude t h a t hyperthyroidism does decrease phrenic
nerve terminal motor l a t e n c y , even though this remains
within t h e normal range. T h i s decreased terminal motor
l a t e n c y which r e p r e s e n t s an i n c r e a s e i n phrenic nerve
conduction v e l o c i t y i s presumably due t o t h e excess of
t h y r o i d hormones, which a r e known t o i n c r e a s e t h e speed
of o t h e r metabolic processes.
.
The Midhurst Medical Research I n s t i t u t e .
Surrey U n i v e r s i t y Research Park, Guildford, Surrey and
The Cardiothoracic I n s t i t u t e . Midhurst, West Sussex
Smoking b e h a v i o u r was r e c o r d e d u s i n g a c i g a r e t t e
h o l d e r i n c o r p o r a t i n g a f l o w r e s i s t i v e e l e m e n t and
measurements were made o f t h e number o f p u f f s p e r
c i g a r e t t e , p u f f d u r a t i o n , p u f f i n t e r v a l , p u f f volume,
maximum flow, peak pressure and l a t e n c y of peak pressure.
M e a s u r e s o f smoke u p t a k e i n c l u d e d pre-smoke
carboxyhaemoglobin (HbCO), HbCO b o o s t , pre-smoke plasma
n i c o t i n e , n i c o t i n e boost and mean plasma cotinine.
45 m a l e s and 65 f e m a l e s were compared u s i n g a n
u n p a i r e d t-test. A l t h o u g h men were on a v e r a g e y o u n g e r ,
and as expected t a l l e r and h e a v i e r than women; t h e r e were
no s i g n i f i c a n t d i f f e r e n c e s i n t h e draw c h a r a c t e r i s t i c s ,
tar y i e l d or t h e number o f c i g a r e t t e s smoked p e r day. The
f o l l o w i n g s i g n i f i c a n t d i f f e r e n c e s were found (p<O.@).
DOES THE MEASUREMENT OF CIRCUMFERENTIAL CHEST EXPANSION RELIABLY IDENTIFY INDIVIDUALS WITH PULMONARY
DISEASE
125
C.R.
SWINBURN
Dept. o f Chest Medicine, Freeman Hospital, Freeman Road,
Newcastle upon Tyne. NE7 7DN
The measurement o f circumferential chest expansion (CCE)
i s s t i l l included i n medical examinations conducted f o r
the l i f e insurance industry, an expansion o f two inches
(5cms) being preferred.
I n order t o determine t h e a b i l i t y
o f CCE t o i d e n t i f y i n d i v i d u a l s w i t h pulmonary disease,