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Growth Hormone Levels at Induced Hypoglycemia Pak Armed Forces Med J 2021; 71 (1): 184-89
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Nida Basharat, Asif Ali Memon, Sumbal Nida, Naveed Asif*, Saima Shakil Malik, Muhammad Tahir Khadim
Armed Forces Institute of Pathology/National University of Medical Sciences (NUMS) Rawalpindi Pakistan, *Combined Military Hospital
Quetta/National University of Medical Sciences (NUMS) Pakistan
ABSTRACT
Objective: To introduce a relatively convenient and effective way of conducting Insulin Tolerance Test for
diagnosis of Growth Hormone deficiency in children with short stature.
Study Design: Cross sectional analytical study.
Place and Duration of Study: Conducted at Department of Chemical Pathology and Endocrinology, Armed
Forces Institute of Pathology, Rawalpindi, from May 2017 to Jul 2018.
Methodology: A total of 185 cases were included. Sample selection was done by non-probability consecutive
sampling technique. Insulin tolerance test was performed by taking basal sample for serum growth hormone
and plasma glucose levels before giving intravenous insulin bolus according to dose of 0.15 IU/kg. Samples for
Growth Hormone level were repeated at time of induced hypoglycemia (defined as plasma glucose level of <2.8
mmol/L), 30 minutes and 60 minutes post induction.
Results: Mean age of the patients was 10 ± 4 years, majority 120 (65%) were males. In the study population, 41
(22%) patients showed adequate response to insulin tolerance test while 144 (78%) showed inadequate response.
At level of induction, mean growth hormone levels were 31.9 ± 18.8 mIU/l and 4.7 ± 4.4 mIU/l in patients sho-
wing adequate and inadequate response respectively (p-value <0.05). Majority 32 (78%) of the patients showing
adequate response had peak growth hormone response (>20 mIU/l) at induction alone, followed by 30 minutes
post induction; reflecting the significance of these two samples in diagnosis of growth hormone deficiency.
Conclusion: We concluded that there is a simpler and effective way of conducting insulin tolerance test based on
two samples (induction and thirty minutes) which are sufficient for diagnosis of growth hormone deficiency.
Keywords: Growth hormone deficiency, Induced hypoglycemia, Insulin tolerance test, Serum growth hormone,
Short stature.
INTRODUCTION
Short stature is a social stigma and cause of
concern among children and their parents. Short
stature is defined as height that is two standard
deviations below the mean height or approxi-
mately below third percentile for that age and
gender1. Common causes of short stature include
malnutrition, anemia, positive family history, hy-
pothyroidism and constitutional delay of growth
and puberty. Growth hormone (GH) deficiency
is still categorized as a rare cause of short stature.
It may be idiopathic or pathological, familial or
sporadic, isolated or linked to pituitary dysfunc-
tion2. GH is usually the first hormone to be affec-
ted in case of any pathology involving pituitary;
presence of additional pituitary hormone disor-
ders reflecting severity of the disease3. The inci-
dence of GH deficiency is estimated to be around
1:4000 to 1:20 0004 with GH deficiency being res-
ponsible for 14% cases of short stature in hospital
setting5.
GH release from pituitary gland is of pul-
satile nature with almost unmeasurable levels
during daytime. As a result, a single basal GH
level is not sufficient enough to discriminate GH
deficient cases from healthy individuals6. The
diagnosis of actual growth hormone deficiency is
still a challenge and requires a multifaceted app-
roach including auxological, biochemical, radio-
logical and rarely genetic investigations. Growth
velocity and degree of short stature are main con-
siderations in deciding to continue evaluation7.
All these confounding factors provided an
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Correspondence: Dr Nida Basharat, Chemical Pathology Dept,
Armed Forces Institute of Pathology, Rawalpindi Pakistan
Received: 25 Feb 2019; revised received: 22 Oct 2019; accepted: 06 Nov
2019 nida_bak@hotmail.com
Original Article
Open Access
Growth Hormone Levels at Induced Hypoglycemia Pak Armed Forces Med J 2021; 71 (1): 184-89
185
impetus for introducing numerous pharmaco-
logical tests over the years to ease the diagnosis
of GH deficiency but their reliability is still under
question.Main reasons are lack of consensus on
cut off values and normal age related references
used, non-standardization of test protocols and
variability among commercially available GH
assays owing to molecular heterogeneity of GH.
Conventionally, atleast two separate provocative
tests are recommended to assess the GH axis with
normal GH peak response of >10 ng/ml8. Diag-
nosis is further strengthened by evaluation of
constant levels of circulating substances such as
IGF-1 and its binding proteins.
