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Effects of Growth Hormone Replacement on Parathyroid
Hormone Sensitivity and Bone Mineral Metabolism
AFTAB M. AHMAD, JOEGI THOMAS, ADRIAN CLEWES, MARION T. HOPKINS, ROSTEM GUZDER,
HISHAM IBRAHIM, BRIAN H. DURHAM, JITEN P. VORA, AND WILLIAM D. FRASER
Departments of Diabetes and Endocrinology and Department of Clinical Chemistry (B.H.D., W.D.F.), Royal Liverpool
University Hospital, Liverpool, United Kingdom L7 8XP
Adult GH deficiency (AGHD) is associated with reduced bone
mineral density, and decreased end-organ sensitivity to the
effects of PTH has been suggested as a possible underlying
mechanism. We investigated the effects of GH replacement
(GHR) on PTH circulating activity and its association with
phosphocalcium metabolism and bone turnover in 16 (8 men
and 8 women) AGHD patients. Half-hourly blood and 3 hourly
urine sampling was performed on each patient over a 24-h
period before GHR and then after 1, 3, 6, and 12 months of
GHR. GH was commenced at a dose of 0.5 IU/d and was titrated
to achieve and maintain an IGF-I
SD score within 2 SD of the
age-related reference range.
The target IGF-I SD score was achieved within 3 months and
was maintained at 12 months after GHR in all patients. Our
results demonstrated a significant decrease in serum PTH at
all visits after GHR compared with baseline values (P < 0.001),
with a concomitant increase in nephrogenous cAMP excretion
at1(P < 0.001) and 3 (P < 0.05) months and increases in serum
calcium (P < 0.001), serum phosphate (P < 0.001), 1,25-dihy-
droxyvitamin D
3
(P < 0.001), type I collagen C-telopeptide (a
bone resorption marker; P < 0.001), and procollagen type I
amino-terminal propeptide (a bone formation marker; P <
0.001). Simultaneously, we observed a significant decrease in
urinary calcium excretion (P < 0.001) and an increase in max-
imum tubular phosphate reabsorption (P < 0.001). Together
these results suggest increased end-organ responsiveness to
the effects of circulating PTH resulting in increased bone
turnover and reduced calcium excretion. Significant circa-
dian rhythms were observed for serum PTH, phosphate, type
I collagen C-telopeptide, and procollagen type I amino-termi-
nal propeptide before and after GHR. However, sustained
PTH secretion was observed between 1400 –2200 h, with a re-
duced nocturnal rise in untreated AGHD patients, whereas
PTH secretion decreased significantly between 1400 –2200 h
(P < 0.001), with a significant increase in nocturnal PTH se-
cretion (P < 0.001) after 12 months of GHR.
Our results demonstrate that GH may have a regulatory
role in bone mineral metabolism, and our data provide a pos-
sible underlying mechanism for the development of osteopo-
rosis in AGHD patients. The changes observed after GHR may
further explain the beneficial effects of GHR on bone mineral
density that have consistently been reported. (J Clin Endo-
crinol Metab 88: 2860 –2868, 2003)
A
DULT GH DEFICIENCY (AGHD) is associated with an
increased prevalence of osteoporosis and reduced
bone mass at different skeletal sites compared with healthy
control subjects (1). The causes of osteoporosis are complex
and multifactorial. Studies have suggested that the GH/
IGF-I axis is one of the major determinants of adult bone mass
(2). An increasing proportion of men and women with ad-
vancing age and no clinical evidence of pituitary pathology
show a decline in GH secretion and serum IGF-I concentra-
tion (3). As AGHD and normal aging are both associated with
a decrease in bone mass, it is possible that reduced GH
secretion and IGF-I concentration in AGHD may account at
least in part for this effect.
Although GH plays an important role in bone metabolism,
the underlying mechanisms remain unclear. Among other
hormones, PTH plays an important role in bone metabolism
and has both catabolic and anabolic effects on bone (4, 5).
These effects are mediated via PTH receptors in bone and
indirectly through regulation of the vitamin D/calcium axis
via receptors in the kidney (6). These receptors in the kidneys
activate mitochondrial vitamin D 1
␣
-hydroxylase, leading to
increased serum 1,25-dihydroxyvitamin D
3
[1,25(OH)
2
D
3
],
which, in turn, is a potent inducer of intestinal calcium ab-
sorption and bone resorption (7), whereas PTH exerts an
antiapoptotic effect on osteoblasts via cAMP-mediated sig-
nals (5). We have previously demonstrated decreased bone
and renal target cell sensitivity to the effects of PTH in un-
treated AGHD patients with significantly lower nephroge-
nous cAMP (NcAMP), bone turnover, and higher calcium
excretion in the presence of significantly higher PTH con-
centrations compared with age- and gender-matched healthy
controls (8).
