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The effect of growth hormone on albumin turnover in man
The Effect of Growth Hormone on Albumin Turnover in Man By K. N. JEEJEEBHOY,* B. J. BOUCHERANDM. HARTOG Albumin turnover has been studied in 3 patients with acromegaly, (one being studied again after partial hypophysectomy), and 4 patients with hypopituitarism before and after the administration of human growth hormone (H.G.H.) Two of the acromegalic patients showed low levels of albumin turnover, which
rose in one patient after hypophysectomy. Three of 4 hypopituitary patients had low levels of albumin turnover; and in 3 of 4 the administration of H.G.H. markedly diminished the rate of albumin turnover. The results obtained in this study suggest that growth hormone lowers albumin turnover.
G
ROWTH HORMONE is known to influence nitrogen balance profoundly, and in this study its effect on albumin turnover in human subjects was observed by following the disappearance of intravenously administered radioiodinated human serum albumin (R.I.H.S.A.). Three subjects with active acromegaly were studied, one of whom was studied again after hypophysectomy. Albumin turnover was also observed in 4 patierits with hypopituitarism before and after the administration of human growth hormone (H.G.H. ). These studies have suggested that H.G.H. reduces albumin turnover. METHODS Details
of the patients
clinical
features
before
treatment
physectomy
or hypopituitarism.
were receiving
any therapy,
was then on stabilized
of the hypopituitary injections of H.G.H.; sin tannate
studied are given in the Appendix.
of acromegaly
and the one patient
maintenance
all showed
therapy
tested
of cortisone
the typical
patients
tested
again after hypo-
12.5 mg. t.d.s.
Three
subjects were maintained on the same therapy before and after the in case 4 this consisted of cortisone 5 mg./day and injections of pitres-
in oil, in case 5, cortisone
case 6, cortisone
They
None of the acromegalic
12.5 mg. q.d.s.
25 mg. t.d.s. and thyroid
4 gr./day.
and thyroxine One patient
0.2 mg./day,
(case
and in
7) was maintained
on cortisone, 12.5 mg. t.d.s. and thyroid 2 gr./day. She acquired a fever with vomiting the day after her injections of H.G.H., which was thought to be due to a viral illness; and was given an injection of 100 mg. hydrocortisone intravenously on that day and cortisone 25 mg. t.d.s. on the next 2 days.
Albumin Turnover Studies ‘I’hesc studies were carried radioiodine Ilsl-R.I.H.S.A. Subsequently,
by the patient’s
out using
thyroid
the
method
was blocked
described
previous1y.V
by the administration
Uptake
of Lugol’s
of
iodine.
was prepared by the method of McFarlanea and was injected intravenously. the radioactivity of daily samples of plasma and aliquots of 24-hour urine
specimens was measured in a well type counter. The 3 acromegalic patients all had single injections of R.I.H.S.A. when being studied before treatment, and the one who was studied again after hypophysectomy
was given a further
injection
1 month after the operation.
The
From the Depvtments of Medicine, West Middlesex Hospital, The London Hospital and Postgraduate LMedkal School of London, London, England. ‘WC& carried out while receiving a grant from the iMedical Research Council. Receked for publication May 13, 1964.
METABOLISM,
VOL. 14, No. 1 (JANUARY),
1965
JEEJEEBHOY,
68 procedure
adopted
to study the effect
of H.G.H.
BOUCHER
in the hypopituitary
AND HABTOG
subjects
was as fol-
lows. In 3 subjects, enough (case
single injections
to permit 4)
turnover
had separate
of R.I.H.S.A.
measurements
injections
were given,
both
of R.I.H.S.A.
and these
before
and after
before
and immediately
were
followed
the H.G.H.
long
One patient
after
the
H.G.H.
In 5 patients, Amberlite resin IRA 400 was given orally in a dose of 5 Gm. 4-hourly, which has the effect of preventing the reabsorption of any degradation products which might have been produced from R.I.H.S.A. lost into the alimentary tract. In these patients 3-day fecal collections were counted in a ring Geiger counter, and provided a measure of gastrointestinal protein catabolism.1~2 From these derived:1
measurements
of the disposal
of the R.I.H.S.A.
the following
values
were
Total exchangeable albumin (T.E.A.), i.e., the sum of the body intra- and extravascular albumin
in which the tracer
the measured
is distributed.
intravascular
pool
The value for the T.E.A.
to the
extravascular
pool
which
is obtained was
by adding
calculated
by the
equilibrium time method.4 At the time when the calculated extravascular activity is maximal (equilibrium time), the protein in the intra- and extravascular protein pools is considered
to be uniformly
labeled,
from the ratio of radioactivity given
only
H.G.H.
single
injections
administration;
and so the mass of extravascular in the 2 pools.
of R.I.H.S.A.,
for the period
the
after
protein
In the 3 hypopituitary T.E.A.
H.G.H.
could
both
Albumin
turnover
which
is measured
as (1)
combined
who were
only be determined
catabolism
therefore calculated on the assumption that there was no significant ing albumin pool during the period of measurements. (2)
can be derived
patients
synthesis
and change
before
synthesis
were
in the circulat-
and transfer
rate,
and
catabolism. (1)
Albumin synthesis and net transfer rate: this is estimated
by Matthews;s this method provides a measure of synthesis from extra- to intravascular pools. Since it does not distinguish we refer
to our results
as “synthesis.”
For
the calculation
by the method
described
plus net transfer of albumin between these two processes, it is necessary
to measure
the
specific activity (&./mg.) of the plasma albumin and the total intra- and extravascular radioactivity, which are derived from the measurements of plasma and urine radioactivity and of the plasma
volume.