ITT (Insulin tolerance test) is still considered
as ‘gold standard’ for diagnosis of GH deficiency,
despite being a full of risk and resource consu-
ming procedure9. Worldwide, common practice
for conducting ITT is to take at least four to five
samples to monitor GH peak response. This prac-
tice of repeated sampling has made it a difficult
and labour intensive procedure; not only for the
patient but also for the pathologist. Despite the
tiered approach for diagnosis of GH deficiency,
pediatric endocrinologists still use ITT as the
main investigatory tool for commencement of
GH therapy in cases of short stature. This evokes
a need to invent a more easy and practical pro-
cedure; yet maintaining its high sensitivity and
specificity. Based on this protocol, we planned to
investigate how predictive are various GH sam-
ples taken during ITT of clinical outcome and to
assess the necessity of repeated sampling in such
cases.
Purpose of this study was to devise a more
practical and effective way of conducting ITT in a
tertiary care setting. As the awareness and dem-
and regarding GH provocative testing is increa-
sing day by day as well as the number of short
stature children visiting pediatric endocrinolo-
gists, it has become vital to strive towards a simp-
ler approach for diagnosing GH deficiency. Al-
though rare, a missed diagnosis is not affordable
as GH therapy is highly efficacious in such cases.
Study reviewed clinical procedures, identified
challenges and provided key recommendations
for best practice for management of growth dis-
orders.
METHODOLOGY
A cross sectional analytical study was
conducted at Endocrine Clinic at Armed Forces
Institute of Pathology, Rawalpindi, to assess the
current sampling protocol of Insulin tolerance
test. Study was approved by Institution Review
Board of AFIP (FC-CHP16-22/READ-IRB/18/
906). A total of 185 patients were studied from
May 2017 to July 2018. Sample size was calcula-
ted using WHO calculator, taking prevalence of
growth hormone deficiency to be 14% in hospital
settings5, with confidence interval of 95% and
margin of error 5%. Sample selection was done
by non-probability consecutive sampling tech-
nique after taking informed consent from parents
of each patient.
All patients below eighteen10 years of age,
fulfilling the criteria of short stature (height lying
<3rd centile on growth chart) and advised ITT
were included in the study.
Patients with a definitive cause of short
stature (hypothyroidism, celiac disease, anemia,
steroid therapy) were excluded from the study.
All those cases with history of epilepsy and heart
disease were also excluded.
Height in cm and weight in kg were measu-
red using digital weighing machine with attached
stadiometer (KERN MPC 250K 100M version 1.3)
KERN and Sohn GmbH with a reproducibility of
+0.2cm and +0.1 kg respectively. These were plot-
ted on Centers for Disease Control and Preven-
tion (CDC) charts and further categorized into
centiles. Each patient was advised blood comp-
lete picture, X-Ray wrist for bone age, serum thy-
roid stimulating hormone (TSH) and anti-tissue
transglutaminase antibody (anti- TTG) to rule out
other causes of short stature like anemia, hypo-
thyroidism and celiac disease. Majority of the
patients had already been evaluated for growth
hormone deficiency, either by exercise stimula-
tion test or levodopa stimulation test and showed
inadequate stimulation.
Growth Hormone Levels at Induced Hypoglycemia Pak Armed Forces Med J 2021; 71 (1): 184-89
186
Children and their parents were briefed
regarding the risks and complications associated
with ITT. Test was performed in the morning (8-
10am) after overnight fasting of at least 10 hours.
Procedure was started after passing an indwell-
ing heparin lock venous cannula for subsequent
sampling. Basal sample was taken for GH and
plasma glucose levels. Patients were given intra-
venous insulin (Regular Humulin) bolus accor-
ding to dose of 0.15 IU/kg. After administration
of insulin, patients were intensively monitored
for plasma glucose levels using glucometer every
5-10 minutes. Sampling for GH was repeated at
time of induced hypoglycemia (defined as plas-
ma glucose levels <2.8 mmol/L or symptoms of
hypoglycemia), followed by a sample 30 minutes
and 60 minutes post induction. Patients were all-
owed oral refreshment immediately at induction
to recover plasma glucose levels and were kept
under observation for one to two hours. Hypo-
glycemia was reverted with 10% dextrose (2ml/
kg) where required.
Samples for growth hormone and plasma
glucose were taken in plain gel tube and sodium
fluoride tube respectively. Serum growth hor-
mone levels were analyzed on Immulite 2000 by
fully automated random access two site Chemi-
lluminescent enzyme labeled immunoassay
(Siemens Healthcare Diagnostics Inc. NY, USA)
with an inter assay and intra assay precision of
5.7%-6.1% and 5.3%-6.5% respectively, linearity
of 120 mIU/L and detection limit of 0.03 mIU/l.
Analysis of glucose was done on fully automated
discrete random access chemistry analyzer
ADVIA 1800 (Siemens Healthcare Diagnostics
Inc. NY, USA) by hexokinase catalyzed method.
