3 8 ~
Medical Research Society
f 0 . 1 pcO.01) and i n micrprolactinoma p a t i e n t s
(+8.3+1.6 vs +5.0+0.6 p<0.005). The rise of TSH
a f t e r DOM was g r e a t e r i n microprolactinoma
p a t i e n t s than i n normals a t each time (1100 h r s
; ATSH +5.0f0.6 v s +1.2?O.a p<O.OOl ; 2300 h r s ;
+8.3tl.6 vs +2.5*0.4 p<O.OOl). Peak increment a l PRL responses t o DOM (%@I&;
30 mins) were
reduced (<loo% b a s a l ) c o n s i s t a n t with t h e
diagnosis of microprolactinoma and d i d not
d i f f e r a t each t i m e . Basal LE l e v e l s (mU/L;
mea&SEM) were s i m i l a r (1100 h r s ; 7 . 3 h . 6
2300 h r s ; 7.723.2 NS) and d i d not change
s i g n i f i c a n t l y from b a s a l over 60 mins a f t e r
DOM
.
In conclusion, p a t i e n t s with micrprolactinomas
have q u a l i t a t i v e l y normal but q u a n t i t a t i v e l y
exaggerated TSE responses t o DA antagonism
with WM. The increased DA i n h i b i t i o n of TSH
r e l e a s e is g r e a t e s t a t 2300 h r s when b a s a l TSH
l e v e l s are a l s o higher. In c o n t r a s t DA i n h i b i t i o n of PRL is reduced and s i m i l a r a t each time
of t h e day.
Anterior p i t u i t a r y and/or median eminence DA
receptor blockade with DOM does not cause an
acute r i s e i n LH l e v e l s a t e i t h e r time of day.
105 BONE LOSS I N OSTMIPOROSIS: REDUCED BONE
FORMATION AND INCREASED RESORPTION INDICES
BOTH CORRELA'IZ WI!tB NEGATIVE CALCIUM
BALANCE
J. REEVE, M. A m , D. SLOVIK, P. HQIME
and P.J. MEUNIER
MRC C l i n i c a l Research Centre, Harrow, UK
and FRA Inserm 234, Lyon, France
Reduced bone mass is t h e hallmark of osteop o r o s i s , and i n a f f e c t e d women increased r a t e s
of bone loss a r e common i n t h e 6 t h and 7 t h decades.
Controversy has surrounded previous h i s t o l o g i c a l and r a d i o i s o t o p i c s t u d i e s designed t o
evaluate whether increased o s t e o c l a s t i c r e s o r p
t i o n o r decreased o s t e o b l a s t i c bone formation i s
c h i e f l y responsible.
We have s t u d i e d 26 p a t i e n t s with 8 mm t r e p h i n e i l i a c b i o p s i e s , preceded
by double t e t r a c y c l i n e l a b e l l i n g , and nearsimultaneous ' I b d a y calcium balance studies.
The balances were designed t o simulate t h e
p a t i e n t s ' normal d i e t a r y i n t a k e of calcium and
phosphate and measured f a e c a l e x c r e t i o n of calcium w a s c o r r e c t e d by means of t h e continuously
administered marker C r 0
Undecalcified
s e c t i o n s of t h e biopsi$s3; r e u a n t i t a t e d f o r
q
o s t e o c l a s t numbers (W mm ), t r a b e c u l a r r e s o r p
t i o n s u r f a c e s (RS $1, t r a b e c u l a r o s t e o i d SUP
f a c e s (0s %) , t r a b e c u l a r o s t e o i d s u r f a c e s t a k i n g
a double l a b e l (DLS %), t h e l i n e a r apposition
r a t e (CR, pm/day) and o s t e o i d t h i c k n e s s index.
The a c t i v i t y of t h e o s t e o b l a s t s in forming new
bone, r e l a t i v e t o t h e o s t e o i d s u r f a c e s they
nourished, was c a l c u l a t e d as (DLS x CR)/OS; (AOB).
AOB, RS and OC all c o r r e l a t e d weakly with calA much b e t t e r regression was
cium balance.
obtained when AOB and RS or AOB and OC were
These
c o r r e l a t e d j o i n t l y with calcium balance.
c o r r e l a t i o n s were s i g n i f i c a n t a t t h e 2 % l e v e l
f o r t h e formation a c t i v i t y index (AOB) and a t
t h e 1 % l e v e l f o r both r e s o r p t i o n i n d i c e s (0s
and RS).
A s e p a r a t e index of t h e duration of
t h e r e v e r s a l phase, during which o s t e o c l a s t s a r e
-2
replaced by o s t e o b l a s t s on t h e r e s o r p t i o n surT h i s did not
f a c e s , was devised (RS/oC).
However, 56 %
c o r r e l a t e with calcium balance.
of p a t i e n t s had a value of AOB more than 1 SD
below Melsen and Mosekilde's normal mean value
Both reduced bone formation
(16 % expected).
and increased r e s o r p t i o n c o n t r i b u t e t o loss of
bone i n p a t i e n t s with osteoporosis.
