The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 21-24
Copyright © 1998 by The Endocrine Society
Effect of Therapy with Recombinant Human Growth Hormone on Insulin-Like Growth Factor System Components and Serum Levels of Biochemical Markers of Bone Formation in Children After Severe Burn Injury1
Gordon L. Klein,
Steven E. Wolf,
Craig B. Langman,
Clifford J. Rosen,
Subburaman Mohan,
Bruce S. Keenan,
Sina Matin,
Christopher Steffen,
Marc Nicolai,
Dawn E. Sailer and
David N. Herndon
Departments of Pediatrics (G.L.K., B.S.K.) and Surgery (S.E.W.,
Si.M., D.N.H.), University of Texas Medical Branch and the Shriners
Burns Institute (G.L.K., S.E.W., Si.M., M.N., D.N.H.), Galveston, Texas
77555; Nephrology Division, Children’s Memorial Hospital and the
Northwestern University Medical School (C.B.L., D.E.S.), Chicago,
Illinois 60614; the Maine Center for Osteoporosis Research, St. Joseph
Hospital (C.J.R., C.S.), Bangor, Maine 04401; and the Jerry L. Pettis
VA Medical Center and Loma Linda University School of Medicine (Su.M.),
Loma Linda, California 92357
Address all correspondence and requests for reprints to: Gordon L. Klein, M.D., Pediatric Gastroenterology Division, Room 3.240B, Children’s Hospital, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0352. E-mail:
gklein{at}utmb.edu
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Abstract
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Burn injury in children is associated with low bone formation and
long-term bone loss. Because recombinant human GH (rHGH) may accelerate
burn wound healing, and because rHGH increases bone formation and
density in GH-deficient patients, we studied the short-term effects of
rHGH on bone formation, reflected by osteocalcin and type I procollagen
propeptide levels in a randomized, double-blind, placebo-controlled
study. Nineteen patients were enrolled and received either rHGH (0.2
mg/kg·day) or an equal volume of saline. Mean burn size and age were
not different between the groups, and test substances were given from
admission to time of wound healing (mean: 43 ± 22 days). At wound
healing, serum levels of insulin-like growth factor (IGF)-1 and IGF
binding protein (IGFBP)-3 in the rHGH group rose to mean values of
229% and 187% of the respective means of the placebo group
(P < 0.025). Serum osteocalcin concentrations
remained below normal in both groups, and type I procollagen propeptide
levels achieved a low normal level. IGFBP-4 levels were twice that of
normal on admission and doubled further at wound healing; IGFBP-5
levels were low on admission but rose to normal at wound healing. We
conclude that large doses of rHGH were ineffective in improving
disordered bone formation despite increasing serum IGF-1 and IGFBP-3.
The rHGH-independent rise in serum levels of the inhibitory binding
protein IGFBP-4 suggests a mechanism by which improved bone formation
is prevented despite successful elevation of IGF-1 and IGFBP-3 in the
burned child.
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Introduction
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BURN INJURY is associated with reduction in
bone formation (1, 2). In children, markedly diminished bone density
(3) and linear growth velocity (4) also have been described. In
previous attempts to improve growth velocity in burned children,
recombinant human GH (rHGH) was administered during the initial
hospitalization. Acceleration of wound healing was previously reported
with this therapy (5, 6), as well as an increase in insulin-like growth
factor (IGF)-1 levels in the blood from low to normal (7). rHGH
increases IGF-1 (8), osteocalcin (8, 9, 10), type I procollagen propeptide
(PICP) (9), and bone density (9), when administered to children with GH
deficiency. Because it also seemed to increase bone formation in
corticosteroid-dependent children, as evaluated by histomorphometric
analysis (11), we hypothesized that rHGH would similarly improve the
low bone turnover state associated with severe burns. Therefore, we
performed a randomized double-blind, placebo-controlled trial of rHGH
to evaluate its effect on bone formation and bone density in burned
children. Furthermore, because the characterization of IGF binding
protein (IGFBP) response to rHGH administration in burned patients had
not been previously studied, measurements of IGFBP-3, -4, and -5 also
were undertaken. Based on the study presented, we conclude that
short-term treatment of this population with rHGH is not effective in
reversing the defects in bone metabolism.