Plasma
volumes
were determined
at the beginning
and end of
each study, and were assumed not to have otherwise changed during the experiment. When the results show a lowering of the rate of “synthesis” together with a decrease in the rate of decline of plasma specific activity, then some decrease in the rate of true albumin synthesis must have occurred though the exact extent of this decrease cannot be established. Since the specific activity of extravascular albumin is higher than that of intravascular albumina after the equilibrium time has been passed, a decrease of net transfer rate from exira- to intravascular pool alone would increase the rate of decline of plasma specific activity. (2) Albumin catabolism: The catabolic rate was derived from the ratio of the intravascular specific activity to the sum of the urinary and fecal radioactivity. Since there is no reason to believe
that these patients
was normal in 5 patients
had any abnormal
in whom it was measured),
gastrointestinal
albumin
only the combined
loss (the value
figures for the total
catabolism are presented in table 1. Two patients (cases 4 and 5) did not receive the Amberlite resin, and in them total catabolism was calculated from urinary radioactivity alone.
The concentration
of ~ZUMWZalbumin
benzeneazo)benzoic acid dye method,7 standards of crystalline human albumin.
was using
estimated by the H.A.B.A. 2-(rl-hydroxyan autoanalyser calibrated against known
Human Growth Hormone This was supplied by .the Clinical Endocrinological Council,
and was prepared
by the Department
Committee of Biochemistry,
of the Medical Research University of Cambridge,
GROWTH
HORMONE
ON ALBUMIN
69
TURNOVER
Table L-Albumin Turnover Studies in Patients Acromegaly and Hypopituiturkm Albumin
cass No.
Diagnosis
Total Exchangeable Albumin (Gm./Kd
Serum AIbumin (Gm./lOO ml.)
Turnover
with
(Untreated T0t.d
catabolism
kns./Ke./dw)
:
+ Tredd)
“Synthesis” (me./Kg./dw)
1
Acromegaly
3.7
-
2
Acromegaly
3.7
-
3
Acromegaly* (before and after
4.3 + 5.2
3.9 + 3.0
156-+ 178
148+ 174
4.8 4.4 3.2 4.3
4.3 4.6 3.5 4.2
4.0 + 3.4 3.3 3.1 3.0 -
207-+ 126 132-a 83 139+ 84 160+ 169
207+ 183 135+ 88 149-+ 75 140’ 140
Normal range ( 17 subjects) $ 3.5 to 5.0 Mean 4.0 (+S.D.) (kO.79)
3.5 to 5.3 4.2 (+0.68)
152to 221 177 (k21.5)
151to 207 176 (k13.1)
-
3.4
-
124
-
131
-
5.3
-
180
-
195
-
hypophysectomy) 4 5 6
Hypopituitarism (before and after H.G.H.)
7f i
+ + + +
*Patient had mild diabetes insipidus after hypophysectomy at the time of study. fPatient was given additional steroid because of a fever (? virus infection). $Jeejeebhoy.l.2 using the Raben technic.* It was given in 3 injections of 10 mg. intramuscularly on successive days in 2 patients (cases 4 and 5), as 2 injections of 12.5 mg. on successive days in one patient (case 6), and as a single injection of 25 mg. in another (case 7 ) . In all the patients a marked fall of blood urea was found after the injections of H.G.H., and this was accompanied by a fall in urinary nitrogen and rise in urine calcium in the 2 patients in whom they were measured. These findings are characteristic of the action of H.G.H. in hypopituitary subjects, and confirm that the material was active.
An example of a single albumin turnover study is shown in figure 1. The results of the albumin turnover studies in all the patients are shown in table 1. Of the 3 acromegalic patients studied before treatment it will be seen that 2 patients (cases 1 and 3) had values for T.E.A. and albumin turnover rates which were either subnormal or at the lower limit of normal, whereas, one patient (case 2) had values which were within the normal range. Another patient (case 3) was studied again 1 month after partial hypophysectomy which had produced a dramatic improvement in the acromegaly. The albumin tumover rate was then found to have risen, the rate of catabolism rising from 156 to 176 mg./Kg./day, and the rate of “synthesis” from 148 to 174 mg./Kg./day. There was also a rise in the plasma albumin from 4.3 to 5.2 Cm. per cent, this rise being associated with a fall in plasma volume from 2,943 to 1,665 ml., which was probably due to diabetes insipidus. Three of the 4 patients with hypopituitarism had basal values for the T.E.A. and.. rates of albumin catabolism and “synthesis” which were either below normal or at the lower limit of normal, the values in one patient (case 4)
JEEJEEBHOY,BOUCHERANDHARTOC
70 O-0
TOTALBODYACTIVITV
M
EXTRAVASCULAR
ACTIVITY
M
IRTRAVASCULAR
SPECIFIC ACTIVIIV
% INJECTED
(‘1. SPECIFIC ACWlTV lllmin. AFTER INJECTION
HGH
l&I
ACTIVITV
)
80 60 GO
CATABOLIC
10 0
RATE ( % INTRAVASCULAR
POOL)
6 G
I
I
I1
1
2
3
4
I
I
5
6
11
I1
7 6 DAYS
Fig. l.-Example of albumin turnover given human growth hormone (H. G. H.), The fall in catabolic rate and the decrease activity of plasma albumin after H.G.H. can
11
11
J
9 10 11 12 13 14 15
data in a patient with hypopituitarism in 2 doses of 12.5 mg. on days 6 and 7.
of the slope of the curve of the specific be seen.