PTH is secreted in a circadian pattern in healthy individ-
uals (8, 9), and although the role of PTH circadian rhythm is
not established, there is accumulating evidence of the im-
portance of the nocturnal rise and subsequent fall in PTH
secretion over 24 h in health and disease (8–12). The PTH
rhythm is lost in patients with primary hyperparathyroidism
and is restored after parathyroid surgery (13). Abnormalities
in circadian rhythms of bone resorption and renal calcium
conservation in women with postmenopausal osteoporosis
are associated with blunting of the nocturnal rise in PTH
secretion (12), suggesting that the dynamics of PTH secretion
Abbreviations: AGHD, Adult GH deficiency: BMD, bone mineral
density; CTX, type I collagen C-telopeptide; CV, coefficient of variance;
1,25(OH)
2
D
3
, 1,25-dihydroxyvitamin D
3
; GFR, glomerular filtration rate;
GHR, GH replacement; MESOR, midline estimate statistic of rhythm;
NcAMP, nephrogenous cAMP; PcAMP, plasma cAMP; PINP, procol-
lagen type I amino-terminal propeptide; SDS, sd score; TmPO
4
/GFR,
tubular phosphate reabsorption.
0013-7227/03/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 88(6):2860–2868
Printed in U.S.A. Copyright © 2003 by The Endocrine Society
doi: 10.1210/jc.2002-021787
2860
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may have an important role in calcium metabolism and bone
remodeling. Animal and human studies have shown the
importance of intermittent PTH injections in increasing tra-
becular bone mass, whereas continuous PTH infusions favor
bone resorption (14–16).
There are as yet no reports of GH regulating PTH secretion,
but a possible role has previously been suggested (17). Al-
though GH replacement (GHR) is consistently reported to
increase bone turnover and phosphocalcium balance (18 –
20), these effects were suggested to be independent of PTH,
as these studies failed to observe any consistent changes in
PTH (21–24). As PTH is secreted in a circadian pattern in
healthy individuals (8, 9), the variability in PTH results may
reflect the single time point methodology used in the pre-
vious studies (21–24).
The effects of GHR on PTH secretory pattern and its as-
sociation with changes in phosphocalcium metabolism and
bone turnover in AGHD patients have not been fully ex-
plained. We investigated the effects of GHR on PTH secre-
tory pattern in AGHD patients and its association with
changes in bone turnover and phosphocalcium homeostasis
in a 12-month prospective study with an aim to determine the
possible underlying mechanisms responsible for changes in
bone metabolism.
Patients and Methods
Patients
Sixteen patients (eight men and eight women) with confirmed AGHD
were recruited for the study. The local ethics committee approved the
study, and all patients provided written informed consent before re-
cruitment. All patients had undergone pituitary surgery, and the orig-
inal diagnoses are presented in Table 1. Severe AGHD was defined as
a peak GH response of less than 9 mU/liter (3
g/liter) to insulin-
induced hypoglycemia (blood glucose, ⬍2.2 mmol/liter) (25). Eight
patients had a peak GH response of less than 0.5 mU/liter (1.6
g/liter)
to provocative tests, with the peak GH response in seven patients be-
tween 0.5–5.0 mU/liter (0.16–1.6
g/liter). All patients required addi-
tional pituitary hormone replacement and were receiving optimal doses
at recruitment (Table 1), which were stable for more than 3 yr before
recruitment. None of these patients previously received GH therapy.
The mean age ⫾ sd at recruitment was 49.5 ⫾ 10.7 yr, and the mean
time ⫾ sd from diagnosis of AGHD to recruitment into the study was
10.7 ⫾ 6.3 yr. All patients were trained in the use of an automated pen
device (Humatrope-Pen II, Eli Lilly & Co., Basingstoke, UK) for sc
self-injection of GH before recruitment. After baseline measurements,
GH (Humatrope, Eli Lilly & Co.) was commenced at a daily dose of 0.5
IU/d (0.17 mg/d), self-injected at 2200 h every night. The GH dose was
titrated at 2 wk after commencement, by increments of 0.25 IU/d (0.085
mg/d), according to the IGF-I concentration with an aim to maintain
IGF-I within the 2 sd score (SDS) of the age-related reference range.
Methods
Subjects were hospitalized at 1300 h for a 25-h period before and after
1, 3, 6, and 12 months of GHR. Blood samples were obtained half-hourly
from 1400–1400 h via indwelling venous cannulae inserted at the time
of admission. Each time 5-ml blood was sampled, and the samples were
immediately centrifuged and separated. Routine analysis was per-
formed on each sample, and then an aliquot was frozen at –20 C before
further analysis.
Urine samples were collected at 3-h intervals between 1400–2300 and
0800–1400 h, and aliquots of the samples were stored at –20 C before
further analysis. During their stay all patients were ambulant during
1400–2300 h and 0800 –1400 h and were recumbent between 2300 –0800
h. Standard hospital meals were served at 1800, 0800, and 1200 h and
were consumed within 30 min.
Biochemistry
Plasma. Serum adjusted calcium, phosphate, creatinine, and albumin
were measured on all samples by the standard autoanalyzer method
(Hitachi 747, Roche, Lewes, UK). Serum calcium was adjusted for al-
bumin (26). Serum adjusted calcium has been shown to strongly cor-
relate with ionized calcium and has been found to be precise in subjects
with calcium and albumin within the reference range (26 –28). Serum
PTH-(1–84) was measured on all samples using a commercial assay
(Nichols Institute Diagnostics, San Juan Capistrano, CA) with a detec-
tion limit of 0.5 pmol/liter and inter- and intra-assay coefficients of
variance (CVs) of less than 7% across the working range.