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Subjects and Methods
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We studied 19 children [9 males and 10 females, ages 5.3
± 3.6 yr (SD)], randomly assigned to receive rHGH (0.2
mg/kg day) sc (GenotropinR, generously provided by
Pharmacia-Upjohn, Kalamazoo, MI and Stockholm, Sweden) or a saline
placebo of equivalent volume. Study drug was administered at 0600
h daily from time of admission, within 72 h of burn, to the time
when wounds were considered 95% healed (indicating that no further
grafting was needed and that the patient was ready for hospital
discharge). All patients received iv fluids according to a standard
formula (12). Enteral nutrition support was instituted within 24 h
of admission, provided as formula, Vivonex TENR or Vivonex
PediatricR (Sandoz Nutrition, Minneapolis, MN), providing
daily: 1200 kcal/m2 as maintenance and an additional 1800
kcal/m2 open-wound area, and 30–50 g
protein/m2. Calcium intake was 0.7–1.2 g/m2
and an additional 0.9–1.8 g/m2 body surface area burned;
phosphate intake was 0.7–1.0 g/m2, plus 0.9 to 1.44
g/m2 surface area burned; magnesium intake was 0.3
g/m2, and 0.36 g/m2, depending on which formula
was used. An age-appropriate regular diet was routinely added as
tolerated.
Blood was obtained on admission and again at wound healing, as defined
above, for the following levels: IGF-1; IGFBP-3, -4, and -5;
osteocalcin; and PICP. Bone mineral density (BMD) of the lumbar spine
was obtained at the time of wound healing.
We determined IGF-1 and IGFBP-3, respectively, by immunoradiometric
assay and RIA kits, purchased from Nichols Laboratories, San Juan
Capistrano, CA (13, 14). For IGF-1, the mean intraassay and interassay
coefficients of variation were 5% and 15%, respectively, for 20
assays. For IGFBP-3, the mean intra- and interassay coefficients of
variation were 
8% and <6.4%, respectively, for 20 assays. IGFBP-4
was analyzed first by Western blot and gel densitometry (15) and then
by RIA (16). Intra- and interassay coefficients of variation,
previously published (16), are <5% and <8.1%, respectively. IGFBP-5
levels also were determined by RIA (17) with intra- and interassay
coefficients of variation, previously published (17), of <4% and
<8%, respectively. Results of the IGFBP-4 and -5 assays were compared
with age-related controls being treated for hypothyroidism and whose
thyroid function was normal at the time of blood sampling. Osteocalcin
and PICP levels were determined by enzyme-linked immunosorbent assay
and compared with the age-related normal range, as determined in the
same laboratory (2, 18). Intra- and interassay coefficients of
variation have been previously published, being 2% and 6% (2), and
4% and 7% (2), respectively.
BMD was determined by dual-energy x-ray absorptiometry using a QDR1000W
absorptiometer (Hologic, Waltham, MA). Results are reported as
z-scores, compared with age and gender-related normals (3).
Sample size was chosen: 1) to exceed that necessary to detect a 40%
difference in serum osteocalcin between rHGH and placebo groups at time
of wound healing, a difference that would place osteocalcin in the
normal range for the rHGH group, with 95% probability at the 
=
0.05 level; and 2) to exceed the number needed to detect the 3-fold
rise in serum levels of IGF-1 previously reported in burned children
(7) who received rHGH therapy.
Statistical methods used for data analysis included linear regression,
paired, and unpaired t tests, as appropriate. Results are
expressed as means and sd unless otherwise noted. This study
was reviewed and approved by the Institutional Review Board of the
University of Texas Medical Branch, Galveston, and by the Shriners
Hospitals for Children, Tampa, FL. Informed consent was obtained from
parents of all study participants.
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Results
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There were 10 patients enrolled in the rHGH group and 9 enrolled
in the placebo group. Samples were obtained from children 5 ± 7
days post burn at baseline, range: 1–23 days (n = 19); and
43 ± 22 days post burn at time of wound healing, range: 12 to 78
days (n = 16). Burn size was comparable between the two groups,
62 ± 15% total body surface area for the rHGH group and 65
± 19% total body surface area in the placebo group.