being within normal limits. In 3 patients (cases 4, 5 and 6) after the administration of H.G.H. the rates of albumin catabolism and “synthesis” fell. In one (case 7) there was no change in albumin turnover rate following H.G.H.; but this observation may have been exceptional because of the fever and the increased steroid therapy given immediately after the injections of H.G.H. DISCUSSION For many years growth hormone has been known to affect protein metabolism. Li and Evans9 reported that chronic treatment of adult rats with purified growth hormone resulted in an increase in the protein and water content of
GROWTH
HORMONE
ON ALBUMIN
TURNOVER
71
the carcass and a decrease in the fat content. Gaebler, Bartlett and Sweeney’” using growth hormone in dogs showed that a marked feature of its action was a fall in the urinary nitrogen excretion. Similar results have been found following the administration in man of H.G.H.ll There is controversy as to whether or not the primary effect of growth hormone is to diminish protein and amino-acid catabolism, or whether it is to stimulate the incorporation of amino acids into protein.12 We have studied the rates of albumin turnover in hypophysectomized patients before and after H.G.H. administration, and in acromegalic patients. In 3 of 4 patients with hypopituitarism the basal measurements showed levels of T.E.A. and rates of albumin turnover which were either subnormal or at the lower limit of normal. Following H.G.H. administration a decreased rate of albumin turnover was found in 3 patients. The one patient who did not show this effect was unusual in that she required increased doses of steroids during the period of study because of an intercurrent viral infection. In view of the known action of corticosteroids in increasing albumin catabolism,13 it seems possible that any growth hormone effect may have been masked. Two of 3 acromegalic patients were found to have levels of T.E.A. and rates of albumin turnover which were either subnormal or at the lower limit of normal. The acromegalic patient in whom these values were normal showed least resistance to insulin in the insulin tolerance test and using this criterion, his condition might be considered to have been less active than in the other 2 patients. One patient was also studied after surgical treatment of her acromegaly when it was found that the rate of albumin turnover had increased. These observations suggest that growth hormone may reduce the rate of albumin turnover; and supporting this hypothesis are the findings in the hypopituitary subjects following administration of H.G.H. In contrast, however are the basal measurements on the patients with hypopituitarism. Hypopituitary patients, however, are subjects who have deficiencies of other hormones known to affect protein metabolism, e.g., the corticosteroids and it seems possible that these deficiencies may counteract the growth hormone effects. There have been previous studies on the effect of hypophysectomy on the metabolism of albumin. Levin and Leatham14 showed that the albumin content of the blood in the rat fell following hypophysectomy, and Goldbert15 and more recently Warner et al. l6 have found the same results in the dog. Only one of our hypopituitary patients had a low plasma albumin concentration; however, this patient had the highest plasma volume (39.5 ml&g.), whereas in the other 3 the plasma volume was markedly lowered (33.9, 27.0 and 27.9 ml&g.), so that a diminished total serum albumin content may have been masked. Such diminutions in plasma volume have previously been recorded in patients who have been hypophysectomized.17 Results of animal experiments on the effect of growth hormone on albumin turnover are rather conflicting. Ulrich, Tarver and Lils assessed albumin turnover in hypophysectomized rats by means of S35-labelled albumin. They found that the exchangeable albumin and the rate of albumin synthesis in-
72
JEEJFSBHOY,
BOUCHER
AND
HAR-
creased markedly following administration of growth hormone. Similarly Bartlett and GaebleP showed that, in dogs fed with N15labelled glycine, the specific activity of the plasma albumin and globulin could be increased by growth hormone. These results are thus in contrast to ours. In hypophysectomized dogs Campbell et al. 2o found that growth hormone produced no change in the total albumin content of the plasma during one course of the hormone, but produced a significant rise during a later course. Warner et al.“’ were unable to produce any rise in serum albumin concentration with growth hormone in hypophysectomized dogs. Gross et aL21 have studied the effect of H.G.H. in a patient with generalized edema and an excessive loss of protein into the gut. On the third day of H.G.H. administration a striking increase in the concentration of albumin in th e p 1asma occurred, which they interpreted as being due to a transient reversal of increased peripheral capillary permeability. The concurrent rise in total plasma radioactivity and in the concentration of plasma albumin could be explained by a transient hemoconcentration which is likely to occur in patients with diarrhea. Gabuzda, Jick and Chalmers22 have obtained similar experimental results to ours. They studied the effect of H.G.H. in patients with cirrhosis and showed that the slope of plasma radioactivity decreased when H.G.H. was given, and interpreted this change as being due partly to decreased albumin catabolism and partly to transfer of protein from the extra- to the intravascular compartment, even though their graphs do not suggest any increase in the rate of decline of extravascular radioactivity. Our results indicate that H.G.H. appears to reduce albumin catabolism, and are ‘perhaps to be expected in the light of the known effects of growth hormone on protein metabolism. The apparent reduction of the rate of “synthesis” is rather surprising and its explanation is uncertain. One possibility is that as growth hormone has been shown to promote synthesis of somatic proteins rather than liver proteins, 23 it therefore increases the utilization of amino acids for somatic protein at the expense of some other proteins such as albumin. ACKNOWLEDGMENTS We are grateful to the Clinical Endocrinology cil and the Department
of Biochemistry,
Committee
University
of the Medical Research
of Cambridge
growth hormone. We are also grateful to Professor Russell Fraser for allowing us to study their patients and for their advice.
Coun-
for supplies of human and Dr. Stuart
Mason
APPENDIX
Description of Patients Studied Case I: A woman aged 52. Symptoms; headaches for many years and enlarging extremities. On examination: acromegalic features, large hands and feet, thick greasy skin. Augmented insulin tolerance test (I.T.T.) ;a4 the insulin resistance index was 177 (normal 62-142) indicating insulin resistance. Glucose tolerance test (G.T.T.) and prednisone stress test resultsas were within normal limits. Skull x-ray revealed enlarged asymmetrical pituitary fossa. Cuss 2:
A man
aged
34.
Symptoms;
right
frontal
headache
and excess
sweating
for
GROWTH
HORMONE
many years.
ON ALBUMIN
On examination:
73
TURNOVER
acromegalic
fdcies,
thick
greasy
skin. Augmented
I.T.T.:
in-
sulin resistance index 134 (i.e., just within normal limits). Normal G.T.T. Prednisone stress test: overnight glycosuria 457 mg./hr. (normal less than 60 mg./hr.) suggesting prediabetes. Skull x-ray revealed asymmetrical pituitary fossa. enlarging extremities and severe paresthesia. Case 3: A woman aged 32. Symptoms; On examination: acromegalic facies, thick greasy skin, marked wasting thenar muscles. Augmented I.T.T.: insulin resistance index: 185 indicating insulin resistance. Normal G.T.T. Skull xlray showed large pituitary fossa. Following surgical exploration and partial hypophysectomy marked improvement megalic facies and skin returning Case 4: A girl aged
with disappearance to normal.