Plasma cAMP (PcAMP) was measured by RIA (BIOTRAC cAMP,
Amersham Pharmacia Biotech, Little Chalfont, UK). The intraassay CV
was less than 8%, and the interassay CV was less than 10% across the
working range, with a detection limit of 5 nmol/liter. NcAMP, which
reflects the circulating activity of PTH (29), was determined according
to the formula: NcAMP ⫽ [S
Cr
⫻ (U
cAMP
/U
Cr
)] – P
cAMP
, where NcAMP
is expressed as nanomoles per liter of glomerular filtration rate (GFR),
S
Cr
is serum creatinine in micromoles per liter, U
cAMP
is urinary cAMP
in micromoles per liter, U
Cr
is urinary creatinine in millimoles per liter,
and P
cAMP
is plasma cAMP in nanomoles per liter. Serum IGF-I was
measured by RIA as previously described (30), and the IGF-I SDS was
then calculated from these values (30).
Serum 1,25-(OH)
2
D
3
was extracted by acetonitrile, purified through
aC
18
-OH reverse phase column, and measured by RIA (kit from Nichols
Institute Diagnostics) with tritiated recovery on each sample. The in-
TABLE 1. Original diagnoses and additional replacement hormones of AGHD patients
Patients Gender Diagnosis Pituitary replacement hormones
1 M Nonfunctioning pituitary adenoma Hydrocortisone, T
4
, desmopressin
2 M Craniopharyngioma Hydrocortisone, T
4
3 M Prolactinoma Hydrocortisone, T
4
, testosterone
4 M Cystic adenoma Hydrocortisone, T
4
5 M Craniopharyngioma Hydrocortisone, T
4
, testosterone
6 M Nonfunctioning pituitary adenoma T
4
, testosterone
7 M Nonfunctioning pituitary adenoma T
4
8 M Nonfunctioning pituitary adenoma Hydrocortisone, T
4
9 F Nonfunctioning pituitary adenoma Hydrocortisone, T
4
10 F Prolactinoma T
4
11 F Epidermal cyst Hydrocortisone, T
4
12 F Nonfunctioning pituitary adenoma Hydrocortisone, T
4
, cycloprognova
13 F Craniopharyngioma Hydrocortisone, T
4
14 F Prolactinoma T
4
, premique
15 F Nonfunctioning pituitary adenoma Hydrocortisone, T
4
, premique
16 F Prolactinoma Hydrocortisone, T
4
M, Male; F, female.
Ahmad et al. • GH, PTH Sensitivity, and Bone Metabolism J Clin Endocrinol Metab, June 2003, 88(6):2860–2868 2861
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traassay CV was less than 9%, and the interassay CV was less than 12%
across the working range, with a detection limit of 15 nmol/liter. Serum
25-hydroxyvitamin-D
3
was measured using an RIA kit (DiaSorin, Inc.,
Stillwater, MN) after acetonitrile extraction. The intraassay CV was less
than 8%, and the interassay CV was less than 11% across the working
range, with a detection limit of 4
mol/liter.
Serum concentrations of type I collagen C-telopeptides (CTX), which
is a bone resorption marker, were measured on all samples using an
electrochemiluminescence assay (ELECSYS, Roche). The intraassay CV
was less than 4%, and the interassay CV was less than 5% across the
working range, with a detection limit of 0.01 ng/ml. Serum concentra-
tions of procollagen type I amino-terminal propeptide (PINP), which is
a bone formation marker, were measured on all samples using an elec-
trochemiluminescence assay (ELECSYS, Roche). The intraassay CV was
less than 2%, and the interassay CV was less than 2.5% across the
working range, with a detection limit of 4
g/liter. All serum samples
obtained from a given individual over a 24-h period were assayed within
a single batch to obviate interassay variability, and the CVs of the assays
were independently determined in our Clinical Chemistry department.
Urine
Urinary creatinine, calcium, and phosphate were analyzed on all
samples according to standard laboratory methods (Roche). The renal
threshold for maximum tubular phosphate reabsorption rate (TmPO
4
/
GFR; millimoles per liter of GFR) was determined from serum and urine
measurements and was derived from the nomogram by Walton and
Bijvoet (31). Urinary cAMP (UcAMP) was measured using an in-house
RIA method that has previously been described (32). The intraassay CV
was less than 8%, and the interassay CV was less than 10% across the
working range, with a detection limit of 0.2
mol/liter. Urinary type I
collagen cross-linked N-telopeptide (NT
x
), which is bone resorption
marker, was measured using chemiluminescence immunoassay
(VITROS ECI, Johnson & Johnson, Amersham, UK) and expressed per
millimole of excreted creatinine (NTX/UCr). The intraassay CV was less
than 4%, and the interassay CV was less than 5%, with a detection limit
of 10 nmol bone collagen equivalent. All urine samples obtained from
a given individual over a 24-h period were assayed within a single batch
to obviate interassay variability, and the CVs of the assays were inde-
pendently determined in our Clinical Chemistry department.
Statistical analysis
Individual and population-mean cosinor analysis, to determine the
circadian rhythm parameters of each variable, was performed using
CHRONOLAB 3.0 (Universdade de Vigo, Vigo, Spain), a software pack-
age for analyzing biological time series by least squares estimation (30,
33). Population-mean cosinor analysis is based on the means of param-
eter estimates obtained from individuals in the study sample The soft-
ware thus provides the following circadian parameters: 1) midline es-
timate statistic of rhythm (MESOR), defined as the rhythm-adjusted
mean or the average value of the rhythmic function fitted to the data;
2) amplitude, defined as half the extent of rhythmic change in a cycle
approximated by the fitted cosine curve (difference between the max-
imum and MESOR of the fitted curve); and 3) acrophase, defined as the
lag between a defined reference time (1400 h of the first day in our study
when the fitted period is 24 h) and time of peak value of the crest time
in the cosine curve fitted to the data.