106
HYPOGONADISM AND OSTEOPOROSIS
R.M.
FRANCIS, M. PEACOCK AND J . THOMPSQN
MRC Mineral Metabolism U n i t , The Genera7
I n f i r m a r y , Leeds LS1 3EX
Osteoporosis i s associated w i t h hypogonadism
though i t s pathogenesis i s unclear. We have
r e c e n t l y i n v e s t i g a t e d 6 men w i t h hypogonadi sm
and v e r t e b r a l crush f r a c t u r e s . A l l had a low
plasma testosterone, 5 had e l e v a t e d gonadot r o p h i n s (primary hypogonadism) and one had low
gonadotrophin concentrations (secondary hypogonadism).
I l i a c c r e s t bone b i o p s y showed a reduced
t r a b e c u l a r bone volume i n a l l cases, a low
m i n e r a l i s a t i o n r a t e i n 4 cases and increased
r e s o r p t i o n i n 2, one o f whom a l s o had a low
mineral i s a t i on r a t e . Radiocalcium absorption
was low, ranging from 0.23 t o 0.56 w i t h a mean
(+SEM) o f 0.35 t 0.05 (normal 0.3
1.3 f r a c t i c n
o f dose/hour), and t h e plasma 1,25( OH)@ was
low i n t h e 3 p a t i e n t s i n whom i t was measured
(39.7, 44.5 and 59.2; normal 75-165 pmol/l).
-
To e s t a b l i s h the a c t i o n o f testosterone on bone
we s t u d i e d t h e e f f e c t i n one p a t i e n t o f t r e a t ment w i t h 250 rng Sustanon i n j e c t i o n s f o r t n i g h t l y . A f t e r a s i n g l e dose t h e r e was a
s i g n i f i c a n t increase i n t h e mean ( B E M ) o f 7
consecutive d a i l y calcium balances from
1.16 mmol/day ( ~ ~ 0 . 0 2 ) .
-3.43 ?: 0.88 t o +0.91
caused b y an increase i n n e t calcium absorpt i o n from 0.37 t o 4.65 m o l l d a y . Radiocalcium
a b s o r p t i o n increased from 0.31 b a s a l l y t o
0.51 a f t e r one week and 0.68 a t 8 weeks, and
plasma 1,25(OH)2D increased from 59.2 t o 83.7
and 112.7 pmol/l r e s p e c t i v e l y . I l i a c c r e s t
bone biopsy was performed b a s a l l y and a t 8 weeks
and showed an increase i n forming surfaces
from 12% t o 39% and a f a l l i n s u r f a c e resorpt i o n from 6% t o 2%.
We conclude t h a t malabsorption o f calcium i s
comnon i n hypogonadal men w i t h osteoporosis
and i s r e l a t e d t o low plasma 1,25(OH)2D
concentrations. Treatment w i t h t e s t o s t e r o n e
improves calcium balance b y i n c r e a s i n g plasma
1,25(OH)2D, calcium a b s o r p t i o n and t h e
m i n e r a l i s a t i o n o f bone.
*
107
HUMAN BONE CELLS I N CULTURE. A NOVEL
SYSTEM FOR THE INVESTIGATION OF BONE CELL
METABOLISM
J . N . BERESFORD, J . A . GALLAGHER, M. GOWEN,
M.K.B. McGUIRE, J. POSER AND R.G.G.
RUSSELL
Dept. Human Metabolism 6 C l i n i c a l Biochemistry,
University of S h e f f i e l d Hedical School, Beech
H i l l Road, S h e f f i e l d , S10 2RX, UK
Studies on transformed c e l l s or i s o l a t e d bone
c e l l s from rodent o r avian t i s s u e s have been
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Medical Research Society
extensively used in the investigation of bone
cell metabolism and its hormonal regulation.
However. It has hecome clear that differences
in hormonal responsiveness occur even between
such closely related species as the rat and
mouse in which glucocorticoids (GC's) modulate
1,25(0H),D3
receptor levels in opposite
directions. In the light of these findings we
have developed a method for the isolation and
long-term culture of cells derived from
explants of human bone. Cells ohtained by this
method exhibit the expected biochemical
characteristics of osteoblasts. They are
responsive to calcium regulating hormones, have
high levels of alkaline phosphatase, syntheutse
collagen (type I and 111) and
glycosaminoglycans (GAG'S).
In addition, they
synthesise the bone speciflc protein,
osteocalcin, the production of which is
absolutely dependent upon 1,25(OH) D3 and
can exceed 90 nglmg cell protein/48 hours.
1,25(OH),D3
treatment also produces a three
fold increase in alkaline phosphatase act vity
(P<O.OOl, ANOVA) and a 25% inhibition of Hglucosamine incorporation into GAG'S.