17. A patient
with
dwarfism
of paresthesia, and other
regression
features
of acro-
of hypopuitarism
due to craniopharyngioma partially excised in 1953. She had been on long-term H.G.H. until 4 months before the present study.
therapy with
Case 5: A man aged 67. He presented with failing vision, and showed loss of body hair, thin skin and other features of hypopituitarism. Skull x-ray revealed a large pituitary fossa, and subsequently, a chromophobe adenoma was removed. The present study was performed 1 month after the operation. Case 6: A woman aged 59 in whom clinical features of hypopituitarism developed following an episode of menorrhagia at age 13. Cease 7: A woman aged 25 who experienced headache and ophthalmoplegia in 1957, and subsequently a chromophobe adenoma was removed. After operation she required replacement therapy for hypopituitarism. REFERENCES 1. Jeejeebhoy,
K.
buminaemia
N.:
Cause
of
hypoal-
with
gastro-
disease.
Lancet
in patients
intestinal and cardiac 1: 343, 1962.
monkey. Science X5:883, 1957. 9. Li, C. H., and Evans, H. M.: The biochemistry mone.
2. -:
Rec.
of
pituitary
Progr.
growth
Hormone
Res.
hor3:
A study of albumin metabolism in gastrointestinal disease. Ph.D. Thesis, London University, 1963. 3. McFarlane, A. S.: Labeling of plasma proteins with radioactive iodine. Bio-
3, 1948. 10. Gaebler, 0. H., Bartlett, P. D., and Sweeney, M. J.: Remarkable effectiveness of small daily doses of growth hormone in dogs. Am. J. Physiol. 165:
them. J. 62:135, 4. Campbell, R. M.,
486, 1951. 11. Clinical Endocrinology
1956. Cuthbertson,
D.
P.,
Matthews, C. M., and McFarlane, A. S.: Behaviour of Cl4 and Ilsi-labeled plasma
proteins
in
the
rat.
Int.
J.
App. Radiat. 1:66, 1956. 5. Matthews, C. M. E.: Effects of plasmaphoresis on albumin pools in rabbits. J. Clin. Invest. 40:603, 1961. 6. Solomon, A. K.: The kinetics of biological processes. Special problems connected with the use of tracers. Adv. Biol. Med. Phys. 3:65, 1953. 7. Rutstein, D. D., Ingenito, E. F., and Reynolds, W. E.: The determination of albumin in human blood plasma and serum. J. Clin. Invest. 33:211, 1954. 8. Raben, M. S.: Preparation of growth hormone from pituitaries of man and
Committee
of
Medical Research Council: The effectiveness in man of human growth hormone. Lancet 1:7, 1959. 12. De Bodo, R. C., and Altszuler,
N.: The
metabolic effects of growth hormone and their physiological significance. Vitam. & Horm. 15:205, 1957. 13. Grossman, J., Yallow, R. A., and Weston, R. E.: Albumin degradation and synthesis as influenced by hydrocortl sone, corticotrophin and infection. Metabolism 9:528, 1960. 14. Levin, L., and Leatham, J. H.: The relation of the pituitary, thyroid and adrenal glands to the maintenance of normal serum albumin and globulin levels. Am. J. Physiol. 136:306, 1942. 15. Goldberg, I.: Action de l’insufllsance
74
JJXJEEBHOY,
hypophysaire sur les proteines du plasma sanguin. C. R. Sot. Biol. (Paris) 128: 1135, 1938. 16. Warner, R. C., Weber, I., De Bodo, R.
21. Gross,
P. A. M.,
P., Shipps,
A.C.T.H.
29:386,
of dogs.
Am. J. Physiol. 190:121, 1957. 17. Falkheden, T., Sjogren, B., and Westling, H.: Studies on the blood volume following hypophysectomy in man. Acta Endocrinol. 42:552, 1963. 18. Ulrich, F., Tarver, H., and Li, C. H.: Effects trophic of
of growth and adrenocorticohormones on the metabolism
albumin
in
hypophysectomised
rats. J. Biol. Chem. 209:117, 1954. 19. Bartlett, P. D., and Gaebler, 0. H.: Studies on mechanism of nitrogen storage. VII. The plasma proteins. Biol. Chem. 196:11, 1952.
J.
20. Campbell, J., Hausler, H. R., Munroe, J. S., and Davidson, I. W. F.: Effects of growth hormone in dogs. crinology 53: 134, 1953.
Endo-
Embree,
tein exudation
J. G., Jick, Human
albumin
permeability Am.
J.
Med.
cirrhosis.
hormone
and
in patients
Metabolism
amino-acids. posium
H., and Chalmers,
growth
metabolism
23. Munroe, N. quirements
12: 1,
with
1963.
H.: Factors affecting refor carbohydrate fats and Paper
given
on Intravenous
at
Sym-
Feeding,
Roy.
&ted. 1962.
24. Fraser,
R., Joplin,
and Rabinowitz, insulin
tolerance
insulin
resistance.
K. J.:
)
pro-
1960.
T. C.:
25. Joplin,
anasarca serum
capillary
hyperanabolism.
22. Gabuzda,
299,
( with
Bally, G. W.:
into the gastrointestinal
increased
and
Sot.
L. J.,
to hypercatabolism,
tract,
proteins
AND HARTOC
J. C., and Thorn,
Hypoalbuminemia due
C., and Wurtz, M.: Effect of hypophysectomy and subsequent administration of growth hormone and on serum
BOUCHER
G. F., D.: test J.
Opie,
The
L.
H.,
augmented
for
detecting
Endocrinol.
25:
1962. G.
F.,
Fraser,
R.,
and
Prednisone-glycosuria
pre-diabetes.
Lancet
2:67,
K. N. Jeejeebhay, M.B., M.R.C.P., Ph.D. (London), Department of Medicine, West Middlesex Hospital, London. Present address: Radiation Medical Centre, Tata Memorial Hospital, Parel, Bombay, India. B. J. Boucher, B.Sc., M.B., M.R.C.P., Department of Medicine (Senior Registrar), The London Hospital, London, England. M. Hartog, B.A., B.M., M.R.C.P., Department of Medicine (Senior Registrar), Postgraduate Medical School, Duane Road, London, England.