The general linear model ANOVA for repeated measures was used
to analyze the data, and a t test for paired data with Bonferroni’s cor-
rection, to allow for multiple comparisons, was then applied to deter-
mine the significance of the differences between visits. Correlations were
sought using Pearson’s linear correlation coefficient. Values are ex-
pressed as the mean ⫾ sem unless otherwise stated. P ⬍ 0.05 was
considered significant.
Results
GH dose and IGF-I levels
The GH dose increased significantly from 0.5 IU/d (0.17
mg/d) to 0.73 IU/d (0.24 mg/d) at 1 month (P ⬍ 0.01), 0.77
IU/d (0.26 mg/d) at 3 months (P ⬍ 0.01), and 0.83 IU/d (0.28
mg/d) at 12 months (P ⬍ 0.01). There were no significant
differences in the GH dose between 1 and 12 months. Target
IGF-I was achieved within 3 months of commencing GHR
and remained within the target range at 12 months in all
patients. Serum IGF-I increased from 7.7 ⫾ 1.1
mol/liter
(58.8 ⫾ 8.4
g/liter) to 24.1 ⫾ 3.3
mol/liter (184.0 ⫾ 25.2
g/liter) within 1 month (P ⬍ 0.001), 26.1 ⫾ 2.4
mol/liter
(199.2 ⫾ 18.3
g/liter) at 3 months (P ⬍ 0.001), and 30.2 ⫾
2.5
mol/liter (230.5 ⫾ 19.1
g/liter) at 12 months (P ⬍
0.001). Similarly, the IGF-I SDS increased from – 4.47 ⫾ 0.86
at baseline to –0.85 ⫾ 0.82 at 1 month (P ⬍ 0.001), 0.27 ⫾ 0.21
at 3 months (P ⬍ 0.001), and 0.61 ⫾ 0.23 at 12 months (P ⬍
0.001).
Serum PTH, calcium, and phosphate
The 24-h mean PTH concentration decreased significantly
after 1 (4.14 ⫾ 0.04 pmol/liter) and 3 (4.10 ⫾ 0.04 pmol/liter)
months of GHR compared with baseline (4.54 ⫾ 0.04 pmol/
liter; P ⬍ 0.001), with a further decrease at 6 months (3.87 ⫾
0.04 pmol/liter) compared with the previous three visits (P ⬍
0.001) that was maintained at 12 months (4.00 ⫾ 0.04 pmol/
liter; P ⬍ 0.001 compared with 0 and 1 months and P ⬍ 0.05
compared with the 3 month visit; Fig. 1A).
The 24-h mean adjusted serum calcium significantly in-
creased at 1 month (2.32 ⫾ 0.002 mmol/liter) and was max-
imal at 3 months (2.36 ⫾ 0.002 mmol/liter) compared with
baseline (2.31 ⫾ 0.002 mmol/liter; P ⬍ 0.001), followed by a
significant decrease to levels below baseline after 6 (2.28 ⫾
0.002 mmol/liter) and 12 (2.28 ⫾ 0.002 mmol/liter; P ⬍ 0.001;
Fig. 1B) months.
Similarly, the 24-h mean serum phosphate concentration
increased significantly after 1 month (1.17 ⫾ 0.006 mmol/
liter) and was maximal at 3 months (1.27 ⫾ 0.006 mmol/liter)
compared with baseline (1.09 ⫾ 0.006 mmol/liter; P ⬍ 0.001),
followed by a significant decrease at 6 (1.20 ⫾ 0.006 mmol/
liter) and 12 (1.20 ⫾ 0.006 mmol/liter) months compared
with 3 months (P ⬍ 0.001), but remained significantly higher
than baseline (P ⬍ 0.001; Fig. 1C).
Urine biochemistry
NcAMP values significantly increased at 1 month (17.59 ⫾
0.77 nmol/liter GFR; P ⬍ 0.001) and 3 months (15.66 ⫾ 0.77
nmol/liter GFR; P ⬍ 0.05) compared with baseline (13.31 ⫾
0.77 nmol/liter GFR), followed by a significant decrease at 6
(12.37 ⫾ 0.79 nmol/liter GFR) and 12 (11.76 ⫾ 0.77 nmol/liter
GFR) months, with values decreasing below baseline (P ⬍
0.001; Fig. 1D).
The 24-h urinary calcium excretion demonstrated a non-
significant decrease at 1 month (2.59 ⫾ 0.40 mmol/liter; P ⫽
NS) and increase at 3 months (3.06 ⫾ 0.42 mmol/liter; P ⫽
NS), followed by a significant decrease at 6 (1.71 ⫾ 0.37
mmol/liter; P ⬍ 0.001) and 12 (1.58 ⫾ 0.26 mmol/liter; P ⬍
0.001) months compared with baseline (2.99 ⫾ 0.46 mmol/
liter; Fig. 1E).