Manufacturer provided controls were run with
each batch of all analytes for internal quality
control. External quality control was assured by
simultaneously analyzing samples from External
Quality Assurance Services (EQAS) and Randox
International Quality Assessment Scheme
(RIQAS). Patients having peak GH response of
>20mIU/l at any point after insulin administra-
tion were categorized as having adequate res-
ponse4,11,12.
Data was analyzed using SPSS version 24.
Qualitative data was expressed as frequency with
percentages whereas quantitative data was sho-
wed as mean ± standard deviation. Non parame-
tric data was expressed as median and inter quar-
tile range (IQR). Data was tested for normality
using Kolmogorove Smirnov test and comparison
between groups was performed by Kruskal
Wallis test for nonparametric data. Statistical sig-
nificance was defined as p-value<0.05.
RESULTS
Total sample size was 185 out of which 120
(65%) were male and 65 (35%) were female. Mean
age of males was 10 ± 4 years and of females was
9.8 ± 3.8 years. Table-I shows the mean bioche-
mical parameters involved in the study.
One hundred and twenty six (68%) of our
study population was from rural area. Anti TTG
levels were performed to rule out celiac disease.
In 6 (3%) of the patients anti TTG was positive.
Only 9 (5%) patients in our study population sho-
wed symptomatic hypoglycemia. Forty one (22%)
patients showed adequate response to ITT and
144 (78%) patients showed inadequate response,
indicating GH deficiency. Therefore, GH defici-
ency was found to be more common cause of
Table-I: Baseline characteristics of study
population (n=185).
Study Variables
Mean ± SD
Age (years)
10 ± 4
Weight (Kg)
23.9 ± 8.7
Height (cm)
120.5 ± 20.8
BMI
22.4 ± 0.38
Hb (g/dl)
11.9 ± 1.2
TSH (mIU/l)
3.1 ± 1.3
Bone Age X-ray (years)
8.3 ± 3.8
Cortisol (nmol/l)
265 (328.5-214.5)
Glucose basal (mmol/L)
4.2 (4.6-3.8)
Glucose at Induction (mmol/L)
1.9 (2.1-1.7)
Glucose at 30 min (mmol/L)
5.5 (6.8-4.3)
Glucose at 60 min (mmol/L)
5.7 (6.8-4.8)
GH Basal (mIU/L)
0.82 (2.6-0.4)
GH at induction (mIU/L)
5.0(14.2-1.4)
GH at 30 min (mIU/L)
2.7(6.2-0.8)
GH at 60 min (mIU/L)
0.97(2.2-0.32)
Growth Hormone Levels at Induced Hypoglycemia Pak Armed Forces Med J 2021; 71 (1): 184-89
187
short stature diagnosed by ITT, as compared to
normal variant of short stature. Among males,
26 (22%) patients showed adequate response.
Among females, 16 (25%) patients showed adeq-
uate response. In patients showing adequate res-
ponse to ITT, 32 (78%) showed peak GH response
(>20 mIU/l) at GH induction alone, 3 (8%) at
thirty minutes post induction, 5 (12%) at both
induction and thirty minutes later and 1 (2%) at
both thirty and sixty minutes post induction.
Figure shows line plot of GH response during
various samples in ITT.
At level of induction, mean GH levels were
31.9 ± 18.8 mIU/l and 4.7 ± 4.4 mIU/l in patients
showing adequate and inadequate response
respectively (p-value <0.05). It was seen that max-
imum patients showed peak GH response at level
of GH induction, followed by thirty minutes post
induction; highlighting the significance of these
two samples during ITT. Significant difference in
GH levels amongst samples taken at induction,
30 minutes and 60 minutes was observed among
patients showing adequate and inadequate res-
ponse (p-value <0.05). Kolmo-gorove Smirnov
test was applied to test the normality of data.
After confirmation of non-normality of data, a
nonparametric Kruskal Wallis test was applied to
test the mean difference between various GH sa-
mples and results are given in table-II for patients
showing adequate and inadequate responses.
DISCUSSION
Purpose of our study was to evaluate the
current sampling protocol followed for diagnosis
of growth hormone deficiency using ITT in our
set up. Current method for ITT is laborious and
time consuming; involving a series of samples
which might become difficult to execute, both for
the patient and pathologist.
Few studies have analyzed the varying
glycemic and GH excursions associated with ITT,
showing a lower mean GH response (5.4 mIU/l)
in GH deficient individuals as compared to nor-
mal individuals (55.5 mIU/l)2,13,14. In this study,
we analyzed the methodology of periodic sam-
pling in ITT based on glucose levels and GH
levels and their effectiveness in diagnosis of
GH deficiency. Result indicated that peak growth
hormone responses which are used for ruling out
GH deficiency mainly exist at induced hypogly-
cemia and thirty minutes post induction, which
raises the question whether interpreting ITT
should take into account the other two samples
included in protocol.