Keeley, test for 1961.
rose in one patient after hypophysectomy. Three of 4 hypopituitary patients had low levels of albumin turnover; and in 3 of 4 the administration of H.G.H. markedly diminished the rate of albumin turnover. The results obtained in this study suggest that growth hormone lowers albumin turnover.
G
ROWTH HORMONE is known to influence nitrogen balance profoundly, and in this study its effect on albumin turnover in human subjects was observed by following the disappearance of intravenously administered radioiodinated human serum albumin (R.I.H.S.A.). Three subjects with active acromegaly were studied, one of whom was studied again after hypophysectomy. Albumin turnover was also observed in 4 patierits with hypopituitarism before and after the administration of human growth hormone (H.G.H. ). These studies have suggested that H.G.H. reduces albumin turnover. METHODS Details
of the patients
clinical
features
before
treatment
physectomy
or hypopituitarism.
were receiving
any therapy,
was then on stabilized
of the hypopituitary injections of H.G.H.; sin tannate
studied are given in the Appendix.
of acromegaly
and the one patient
maintenance
all showed
therapy
tested
of cortisone
the typical
patients
tested
again after hypo-
12.5 mg. t.d.s.
Three
subjects were maintained on the same therapy before and after the in case 4 this consisted of cortisone 5 mg./day and injections of pitres-
in oil, in case 5, cortisone
case 6, cortisone
They
None of the acromegalic
12.5 mg. q.d.s.
25 mg. t.d.s. and thyroid
4 gr./day.
and thyroxine One patient
0.2 mg./day,
(case
and in
7) was maintained
on cortisone, 12.5 mg. t.d.s. and thyroid 2 gr./day. She acquired a fever with vomiting the day after her injections of H.G.H., which was thought to be due to a viral illness; and was given an injection of 100 mg. hydrocortisone intravenously on that day and cortisone 25 mg. t.d.s. on the next 2 days.
Albumin Turnover Studies ‘I’hesc studies were carried radioiodine Ilsl-R.I.H.S.A. Subsequently,
by the patient’s
out using
thyroid
the
method
was blocked
described
previous1y.V
by the administration
Uptake
of Lugol’s
of
iodine.
was prepared by the method of McFarlanea and was injected intravenously. the radioactivity of daily samples of plasma and aliquots of 24-hour urine
specimens was measured in a well type counter. The 3 acromegalic patients all had single injections of R.I.H.S.A. when being studied before treatment, and the one who was studied again after hypophysectomy
was given a further
injection
1 month after the operation.
The
From the Depvtments of Medicine, West Middlesex Hospital, The London Hospital and Postgraduate LMedkal School of London, London, England. ‘WC& carried out while receiving a grant from the iMedical Research Council. Receked for publication May 13, 1964.
METABOLISM,
VOL. 14, No. 1 (JANUARY),
1965
JEEJEEBHOY,
68 procedure
adopted
to study the effect
of H.G.H.
BOUCHER
in the hypopituitary
AND HABTOG
subjects
was as fol-
lows. In 3 subjects, enough (case
single injections
to permit 4)
turnover
had separate
of R.I.H.S.A.
measurements
injections
were given,
both
of R.I.H.S.A.
and these
before
and after
before
and immediately
were
followed
the H.G.H.
long
One patient
after
the
H.G.H.
In 5 patients, Amberlite resin IRA 400 was given orally in a dose of 5 Gm. 4-hourly, which has the effect of preventing the reabsorption of any degradation products which might have been produced from R.I.H.S.A. lost into the alimentary tract. In these patients 3-day fecal collections were counted in a ring Geiger counter, and provided a measure of gastrointestinal protein catabolism.1~2 From these derived:1
measurements
of the disposal
of the R.I.H.S.A.
the following
values
were
Total exchangeable albumin (T.E.A.), i.e., the sum of the body intra- and extravascular albumin
in which the tracer
the measured
is distributed.
intravascular
pool
The value for the T.E.A.
to the
extravascular
pool
which
is obtained was
by adding
calculated
by the
equilibrium time method.4 At the time when the calculated extravascular activity is maximal (equilibrium time), the protein in the intra- and extravascular protein pools is considered
to be uniformly
labeled,
from the ratio of radioactivity given
only
H.G.H.
single
injections
administration;
and so the mass of extravascular in the 2 pools.
of R.I.H.S.A.,
for the period
the
after
protein
In the 3 hypopituitary T.E.A.
H.G.H.
could
both
Albumin
turnover
which
is measured
as (1)
combined
who were
only be determined
catabolism
therefore calculated on the assumption that there was no significant ing albumin pool during the period of measurements. (2)
can be derived
patients
synthesis
and change
before
synthesis
were
in the circulat-
and transfer
rate,
and
catabolism. (1)
Albumin synthesis and net transfer rate: this is estimated
by Matthews;s this method provides a measure of synthesis from extra- to intravascular pools. Since it does not distinguish we refer
to our results
as “synthesis.”
For
the calculation
by the method
described
plus net transfer of albumin between these two processes, it is necessary
to measure
the
specific activity (&./mg.) of the plasma albumin and the total intra- and extravascular radioactivity, which are derived from the measurements of plasma and urine radioactivity and of the plasma
volume.
Plasma
volumes
were determined
at the beginning
and end of
each study, and were assumed not to have otherwise changed during the experiment. When the results show a lowering of the rate of “synthesis” together with a decrease in the rate of decline of plasma specific activity, then some decrease in the rate of true albumin synthesis must have occurred though the exact extent of this decrease cannot be established. Since the specific activity of extravascular albumin is higher than that of intravascular albumina after the equilibrium time has been passed, a decrease of net transfer rate from exira- to intravascular pool alone would increase the rate of decline of plasma specific activity. (2) Albumin catabolism: The catabolic rate was derived from the ratio of the intravascular specific activity to the sum of the urinary and fecal radioactivity. Since there is no reason to believe
that these patients
was normal in 5 patients
had any abnormal
in whom it was measured),
gastrointestinal
albumin
only the combined
loss (the value
figures for the total
catabolism are presented in table 1. Two patients (cases 4 and 5) did not receive the Amberlite resin, and in them total catabolism was calculated from urinary radioactivity alone.