Similarly, a significant decrease in 24-h urinary phosphate
excretion was observed after 6 (10.4 ⫾ 2.2 mmol/liter) and
12 (2.7 ⫾ 0.4 mmol/liter) months of GHR compared with
baseline (15.9 ⫾ 2.3 mmol/liter; P ⬍ 0.001; Fig. 1F). Concom-
itantly, TmPO
4
/GFR progressively increased at 1 (1.03 ⫾ 0.02
2862 J Clin Endocrinol Metab, June 2003, 88(6):2860 –2868 Ahmad et al. • GH, PTH Sensitivity, and Bone Metabolism
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 November 2015. at 18:37 For personal use only. No other uses without permission. . All rights reserved.
mmol/liter GFR), 3 (1.08 ⫾ 0.02 mmol/liter GFR), 6 (1.07 ⫾
0.02 mmol/liter GFR), and 12 (1.13 ⫾ 0.02 mmol/liter GFR)
months compared with baseline (0.92 ⫾ 0.02 mmol/liter
GFR; P ⬍ 0.001 compared with each visit; Fig. 1I).
Serum vitamin D metabolites
There was a significant increase in 1,25-(OH)
2
D
3
after 1
month of GHR (100.25 ⫾ 4.24 nmol/liter; P ⬍ 0.001), and
these levels were maintained at 3 (95.63 ⫾ 3.43 nmol/liter)
and 12 (99.81 ⫾ 3.86 nmol/liter) months compared with
baseline (60.56 ⫾ 2.23 nmol/liter; P ⬍ 0.001; Fig. 1G). There
were no significant changes in 1,25(OH)
2
D
3
concentrations
between 1, 3, and 12 months. Serum 25-hydroxyvitamin D
3
increased significantly after 3 months (41.13 ⫾ 3.26
mol/
liter; P ⬍ 0.001) and was maintained after 12 months (38.13 ⫾
3.83
mol/liter; P ⬍ 0.01) of GHR compared with baseline
FIG. 1. Twenty-four-hour mean ⫾ SEM for serum PTH (A), serum calcium (B), serum phosphate (C), NcAMP excretion (D), urinary calcium
excretion rate (E), urinary phosphate excretion rate (F), serum 1,25-(OH)
2
D
3
(G), 25 (OH) D
3
(H), TmPO
4
/GFR (I), serum CTx (J), serum PINP
(K), and urinary NTx/Cr (L), before GHR at 0 months and then at 1, 3, 6, and 12 months of GHR. *, P ⬍ 0.001; †, P ⬍ 0.05.
Ahmad et al. • GH, PTH Sensitivity, and Bone Metabolism J Clin Endocrinol Metab, June 2003, 88(6):2860–2868 2863
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(30.19 ⫾ 3.01
mol/liter), with no significant differences
between pretreatment and 1 month (31.25 ⫾ 3.17
mol/liter;
P ⫽ NS) values (Fig. 1H).
Bone markers
The changes in 24-h serum CTX and PINP as well as
NTX/Cr are presented in Fig. 1, J–L. The 24-h mean CTX
increased significantly at 1 month (0.21 ⫾ 0.005 ng/ml; P ⬍
0.001) and increased further at 3 (0.33 ⫾ 0.005 ng/ml; P ⬍
0.001) and 12 months (0.37 ⫾ 0.005 ng/ml; P ⬍ 0.001) com-
pared with baseline (0.18 ⫾ 0.005 ng/ml).
Similarly, there was a significant increase in PINP at 1
month (52.01 ⫾ 1.25
g/liter; P ⬍ 0.001) that increased fur-
ther at 3 (62.32 ⫾ 1.25
g/liter; P ⬍ 0.001) and 12 (70.82 ⫾
1.27
g/liter; P ⬍ 0.001) months compared with baseline
(40.76 ⫾ 1.23
g/liter).
The 24-h urinary NTX/Cr (nanomoles of bone collagen
equivalent per millimoles of creatinine) progressively in-
creased after GHR and was significant at 3 months (38.44 ⫾
6.01; P ⬍ 0.001), then increased further at 6 (43.03 ⫾ 6.78, P ⬍
0.001) and 12 (53.53 ⫾ 7.57, P ⬍ 0.001) months compared with
baseline (29.58 ⫾ 3.80).
Circadian rhythms
PTH, calcium, and phosphate. All individuals demonstrated
significant circadian rhythms for PTH and phosphate (P ⬍
0.001), with no rhythmicity detected for adjusted serum cal-
cium (P ⫽ NS). Cosinor-derived population mean circadian
rhythm parameter estimates are presented in Fig. 2. There
was a significant decrease in PTH MESOR at 3 months
(4.13 ⫾ 0.44 pmol/liter; P ⬍ 0.05) compared with baseline
(4.56 ⫾ 0.41 pmol/liter) that was maintained at 12 months
(4.01 ⫾ 0.31 pmol/liter; P ⬍ 0.05). The amplitude decreased
significantly at 6 months compared with baseline (0.62 ⫾ 0.09
vs. 0.87 ⫾ 0.10 pmol/liter; P ⬍ 0.05), with no significant
change in acrophase.