Majority 144 (78%) patients in our study
were labeled GH deficient, which is in close
proximity to a retrospective study conducted by
Abdul moein et al. in 2011 in Jeddah15, where
frequency of GH deficiency was around 69.4%
among 650 patients. Study showed female pre-
Table-II: Comparison of GH levels using Kruskal Wallis test in patients showing adequate and inadequate
responses (n=185).
Adequate
Inadequate
Median
p-value
Median
p-value
GH Levels
<0.001
GH Levels
<0.001
0 mint
33
0 mint
59
30 mint
39
30 mint
53
60 mint
40
60 mint
52
induction
40
induction
52
Figure: Line plot of mean GH values at different GH
levels (n=185).
Growth Hormone Levels at Induced Hypoglycemia Pak Armed Forces Med J 2021; 71 (1): 184-89
188
ponderance which is contrary to current study.
However, another study by Yousaf et al11 conduc-
ted among Pakistani population in 2016 showed
majority (66.6%) patients of short stature to be
males, which is comparable to current study. Fur-
thermore, another multi centric study by Sukran
et al16 conducted in Turkey in 2015 involving 44
Turkish pediatric centres showed 50% of short
stature children with normal growth velocity as
GH deficient, using GH cut off of 30 mIU/l in
ITT. Similar to current study, the most frequently
used assay in this study was chemilluminescence
immunoassay.
In another study conducted by Asif et al in
Rawalpindi, Pakistan in 20095, frequency of GH
deficiency based on ITT was around 60% which
was also comparable to result of current study.
There is still ambiguity regarding the cut off
values used for diagnosis of GH deficiency in
provocative tests. Canadian Pediatric Endocrine
group has defined a cut off of 24 mIU/l for
diagnosis of GH deficiency using Immulite 2000.
Meanwhile, most centers around UK use a cutoff
of 21 mIU/l in GH stimulation tests17. In our
study, we used 20 mIU/l as definitive cutoff,
which was close to international studies. On the
other hand Bonfig et al14 in 2008 stated the highest
accuracy of ITT with a cutoff of 15 mIU/l.
In another study conducted by Kevin et al in
USA in 201218, which was based on glucagon
stimulation test, it was seen that BMI and peak
and nadir responses of glucose correlated nega-
tively with GH levels. Our study was based on
ITT but still reflected the same findings. Our
study was not designed to determine specific GH
cut points for ITT based on age and BMI, but does
highlight the need for additional prospective stu-
dies based on control groups to provide defini-
tive evidence in establishing cut off values.
Even though ITT remains the gold standard
for diagnosis of GHD, there are not many studies
to assess its reproducibility, reliability and samp-
ling protocol to give the best clinical decision in
case of short stature patients. Majority of short
stature children may present with normal height
velocity and show adequate response during
ITT12. Furthermore, there is still lack of consensus
regarding the most appropriate GH assay avail-
able, with inter assay standardization and com-
parability as well as harmonization of cutoff val-
ues being the essential aspects to reduce variabi-
lity19. Diagnosis of short stature requires a dyna-
mic approach, especially while interpreting a
blunted response to GH provocative tests in case
of children presenting with normal growth rate.
There is also a need to introduce new tests like
GHRH plus arginine test to identify false GHD12.
Diagnosis of short stature includes discrimination
of normal growth variants like Constitutional
Delay of Growth and Puberty (CDGP), familial
short stature, Small for gestational age (SGA)
with catch up growth from pathological growth
impairment as both require different approach
towards management. In our opinion, purpose of
dynamic gold standard tests like ITT should be to
identify those patients in need of GH replacement
correctly, rather than identifying clinically insig-
nificant defects. There are still grey areas in case
of borderline cases, even after conducting ITT, so
clinical judgement and follow up is still mainstay
for assessment in such patients.
This study had certain limitations. Firstly, we
did not include IGF-1 and IGF-BP3 levels in our
diagnostic workup. Secondly, there is a need to
conduct multi center studies before introducing
the new strategy into clinical and laboratory
practice.
ACKNOWLEDGEMENT
We acknowledge Ms Sumaira Mubarik for
her useful contribution in statistical analysis.
CONCLUSION
We concluded that there is a simpler and
convenient way of conducting ITT comprising of
two samples (induction and thirty minutes post
induction) which are sufficient for diagnosis of
GH deficiency in case of short stature children.
There is a need to inculcate this methodology in
future diagnostic practice for the benefit of
patient, treating physician and pathologist.
Growth Hormone Levels at Induced Hypoglycemia Pak Armed Forces Med J 2021; 71 (1): 184-89
189
CONFLICT OF INTEREST
This study has no conflict of interest to be
declared by any author.
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