The concentration
of ~ZUMWZalbumin
benzeneazo)benzoic acid dye method,7 standards of crystalline human albumin.
was using
estimated by the H.A.B.A. 2-(rl-hydroxyan autoanalyser calibrated against known
Human Growth Hormone This was supplied by .the Clinical Endocrinological Council,
and was prepared
by the Department
Committee of Biochemistry,
of the Medical Research University of Cambridge,
GROWTH
HORMONE
ON ALBUMIN
69
TURNOVER
Table L-Albumin Turnover Studies in Patients Acromegaly and Hypopituiturkm Albumin
cass No.
Diagnosis
Total Exchangeable Albumin (Gm./Kd
Serum AIbumin (Gm./lOO ml.)
Turnover
with
(Untreated T0t.d
catabolism
kns./Ke./dw)
:
+ Tredd)
“Synthesis” (me./Kg./dw)
1
Acromegaly
3.7
-
2
Acromegaly
3.7
-
3
Acromegaly* (before and after
4.3 + 5.2
3.9 + 3.0
156-+ 178
148+ 174
4.8 4.4 3.2 4.3
4.3 4.6 3.5 4.2
4.0 + 3.4 3.3 3.1 3.0 -
207-+ 126 132-a 83 139+ 84 160+ 169
207+ 183 135+ 88 149-+ 75 140’ 140
Normal range ( 17 subjects) $ 3.5 to 5.0 Mean 4.0 (+S.D.) (kO.79)
3.5 to 5.3 4.2 (+0.68)
152to 221 177 (k21.5)
151to 207 176 (k13.1)
-
3.4
-
124
-
131
-
5.3
-
180
-
195
-
hypophysectomy) 4 5 6
Hypopituitarism (before and after H.G.H.)
7f i
+ + + +
*Patient had mild diabetes insipidus after hypophysectomy at the time of study. fPatient was given additional steroid because of a fever (? virus infection). $Jeejeebhoy.l.2 using the Raben technic.* It was given in 3 injections of 10 mg. intramuscularly on successive days in 2 patients (cases 4 and 5), as 2 injections of 12.5 mg. on successive days in one patient (case 6), and as a single injection of 25 mg. in another (case 7 ) . In all the patients a marked fall of blood urea was found after the injections of H.G.H., and this was accompanied by a fall in urinary nitrogen and rise in urine calcium in the 2 patients in whom they were measured. These findings are characteristic of the action of H.G.H. in hypopituitary subjects, and confirm that the material was active.
An example of a single albumin turnover study is shown in figure 1. The results of the albumin turnover studies in all the patients are shown in table 1. Of the 3 acromegalic patients studied before treatment it will be seen that 2 patients (cases 1 and 3) had values for T.E.A. and albumin turnover rates which were either subnormal or at the lower limit of normal, whereas, one patient (case 2) had values which were within the normal range. Another patient (case 3) was studied again 1 month after partial hypophysectomy which had produced a dramatic improvement in the acromegaly. The albumin tumover rate was then found to have risen, the rate of catabolism rising from 156 to 176 mg./Kg./day, and the rate of “synthesis” from 148 to 174 mg./Kg./day. There was also a rise in the plasma albumin from 4.3 to 5.2 Cm. per cent, this rise being associated with a fall in plasma volume from 2,943 to 1,665 ml., which was probably due to diabetes insipidus. Three of the 4 patients with hypopituitarism had basal values for the T.E.A. and.. rates of albumin catabolism and “synthesis” which were either below normal or at the lower limit of normal, the values in one patient (case 4)
JEEJEEBHOY,BOUCHERANDHARTOC
70 O-0
TOTALBODYACTIVITV
M
EXTRAVASCULAR
ACTIVITY
M
IRTRAVASCULAR
SPECIFIC ACTIVIIV
% INJECTED
(‘1. SPECIFIC ACWlTV lllmin. AFTER INJECTION
HGH
l&I
ACTIVITV
)
80 60 GO
CATABOLIC
10 0
RATE ( % INTRAVASCULAR
POOL)
6 G
I
I
I1
1
2
3
4
I
I
5
6
11
I1
7 6 DAYS
Fig. l.-Example of albumin turnover given human growth hormone (H. G. H.), The fall in catabolic rate and the decrease activity of plasma albumin after H.G.H. can
11
11
J
9 10 11 12 13 14 15
data in a patient with hypopituitarism in 2 doses of 12.5 mg. on days 6 and 7.
of the slope of the curve of the specific be seen.