The phosphate MESOR, similar to the 24-h phosphate
mean, showed a significant increase at 1 month compared
with baseline (1.16 ⫾ 0.02 vs. 1.08 ⫾ 0.02 mmol/liter, re-
spectively; P ⬍ 0.001) that increased further at 3 months
(1.26 ⫾ 0.05 mmol/liter; P ⬍ 0.001 compared with baseline;
P ⬍ 0.05 compared with 1 month), and although there was
a nonsignificant decrease at 6 (1.18 ⫾ 0.03 mmol/liter) and
12 (1.21 ⫾ 0.03 mmol/liter) months compared with 3 months,
the values remained significantly higher than baseline (P ⬍
0.001). The amplitude increased at 1 month (0.12 ⫾ 0.01
mmol/liter; P ⬍ 0.05) and remained significantly higher at 3
months (0.13 ⫾ 0.01 mmol/liter; P ⬍ 0.05), 6 months (0.12 ⫾
0.01 mmol/liter; P ⬍ 0.05), and 12 months (0.14 ⫾ 0.02 mmol/
liter; P ⬍ 0.001) compared with baseline (0.09 ⫾ 0.01 mmol/
liter). There was a nonsignificant backward shift in acrophase
from 0217 h at baseline to 0031 h after 12 months.
Although PTH circadian rhythmicity was maintained in
AGHD patients, both before and after GHR, we observed a
sustained increase in PTH secretion between 1400 –2200 h,
with a less pronounced nocturnal rise in untreated AGHD
compared with treated AGHD patients (Fig. 2). We therefore
analyzed the difference in the percent increase in PTH se-
cretion during the periods 1400–2200 h and 2230 – 0800 h
between visits. The percent increase in PTH secretion be-
tween 1400 –2200 h was 19.3% before commencing GHR,
then significantly decreased to 8.1%, 6.4%, 12.9%, and 6.6%
after 1, 3, 6, and 12 months of GHR, respectively (P ⬍ 0.001).
Concomitantly, nocturnal PTH secretion between 2230 – 0800
h increased from 9.0% before GHR to 9.4% (P ⫽ NS), 10.1%
(P ⫽ NS), 15.8% (P ⬍ 0.001), and 16.4% (P ⬍ 0.001) after 1,
3, 6, and 12 months of GHR, respectively. These significant
changes may explain the change in PTH amplitude observed
after GHR and possibly suggest that GHR in AGHD patient
plays an important role in the PTH secretory pattern. As most
previous studies using single time point measurements
would have invariably measured PTH between 0800–1700 h,
we analyzed the difference in PTH concentration at each time
point between 0800 –1700 h after GHR. We found no signif-
icant differences in PTH concentrations at each time point.
Further analysis demonstrated that the PTH concentration in
blood samples drawn between 0800 – 0930 h was significantly
higher than that in samples drawn between 1000–1130 and
1200–1330 h (P ⬍ 0.001) and significantly lower than that
from samples drawn between 1400 –1530 and 1600–1730 h. It
was also apparent from our data that most changes in PTH
occur during the evening and early morning after GHR
(Fig. 2).
Bone markers. All individuals demonstrated a significant cir-
cadian rhythm for CTX and PINP, both before and after GHR
(P ⬍ 0.001). Cosinor-derived population mean circadian
rhythm parameter estimates for CTX and PINP are presented
in Fig. 2. There was a significant increase in the CTX (0.21 ⫾
0.01 ng/ml; P ⬍ 0.01) and PINP (52.1 ⫾ 5.0
g/liter; P ⬍ 0.01)
MESORs after 1 month of GHR compared with baseline
(0.18 ⫾ 0.01 ng/liter and 40.8 ⫾ 3.2
g/liter, respectively);
these progressively increased after 3 (0.33 ⫾ 0.02 ng/ml and
62.5 ⫾ 4.9
g/liter, respectively; P ⬍ 0.001) and 12 (0.37 ⫾
0.02 ng/liter and 70.8 ⫾ 6.4
g/liter, respectively; P ⬍ 0.001)
months. There was a significant increase in CTX amplitude
after 1 month of GHR (0.10 ⫾ 0.01 ng/ml; P ⬍ 0.05) that
increased further at 3 (0.13 ⫾ 0.01 ng/ml; P ⬍ 0.001) and 12
(0.15 ⫾ 0.01 ng/ml; P ⬍ 0.001) months compared with base-
line (0.07 ⫾ 0.01 ng/ml), with no significant differences be-
tween 3 and 12 months. PINP amplitude increased signifi-
cantly after 3 months (5.9 ⫾ 0.5
g/liter; P ⬍ 0.01) of GHR
compared with baseline (2.6 ⫾ 0.6
g/liter) and was main-
tained after 12 months (5.4 ⫾ 0.6
g/liter; P ⬍ 0.01), with no
significant differences between 3 and 12 months. There were
no significant differences in acrophase between visits for
either parameter.
Discussion
Our data demonstrate not only a significant decrease in
PTH concentration, but also a change in the PTH secretory
pattern with a concomitant increase in NcAMP after GHR,
suggesting an increase in PTH target cell sensitivity (34).