Serum IGF-1 levels in the serum were not significantly different at
admission between the rHGH and placebo groups, although they were low
in one third of the children. However, at wound healing, IGF-1 levels
were significantly higher in the rHGH group (Fig. 1
). Serum concentrations of IGFBP-3 were
not different between the groups on admission but were low in 37% of
the children. However, serum IGFBP-3 levels rose to be
significantly higher in the rHGH group by time of wound healing (Fig. 2).
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Figure 1. Serum concentrations of IGF-1 in the rHGH
and placebo groups at baseline (admission) and at time of wound
healing. Data are given as mean ± SD. Numbers of
patients in each group are shown at the top of each
bar. Normal range from ages 2 months to 5 yr is 17–248
ng/mL; and from 6–12 yr, from 88–1096 ng/mL.
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Figure 2. Serum concentrations of IGFBP-3 in the rHGH
and placebo groups at baseline (admission) and at time of wound
healing. Data are given as mean ± SD. Numbers of
patients in each group are shown at the top of each
bar. Normal range in children up to age 4 yr is
0.66–3.77 µg/mL; and from ages 4–12 yr, 1.16–5.46 µg/mL.
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Western ligand blot analysis revealed the presence of IGFBP-4 in nearly
all sera. Scanning densitometry of the gels, reported as the ratio of
percent area of IGFBP-4 in study subjects over the control value was
2.4 ± 0.8 for the rHGH group (n = 6) and 2.17 ± 0.2
for the placebo group (n = 8). RIA results (Fig. 3) revealed elevated serum levels of
IGFBP-4 on admission, when compared with age-matched controls. These
levels increased significantly, irrespective of rHGH administration,
from admission to wound healing, thus further exceeding the normal
range. Serum concentrations of IGFBP-5 (Fig. 4) were uniformly low at admission but
normal at time of wound healing. There were no significant differences
between the GH and control groups.
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Figure 3. Serum concentrations of IGFBP-4 are shown at
baseline (admission) and at wound healing for individual members of
both rHGH and placebo groups. Because there were no statistically
significant differences between the patients receiving rHGH (n =
7) and the patients receiving placebo (n = 8), the observations
were combined. Lines connect the dots for
each patient from baseline to healed periods. Data obtained from 9
age-matched control children are given as mean ± SD.
Normal range was 177–368 ng/mL. The dotted line extends
the mean value of the normal group across the length of the
horizontal axis.
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Figure 4. Serum concentrations of IGFBP-5 are shown at
baseline (admission) and at wound healing for individual members of the
rHGH and placebo groups. Because there were no statistically
significant differences between the patients receiving rHGH (n =
7) and the patients receiving placebo (n = 8), the observations
were combined. Lines connect the dots for
each patient from baseline to healed periods. Data obtained from 9
age-matched control children are given as mean ± SD.
Normal range was 184–248 ng/mL. The dotted line extends
the mean value of the normal group across the length of the
horizontal axis.
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Serum concentrations of osteocalcin (Fig. 5) and PICP did not differ between the
two groups either at admission or at wound healing. Serum levels of
both markers of bone formation rose at the time of healing. Baseline
serum levels of PICP were low in the rHGH group (n = 3, 115
± 28 ng/mL) and low normal in the placebo group (n = 4, 233
± 183 ng/mL). Serum PICP levels rose in both groups at time of healing
to 296 ± 79 ng/mL (rHGH) and 283 ± 74ng/mL (placebo).
Normal range is 200–700 ng/mL.
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Figure 5. Serum concentrations of osteocalcin are
shown for the rHGH and placebo groups at baseline (admission) and at
wound healing. Data are given as mean ± SD. The lower
limit of normal is designated by a dotted line at the
top of the figure. Numbers of patients in each group are
given at the top of the bars.
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Lumbar spine BMD was obtained in four of the patients in the rHGH group
and five patients in the placebo group. Z-scores were -0.5 ± 1.0
(range: -1.26 to + 0.87) in the rHGH group and -0.7 ± 1.2
(range: -1.75 to +1.20) in the placebo group, respectively
(P = not significant).