being within normal limits. In 3 patients (cases 4, 5 and 6) after the administration of H.G.H. the rates of albumin catabolism and “synthesis” fell. In one (case 7) there was no change in albumin turnover rate following H.G.H.; but this observation may have been exceptional because of the fever and the increased steroid therapy given immediately after the injections of H.G.H. DISCUSSION For many years growth hormone has been known to affect protein metabolism. Li and Evans9 reported that chronic treatment of adult rats with purified growth hormone resulted in an increase in the protein and water content of
GROWTH
HORMONE
ON ALBUMIN
TURNOVER
71
the carcass and a decrease in the fat content. Gaebler, Bartlett and Sweeney’” using growth hormone in dogs showed that a marked feature of its action was a fall in the urinary nitrogen excretion. Similar results have been found following the administration in man of H.G.H.ll There is controversy as to whether or not the primary effect of growth hormone is to diminish protein and amino-acid catabolism, or whether it is to stimulate the incorporation of amino acids into protein.12 We have studied the rates of albumin turnover in hypophysectomized patients before and after H.G.H. administration, and in acromegalic patients. In 3 of 4 patients with hypopituitarism the basal measurements showed levels of T.E.A. and rates of albumin turnover which were either subnormal or at the lower limit of normal. Following H.G.H. administration a decreased rate of albumin turnover was found in 3 patients. The one patient who did not show this effect was unusual in that she required increased doses of steroids during the period of study because of an intercurrent viral infection. In view of the known action of corticosteroids in increasing albumin catabolism,13 it seems possible that any growth hormone effect may have been masked. Two of 3 acromegalic patients were found to have levels of T.E.A. and rates of albumin turnover which were either subnormal or at the lower limit of normal. The acromegalic patient in whom these values were normal showed least resistance to insulin in the insulin tolerance test and using this criterion, his condition might be considered to have been less active than in the other 2 patients. One patient was also studied after surgical treatment of her acromegaly when it was found that the rate of albumin turnover had increased. These observations suggest that growth hormone may reduce the rate of albumin turnover; and supporting this hypothesis are the findings in the hypopituitary subjects following administration of H.G.H. In contrast, however are the basal measurements on the patients with hypopituitarism. Hypopituitary patients, however, are subjects who have deficiencies of other hormones known to affect protein metabolism, e.g., the corticosteroids and it seems possible that these deficiencies may counteract the growth hormone effects. There have been previous studies on the effect of hypophysectomy on the metabolism of albumin. Levin and Leatham14 showed that the albumin content of the blood in the rat fell following hypophysectomy, and Goldbert15 and more recently Warner et al. l6 have found the same results in the dog. Only one of our hypopituitary patients had a low plasma albumin concentration; however, this patient had the highest plasma volume (39.5 ml&g.), whereas in the other 3 the plasma volume was markedly lowered (33.9, 27.0 and 27.9 ml&g.), so that a diminished total serum albumin content may have been masked. Such diminutions in plasma volume have previously been recorded in patients who have been hypophysectomized.17 Results of animal experiments on the effect of growth hormone on albumin turnover are rather conflicting. Ulrich, Tarver and Lils assessed albumin turnover in hypophysectomized rats by means of S35-labelled albumin. They found that the exchangeable albumin and the rate of albumin synthesis in-
72
JEEJFSBHOY,
BOUCHER
AND
HAR-
creased markedly following administration of growth hormone. Similarly Bartlett and GaebleP showed that, in dogs fed with N15labelled glycine, the specific activity of the plasma albumin and globulin could be increased by growth hormone. These results are thus in contrast to ours. In hypophysectomized dogs Campbell et al. 2o found that growth hormone produced no change in the total albumin content of the plasma during one course of the hormone, but produced a significant rise during a later course. Warner et al.“’ were unable to produce any rise in serum albumin concentration with growth hormone in hypophysectomized dogs. Gross et aL21 have studied the effect of H.G.H. in a patient with generalized edema and an excessive loss of protein into the gut. On the third day of H.G.H. administration a striking increase in the concentration of albumin in th e p 1asma occurred, which they interpreted as being due to a transient reversal of increased peripheral capillary permeability. The concurrent rise in total plasma radioactivity and in the concentration of plasma albumin could be explained by a transient hemoconcentration which is likely to occur in patients with diarrhea. Gabuzda, Jick and Chalmers22 have obtained similar experimental results to ours. They studied the effect of H.G.H. in patients with cirrhosis and showed that the slope of plasma radioactivity decreased when H.G.H. was given, and interpreted this change as being due partly to decreased albumin catabolism and partly to transfer of protein from the extra- to the intravascular compartment, even though their graphs do not suggest any increase in the rate of decline of extravascular radioactivity. Our results indicate that H.G.H. appears to reduce albumin catabolism, and are ‘perhaps to be expected in the light of the known effects of growth hormone on protein metabolism. The apparent reduction of the rate of “synthesis” is rather surprising and its explanation is uncertain. One possibility is that as growth hormone has been shown to promote synthesis of somatic proteins rather than liver proteins, 23 it therefore increases the utilization of amino acids for somatic protein at the expense of some other proteins such as albumin. ACKNOWLEDGMENTS We are grateful to the Clinical Endocrinology cil and the Department
of Biochemistry,
Committee
University
of the Medical Research
of Cambridge
growth hormone. We are also grateful to Professor Russell Fraser for allowing us to study their patients and for their advice.
Coun-
for supplies of human and Dr. Stuart
Mason
APPENDIX
Description of Patients Studied Case I: A woman aged 52. Symptoms; headaches for many years and enlarging extremities. On examination: acromegalic features, large hands and feet, thick greasy skin. Augmented insulin tolerance test (I.T.T.) ;a4 the insulin resistance index was 177 (normal 62-142) indicating insulin resistance. Glucose tolerance test (G.T.T.) and prednisone stress test resultsas were within normal limits. Skull x-ray revealed enlarged asymmetrical pituitary fossa. Cuss 2:
A man
aged
34.
Symptoms;
right
frontal
headache
and excess
sweating
for
GROWTH
HORMONE
many years.
ON ALBUMIN
On examination:
73
TURNOVER
acromegalic
fdcies,
thick
greasy
skin. Augmented
I.T.T.:
in-
sulin resistance index 134 (i.e., just within normal limits). Normal G.T.T. Prednisone stress test: overnight glycosuria 457 mg./hr. (normal less than 60 mg./hr.) suggesting prediabetes. Skull x-ray revealed asymmetrical pituitary fossa. enlarging extremities and severe paresthesia. Case 3: A woman aged 32. Symptoms; On examination: acromegalic facies, thick greasy skin, marked wasting thenar muscles. Augmented I.T.T.: insulin resistance index: 185 indicating insulin resistance. Normal G.T.T. Skull xlray showed large pituitary fossa. Following surgical exploration and partial hypophysectomy marked improvement megalic facies and skin returning Case 4: A girl aged
with disappearance to normal.