These changes were associated with an increase in 1,25-
(OH)
2
D
3
and a simultaneous increase in serum calcium that
may be the result of increased intestinal calcium absorption
(35) and renal tubular calcium reabsorption (6). The increase
in serum phosphate and TMPO
4
/GFR observed may reflect
a direct antiphosphaturic effect of GH (36, 37) or augmented
2864 J Clin Endocrinol Metab, June 2003, 88(6):2860 –2868 Ahmad et al. • GH, PTH Sensitivity, and Bone Metabolism
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intestinal absorption mediated by an increased 1,25-(OH)
2
D
3
production after GHR (38). The simultaneous increases in
bone resorption and bone formation markers suggest sig-
nificantly increased bone turnover that may also have con-
tributed to the increase in serum calcium and phosphate.
These changes were maximal at 3 months, with serum PTH,
calcium, phosphate, and NcAMP excretion decreasing to
pretreatment levels, possibly reflecting a new equilibrium
state with further contribution from the increased calcium
and phosphate uptake by bone, as bone turnover continued
at 6 and 12 months. Target IGF-I was achieved in all patients
within 3 months and was maintained at 12 months of GHR.
There remains controversy about the mechanism and ef-
fects of GHR on phosphocalcium and bone mineral metab-
olism. PTH, which is mainly regulated by calcium, phos-
phate, and 1,25-(OH)
2
D
3
, has been reported to decrease (21,
22), not change (24, 34, 35), or increase (39) after GHR. Sim-
ilarly, reports on the effects of GHR on 1,25-(OH)
2
D
3
vary
from a decrease (35), to no change (21, 24, 40), to an increase
(22, 34) after therapy. Most previous studies have consis-
tently shown an increase in serum calcium and phosphate
after GHR (21, 23, 24, 40). GH administered for3dinpost-
menopausal women resulted in increased in calcium excre-
tion and calcitriol levels, with an increase in serum phos-
phate observed after 5 wk of GH, whereas calcitriol levels
returned to baseline, and PTH showed no changes (41). Other
studies that have shown an increase in serum calcium and
phosphate after GHR, with variable effects on PTH and 1,25-
(OH)
2
D
3
, have suggested the changes to be either due to
increased bone turnover and enhanced mobilization of skel-
etal calcium or increased calcium absorption (21, 23, 24, 40).
None of these studies (21, 23, 24, 35, 40) measured NcAMP,
which reflects the circulating activity of PTH in both phys-
iological and pathophysiological states and is a reliable index
of PTH function (29). In contrast, Burstein et al. (34) reported
an increase in 1,25-(OH)
2
D
3
, with no significant change in
PTH or serum calcium in GHD patients, and a significant
increase in NcAMP excretion after GHR, suggesting a pos-
sible increase in renal sensitivity to PTH after GHR.
Our findings differ from each of these studies and prob-
ably provide a rationalization for the disparity between the
results obtained. We have demonstrated a decline in PTH
concentration, with a reciprocal increase in NcAMP excretion
after 1 and 3 months of GHR. These changes were associated
with a simultaneous increase in serum 1,25-(OH)
2
D
3
, cal
-
cium, and phosphate, followed by a decrease in 24-h urinary
FIG. 2. Cosinor-derived circadian rhythmometry (CHRONOLAB) for PTH (A), serum phosphate (B), CTX (C), and PINP (D) before (dotted lines)
and after 12 months of GHR (bold lines). Arrows represent the acrophase.
Ahmad et al. • GH, PTH Sensitivity, and Bone Metabolism J Clin Endocrinol Metab, June 2003, 88(6):2860–2868 2865
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calcium and phosphate excretion after 6 months of GHR. As
NcAMP excretion parallels changes in PTH secretion (42), a
reciprocal increase in NcAMP excretion with decreasing PTH
concentration, as observed in our study, would suggest in-
creased renal sensitivity to the effects of PTH, as suggested
by Burstein et al. (34). The increased renal sensitivity to PTH
would result in increased 1
␣
-hydroxylase activity with in-
creased 1,25-(OH)
2
D
3
production (41, 43, 44). This, in turn,
leads to increased intestinal calcium absorption (35) and
renal calcium reabsorption as a direct effect of PTH (6) due
to restoration of renal sensitivity (34). This would in part
explain the increase in serum calcium observed in our study.
Renal insensitivity to PTH in AGHD is further supported by
the high 24-h urinary calcium excretion in the presence of a
relatively high PTH concentration before GHR that is re-
duced significantly after 6 months of treatment. The rela-
tively high urinary calcium excretion at 1 and 3 months is in
agreement with previous studies (40) and reflects a higher
filtered calcium load due to increasing serum calcium ob-
served at concurrent visits. It is unlikely that the observed
changes in serum calcium and PTH are a result of relative
intestinal insensitivity to 1,25-(OH)
2
D
3
(35), as we would
expect to see a decline in 1,25-(OH)
2
D
3
concentrations with
restoration of sensitivity, which is clearly not the case in our
study.
GH has been shown to support phosphate retention by
increasing the renal threshold for phosphate excretion (45,
46), and this effect occurs independently of PTH, vitamin D,
or urinary cAMP and is mediated by IGF-I (45, 47). Such an
effect derives from an increase in TMPO
4
/GFR (45, 46),
which is found to be in the lower limit of normal (46) or
reduced (48) in GHD and increases with GHR (46, 48). In this
regard, the increases in TMPO
4
/GFR and serum phosphate
concentration may reflect a direct antiphosphaturic effect of
GH (36, 37), which is supported by the decrease in 24-h
urinary phosphate excretion. The increase in serum phos-
phate is also mediated by increased intestinal absorption
(45), which may be due to the increased production of 1,25-
(OH)
2
D
3
(38).