In
contrast, parathyroid hormone (PTH) stimulates
GAG synthesis in a dose-dependent manner and
stimulates plasminogen activator activity, a
protease which may be involved in matrix
turnover. We have employed this system to
investigate the effects on human bone cells of
the following agents: FTH, 1,25(OH),D3,
interleukin 1 and related factors, anabolic
steroids and novel glucocorttcoid analogues.
Parameters followed have included prostaglandin
production, extracellular enzyme activities,
production of matrix components and osteocalcin
synthesis. Our results suggest that this
novel, human cell culture system may be
valuable for the investigation of factors
affecting bone formation in vitro.
4
108 SECRETION OF A S O D I M TRANSPORT INHIBITOR
(INHIBITIN) BY CULTURED LE-IC
PRCMYELOCYTES
K. MOROAN and M.A. MIR
Department of Medicine, University Hospital of
Wales, Cardiff
Patients with acute myeloid leukaemia develop
multiple electrolyte disturbances and leukaemic
plasma has been shown toexert an inhibitory
effect on sodium efflux from erythrocytes (Mir &
Bobinski, Clin. Sci. & Mol. Ped. 1975, 48, 213).
Our recent studies have shown that the supernatant from sonicated leukaemic blast cells
1nhIbits.ousbsin-insensitive and frusemideinsensitive sodium efflux. In an effort to
identify the source of this inhibitory factor we
cultured leukaemic promyelocytes and an extract
of these cells significantly reduced (P< 0.001)
the passive sodium efflux rate constant fran
0.096
0.009 to 0.056 f 0.003 (n=10; f I.S.D.)
Using the inhibition of ouabain-insensitive
sodium transport as a marker we subjected the
C N d e extract (sonicated cells) to a purificntion
regime of gel filtration (Sephadex G25B10) and
ion-exchange (Whatman DE32). The final product
eluted in a symmetrical peak at the void volume
of G10 and when radiolabelled with Bolton and
Hunter reagent (Bolton & Hunter, Biochem. J. 133
*
529-539,11973) exhibited a single peak on S . D . S .
polyacrylamide gel electrophoresis. This material reduced sodium influx from 2.39 f 0.13 mmol/
h-l 1-1 erythrocytes to 1.90 f 0.23 mmol h-l 1-1
erythrocytes (P< 0.001). Aminoacid analysis of
the acid hydrolysed final produce showed that
over 708 was accounted for by seven aminoacids.
The inhibitory activity survived heating at 80°C
for 30 min but was destroyed by acid hydrolysis;
the factor did not absorb at 280 nm but showed
strong absorbances at 254 nm and 206 nm. These
characteristics suggest that the inhibitory
factor we refer to as inhibitin is a peptide.
log
ALCOHOL INGESTION ERTURBS
ELATIONSEIP E T r E X N ALM)ST3FiONY AND RENIN
FGL'??SI;: IN NORMAL PEOPIZ
JEN"
EARN, J.D.H.SLATZI
AND R.MVl3S
BarEiac Department, Whittington Hospital
anri Cobbold Laboratories, Middlesex
Hospital Medical School, London
Several recent studies have suggested a
causal relationship between excessive alcohol
consumption and high blood pressure. 30wever.
the precise mechanisms whereby alcohol affects
blood pressure remain obscure. One possible
mechanism of its effect on blood pressure is
through stimulation of the renin-aldosterone
axis. In this etudy therefore, we have examinad the acute effect of an oral alcohol load on
plasma aldosterone and on certain of the factors known to control its releaae.
Nine normal males participated in the study.
Control plasma samples were obtained at 13.30
hours after a six hour period of abstinence from
food, drink and tobacco. The subjects then
consumed an alcohol load consisting of 2.5 mls/
kg body weight of Scotch whisky over a ten
minute period. Plasma samples were obtained at
hourly intervals for six hours. Plasma aldosteroTe fell consistently from a control value of
221
9.3 pmo)/L (mean SEN) to a minimum
value of 140 0.7 pmol/L at four hens; paradoxically, plasma ronin activity Tose progressively and consistently 9 o m 1.54 0.18 pmol/hr/
ml to a maximum of 2.12
0.30 pmol/hr/ml a$
five hours. P l a y cortisol fell from 416
d o nmol/L to 255
43 nmol/L at six hours. Plasma sodium concentration did not vary but there
was a slight but sieificantin plasma
potassium throughout the course of the study.
In this study, we have demonstrated a paradoxical effect of alcohol on plasma renin and
plasma aldosterone and this effect appears to
be independent of the other factors known to
modulate aldosterone release, namely serum
potasaium concentration and ACTH.
-
-
-
-
-
-
-
l o CEANGES IN LEUcocrpE MEMBRANE ELECl'ROLW
EANDLING WITHOUT ACCOMPANYING ALTERATION IN
BLOOD PRESSURE
A.M.HEA&RTY,M.MIL,R.F.BING,J.D.SWALS,
H.THURSTON,F.D.ROSE~,B.P.O'~Y
Department of Medicine,Leicester Royal Infirmary
Reduced leucocyte Na/K ATP-ase activity has
been demonstrated in essential hypertension.This