17. A patient
with
dwarfism
of paresthesia, and other
regression
features
of acro-
of hypopuitarism
due to craniopharyngioma partially excised in 1953. She had been on long-term H.G.H. until 4 months before the present study.
therapy with
Case 5: A man aged 67. He presented with failing vision, and showed loss of body hair, thin skin and other features of hypopituitarism. Skull x-ray revealed a large pituitary fossa, and subsequently, a chromophobe adenoma was removed. The present study was performed 1 month after the operation. Case 6: A woman aged 59 in whom clinical features of hypopituitarism developed following an episode of menorrhagia at age 13. Cease 7: A woman aged 25 who experienced headache and ophthalmoplegia in 1957, and subsequently a chromophobe adenoma was removed. After operation she required replacement therapy for hypopituitarism. REFERENCES 1. Jeejeebhoy,
K.
buminaemia
N.:
Cause
of
hypoal-
with
gastro-
disease.
Lancet
in patients
intestinal and cardiac 1: 343, 1962.
monkey. Science X5:883, 1957. 9. Li, C. H., and Evans, H. M.: The biochemistry mone.
2. -:
Rec.
of
pituitary
Progr.
growth
Hormone
Res.
hor3:
A study of albumin metabolism in gastrointestinal disease. Ph.D. Thesis, London University, 1963. 3. McFarlane, A. S.: Labeling of plasma proteins with radioactive iodine. Bio-
3, 1948. 10. Gaebler, 0. H., Bartlett, P. D., and Sweeney, M. J.: Remarkable effectiveness of small daily doses of growth hormone in dogs. Am. J. Physiol. 165:
them. J. 62:135, 4. Campbell, R. M.,
486, 1951. 11. Clinical Endocrinology
1956. Cuthbertson,
D.
P.,
Matthews, C. M., and McFarlane, A. S.: Behaviour of Cl4 and Ilsi-labeled plasma
proteins
in
the
rat.
Int.
J.
App. Radiat. 1:66, 1956. 5. Matthews, C. M. E.: Effects of plasmaphoresis on albumin pools in rabbits. J. Clin. Invest. 40:603, 1961. 6. Solomon, A. K.: The kinetics of biological processes. Special problems connected with the use of tracers. Adv. Biol. Med. Phys. 3:65, 1953. 7. Rutstein, D. D., Ingenito, E. F., and Reynolds, W. E.: The determination of albumin in human blood plasma and serum. J. Clin. Invest. 33:211, 1954. 8. Raben, M. S.: Preparation of growth hormone from pituitaries of man and
Committee
of
Medical Research Council: The effectiveness in man of human growth hormone. Lancet 1:7, 1959. 12. De Bodo, R. C., and Altszuler,
N.: The
metabolic effects of growth hormone and their physiological significance. Vitam. & Horm. 15:205, 1957. 13. Grossman, J., Yallow, R. A., and Weston, R. E.: Albumin degradation and synthesis as influenced by hydrocortl sone, corticotrophin and infection. Metabolism 9:528, 1960. 14. Levin, L., and Leatham, J. H.: The relation of the pituitary, thyroid and adrenal glands to the maintenance of normal serum albumin and globulin levels. Am. J. Physiol. 136:306, 1942. 15. Goldberg, I.: Action de l’insufllsance
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hypophysaire sur les proteines du plasma sanguin. C. R. Sot. Biol. (Paris) 128: 1135, 1938. 16. Warner, R. C., Weber, I., De Bodo, R.
21. Gross,
P. A. M.,
P., Shipps,
A.C.T.H.
29:386,
of dogs.
Am. J. Physiol. 190:121, 1957. 17. Falkheden, T., Sjogren, B., and Westling, H.: Studies on the blood volume following hypophysectomy in man. Acta Endocrinol. 42:552, 1963. 18. Ulrich, F., Tarver, H., and Li, C. H.: Effects trophic of
of growth and adrenocorticohormones on the metabolism
albumin
in
hypophysectomised
rats. J. Biol. Chem. 209:117, 1954. 19. Bartlett, P. D., and Gaebler, 0. H.: Studies on mechanism of nitrogen storage. VII. The plasma proteins. Biol. Chem. 196:11, 1952.
J.
20. Campbell, J., Hausler, H. R., Munroe, J. S., and Davidson, I. W. F.: Effects of growth hormone in dogs. crinology 53: 134, 1953.
Endo-
Embree,
tein exudation
J. G., Jick, Human
albumin
permeability Am.
J.
Med.
cirrhosis.
hormone
and
in patients
Metabolism
amino-acids. posium
H., and Chalmers,
growth
metabolism
23. Munroe, N. quirements
12: 1,
with
1963.
H.: Factors affecting refor carbohydrate fats and Paper
given
on Intravenous
at
Sym-
Feeding,
Roy.
&ted. 1962.
24. Fraser,
R., Joplin,
and Rabinowitz, insulin
tolerance
insulin
resistance.
K. J.:
)
pro-
1960.
T. C.:
25. Joplin,
anasarca serum
capillary
hyperanabolism.
22. Gabuzda,
299,
( with
Bally, G. W.:
into the gastrointestinal
increased
and
Sot.
L. J.,
to hypercatabolism,
tract,
proteins
AND HARTOC
J. C., and Thorn,
Hypoalbuminemia due
C., and Wurtz, M.: Effect of hypophysectomy and subsequent administration of growth hormone and on serum
BOUCHER
G. F., D.: test J.
Opie,
The
L.
H.,
augmented
for
detecting
Endocrinol.
25:
1962. G.
F.,
Fraser,
R.,
and
Prednisone-glycosuria
pre-diabetes.
Lancet
2:67,
K. N. Jeejeebhay, M.B., M.R.C.P., Ph.D. (London), Department of Medicine, West Middlesex Hospital, London. Present address: Radiation Medical Centre, Tata Memorial Hospital, Parel, Bombay, India. B. J. Boucher, B.Sc., M.B., M.R.C.P., Department of Medicine (Senior Registrar), The London Hospital, London, England. M. Hartog, B.A., B.M., M.R.C.P., Department of Medicine (Senior Registrar), Postgraduate Medical School, Duane Road, London, England.
Keeley, test for 1961.