Bone remodeling requires bone resorption and formation,
which is reliably assessed by measuring biochemical markers
of bone resorption and formation (49). A negative equilib-
rium between bone resorption and formation has been sug-
gested to explain the low bone mass in AGHD patients (50).
GHR simultaneously increases markers of resorption and
formation, demonstrating that GH reactivates bone remod-
eling in AGHD patients (40, 50). It is still unclear whether the
changes in bone turnover markers and bone mineral density
(BMD) are a direct GH effect or an indirect effect mediated
via changes in PTH and 1,25-(OH)
2
D
3
. PTH increases renal
tubular reabsorption of calcium, bone resorption, and 1,25-
(OH)
2
D
3
production, factors important for the positive bone
remodeling during GH treatment (6). Inconsistent reports of
changes in PTH concentrations, possibly due to the single
time point PTH measurements used in studies (18, 40, 51)
have led to the suggestion that changes in bone turnover and
BMD are associated with a direct GH effect or an effect via
IGF-I. We have previously demonstrated decreased bone and
renal target cell sensitivity to the effects of PTH, with sig-
nificantly lower NcAMP and higher PTH concentration in
untreated AGHD patients compared with healthy controls
leading to decreased bone resorption and formation, de-
creased 1,25-(OH)
2
D
3
production, and increased calcium ex
-
cretion (8). Our present data support our concept of de-
creased end-organ sensitivity to the effects of PTH in
untreated AGHD patients, which is restored after GHR, re-
sulting in a simultaneous increase in bone turnover markers,
1,25-(OH)
2
D
3
, and serum calcium absorption/reabsorption
that will contribute to the previously reported increase in
BMD after GHR (18).
Circadian rhythm is known to exist for many hormones
(52, 53), and the changes in normal concentrations appear to
be important for their physiological and pathophysiological
effects. Detailed analysis of the PTH rhythms in our study
demonstrated a sustained increase between 1400–2200 h,
with a reduced nocturnal rise in untreated AGHD, a pattern
similar to that observed in osteoporotic women (10). After
GHR, PTH secretion decreased significantly between 1400 –
2200 h, with a pronounced increase in nocturnal PTH secre-
tion, a pattern previously observed in healthy individuals
and nonosteoporotic women (9, 12, 42). These findings may
suggest a possible role for GH regulation of PTH secretory
pattern. Increased PTH secretion with reduced NcAMP is
characteristic of patients with pseudohypoparathyroidism,
who have target tissue unresponsiveness to the biological
actions of PTH (54). It is possible that decreased target tissue
responsiveness to PTH, as suggested by our present data and
in part by Burstein et al. (34), is responsible for the observed
sustained increase in PTH secretion in untreated AGHD.
Restoration of the PTH secretory pattern may be an impor-
tant factor contributing to increased bone remodeling and
BMD observed in AGHD after GHR (18). However, it is
important to recognize that changes observed after exoge-
nous PTH administration may not necessarily be the same as
those occurring in response to enhanced skeletal sensitivity.
As PTH is secreted in a circadian pattern with significant
day-night variability (9, 42), the inconsistency in previous
reports regarding changes in PTH (21–23) may reflect the
single time point sampling methodology used in the studies.
We have shown that variability in the time of sampling by
2 h, as may be the case in previous studies using single time
point measurements, would result in significant differences
in PTH concentration. Our data show that most changes in
PTH occur during the evening and early morning after GHR,
whereas single PTH measurements would have been per-
formed between 0800–1700 h. These observations emphasize
that single time point PTH measurements may not be ap-
propriate to detect changes in PTH and help explain the
previously reported PTH variability.
In conclusion, our results suggest that AGHD leads to both
renal and bone insensitivity to the effects of PTH. These
changes may explain the underlying mechanisms that lead
to decreased BMD in untreated AGHD patients and even-
tually osteoporosis. GHR restores renal and bone sensitivity
to PTH, resulting in increased bone turnover and the increase
in BMD reported in previous studies. We suggest that future
studies investigating the effects of GHR on PTH should use
frequent blood sampling to accurately report the changes in
this fluctuant hormone.
2866 J Clin Endocrinol Metab, June 2003, 88(6):2860 –2868 Ahmad et al. • GH, PTH Sensitivity, and Bone Metabolism
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 04 November 2015. at 18:37 For personal use only. No other uses without permission. . All rights reserved.
Acknowledgments
We thank Eli Lilly and Prof. Blum (Endokrinologisches Labor, Gies-
sen, Germany) for analysis of IGF-I. We are grateful to Eli Lilly and
Pharmacia & Upjohn for the help and support they have provided. We
also thank the nursing staff on Ward 2-C, Royal Liverpool University
Hospital, for their help. We are grateful to N. Hoyle (Roche, Lewes, UK)
for the supply of bone marker kits.
Received November 13, 2002. Accepted March 5, 2003.
Address all correspondence and requests for reprints to: Dr. A. M.
Ahmad, Link 7-C, Department of Diabetes and Endocrinology, Royal
Liverpool University Hospital, Prescot Street, Liverpool, United King-
dom L7 8XP. E-mail: draahmad@yahoo.com.
This work was supported in part by the Research and Development
Department of the Royal Liverpool and Broadgreen University Hospital
Trust.
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