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Discussion
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Our data, reported herein, show that rHGH does not increase bone
formation or bone density in the short term for burned children. This
failure is not caused by a dose-response effect, because we
administered a daily dose of rHGH that is four times that given for GH
deficiency, in which measurable increases in serum levels of PICP and
osteocalcin occur (8, 9, 10).
The efficacy of rHGH therapy is generally monitored biochemically by
demonstration of a rise in serum levels of IGF-1, and in our study, we
observed increases in both IGF-1 and IGFBP-3 levels in the serum of the
burned patients. IGF-1 is reported to be anabolic to bone (19), and
serum levels of IGFBP-3 are closely associated with increased lumbar
spine BMD in adult males (20). Although rHGH administration in GH
deficiency has been reported to increase serum levels of IGFBP-5, a
binding protein that may fix IGF-1 to bone (21), no such effect of rHGH
was seen in the burn patients, perhaps because of other, as yet
unspecified, metabolic abnormalities. However, serum IGFBP-5 levels
rose to normal in these children, regardless of GH administration, at
the time of wound healing.
One possible explanation for the apparent resistance of bone to
increased circulating levels of IGF-1, as well as for the previous
finding of retardation in growth velocity post burn, may be
insufficient length of treatment. Data from Saggese et al.
(9) in GH-deficient patients demonstrated that rHGH produces a
significant rise in serum levels of PICP after 1 week of treatment with
rHGH, and a significant increase occurred in serum osteocalcin
concentration by 3 months. Our patients exhibited little change in PICP
levels and osteocalcin levels in serum during the study period, which
lasted a mean of 6 weeks from time of burn injury, with an upper range
of 11 weeks, using doses that were four times those given to
GH-deficient children. Though the possibility exists that treatment
length was insufficient, it would not be unreasonable to expect
increases in serum PICP or osteocalcin levels in at least some of the
patients.
To explain resistance to rHGH in bone, an alternative to insufficient
time of therapy is the rise in serum concentration of IGFBP-4, present
on admission and rising further by an apparently
growth-hormone-independent mechanism by the time of wound healing.
IGFBP-4 is reported to inhibit the anabolic effects of IGF-1 on bone
and other tissues, perhaps by decreasing the bioavailability of IGF-1
to the local tissue IGF-1 receptor (16). IGFBP-4 is produced by bone
cells (16) and by keratinocytes (22). Its production is stimulated by
both 1,25-dihydroxyvitamin D (calcitriol) (15) and by PTH (16, 23).
However, serum levels of calcitriol have been reported as either low or
normal in burn patients (1). Similarly, burned children are
hypoparathyroid, as evidenced by subnormal PTH response to the commonly
observed low serum concentrations of ionized calcium in these patients
(24). Therefore, neither can be considered a stimulus for increased
IGFBP-4 production in our patients.
We speculate that IGFBP-4 levels are increased by proliferating
keratinocytes in the burned child during the process of wound healing.
Such increases in IGFBP-4 may divert the anabolic activities of IGF-1
from bone to promote the cutaneous wound healing effects of rHGH in the
burned patient (5, 6, 7) or other functions, as yet unspecified.
Our data demonstrate further that the long-lasting effects of the burn
injury on the metabolic aspects of bone function are not easily
reversed. The exact mechanism remains uncertain but now can be extended
to include the GH-IGF-1 axis, including an important role for IGF-1
binding proteins.
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Footnotes
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1 This work was presented, in part, at the 19th Annual Meeting of The
American Society for Bone and Mineral Research, Cincinnati, OH,
September 10–14, 1997. Funding for this work was obtained, in part,
from Shriners Hospitals for Children Grants 8770 and 8680 (to G.L.K.);
NIH Grant AR-31062 (to S.M.); NIH, NIA Grant AG-1094–01 (to C.J.R.);
and NIH Grant RR-00078 (to C.B.L.). 
Received August 18, 1997.
Revised September 25, 1997.
Accepted October 6, 1997.
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Abstract
Introduction
