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\begin{document}

\title{A Comparison of L-Dopa and Clonidine Growth Hormone Stimulation Tests in
Children with Short Stature}



\author[1]{Semih Bolu}%
\author[1]{Abdulvahit Aşık}%
\affil[1]{Adiyaman Universitesi Egitim ve Arastirma Hastanesi}%


\vspace{-1em}



  
  \date{May 05, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Growth hormone (GH) release is pulsatile, and daytime GH
levels are low. GH stimulation tests are therefore needed in cases
requiring GH level investigation. The purpose of this study was to
compare the results of L-dopa and clonidine GH stimulation tests applied
in children with short stature and to identify which of these tests
should be primarily selected. Methods: The records of 68 patients aged
between 2.5 and 16.6 years presenting to the pediatric endocrinology
clinic with short stature and undergoing GH stimulation tests between
September 2016 and February 2021 were evaluated retrospectively. Cases
with GH levels \textless{}10 ng/dl following the first GH stimulation
test then underwent the other GH stimulation test. Thirty-four (50\%) of
the cases in the study consisted of individuals beginning with the
clonidine test, while the other 34 (50\%) started with the L-dopa test.
Results: Seventeen (50\%) individuals in whom clonidine was employed in
the first test had low GH responses, while a low GH response was
determined at the second, L-dopa test, in 15 (88.2\%) of these
individuals, significant variation being observed between the groups
(p\textless{} 0.001). Conclusion: GH stimulation tests performed to
investigate GHD are laborious and time-consuming. The first stimulation
test to be applied to differentiate GHD from ISS must therefore be well
selected. The clonidine stimulation test, with higher sensitivity than
but similar specificity to the L-dopa test, can be employed as the first
test.%
\end{abstract}%



\sloppy


\selectlanguage{polish}\textbf{A Comparison of L-Dopa and Clonidine Growth Hormone Stimulation
Tests ın Children with Short Stature}\selectlanguage{english}

\textbf{ABSTRACT}

\textbf{Background:} Growth hormone (GH) release is pulsatile, and
daytime GH levels are low. GH stimulation tests are therefore needed in
cases requiring GH level investigation. The purpose of this study was to
compare the results of L-dopa and clonidine GH stimulation tests applied
in children with short stature and to identify which of these tests
should be primarily selected.

\textbf{Methods:} The records of 68 patients aged between 2.5 and 16.6
years presenting to the pediatric endocrinology clinic with short
stature and undergoing GH stimulation tests between September 2016 and
February 2021 were evaluated retrospectively. Cases with GH levels
\textless{}10 ng/dl following the first GH stimulation test then
underwent the other GH stimulation test. Thirty-four (50\%) of the cases
in the study consisted of individuals beginning with the clonidine test,
while the other 34 (50\%) started with the L-dopa test.

\textbf{Results} : Seventeen (50\%) individuals in whom clonidine was
employed in the first test had low GH responses, while a low GH response
was determined at the second, L-dopa test, in 15 (88.2\%) of these
individuals, significant variation being observed between the groups
(\emph{p} \textless{} 0.001).

\textbf{Conclusion:} GH stimulation tests performed to investigate GHD
are laborious and time-consuming. The first stimulation test to be
applied to differentiate GHD from ISS must therefore be well selected.
The clonidine stimulation test, with higher sensitivity than but similar
specificity to the L-dopa test, can be employed as the first test.

\textbf{Keywords} : Growth hormone stimulation test, Clonidine, L-Dopa\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
\selectlanguage{polish}\textbf{WHAT İS ALREADY KNOWN?}\selectlanguage{english} The first test to be employed for
differentiating GHD from ISS must carefully selected \textbf{WHAT THIS
STUDY ADDS?} The application of clonidine stimulation test, which has
higher sensitivity and similar specificity in demonstrating GHD compared
to L-dopa test, as the first test may provide some advantages for both
the child, the family and the clinician.\tabularnewline
\bottomrule
\end{longtable}

\textbf{INTRODUCTION}

Short stature is an important problem frequently encountered in
pediatric clinics, and one which causes anxiety in children and
parents.\textsuperscript{1} The majority of children presenting with
short stature represent the idiopathic variant, and pathological short
stature constitute a smaller proportion of children presenting with that
complaint. Short stature more than two standard deviations below
standard community values for age and gender, with no systemic,
nutritional, chromosomal, or endocrine cause, is known as idiopathic
short stature.\textsuperscript{2,3} Growth hormone deficiency (GHD), one
form of pathological short stature, is an important cause of endocrine
short stature seen in one in every 3500-4000 live
births.\textsuperscript{4} Early detection of GHD can contribute to
patients receiving prompt growth hormone therapy and thus achieving
target heights.\textsuperscript{5} No benefit has been shown from
investigating basal growth hormone levels when deficiency is suspected.
Growth hormone release is pulsatile, and daytime levels are low. Growth
hormone stimulation tests are therefore needed in cases requiring growth
hormone level investigation\textbf{.\textsuperscript{6}}

The purpose of this study was to compare the superiorities, sensitivity,
and specificity of the L-dopa and clonidine stimulation tests used in
the differential diagnosis of idiopathic short stature (ISS) and GHD.

\textbf{METHODS}

Clinical and laboratory data were elicited through a retrospective
examination of the files of 68 patients presenting to the pediatric
endocrinology clinic with short stature between September 2016 and
February 2021 and undergoing growth hormone stimulation tests following
clinical and laboratory evaluation. Patients with other systemic and
hormonal disorder affecting growth hormone levels or with syndromic
short stature were excluded from the study. Anthropometric and
measurements and biochemical values in terms of systemic diseases were
recorded from patients' old records. Pubertal examinations were
evaluated based on Tanner staging.\textsuperscript{7} Thirty-four (50\%)
of the cases in the study consisted of individuals commencing with the
clonidine test, while the other 34 (50\%) started with the L-dopa test.
The 34-member group undergoing the L-dopa test as the first test was
randomly selected from among 164 cases.

Growth hormone stimulation tests began between 08:00 and 09:00 after
fasting of at least 8 h. Intravenous access was established before the
tests commenced. Following collection of the first blood specimen, a
tablet containing 125 \selectlanguage{greek}μ\selectlanguage{english}g dopamine was given to patients weighing less
than 15 kg, containing 250 \selectlanguage{greek}μ\selectlanguage{english}g dopamine to patients weighing 15-30 kg,
and containing 500 \selectlanguage{greek}μ\selectlanguage{english}g to children weighing more than 30 kg for the
L-dopa tests, while tablets containing 150
mcg/m\textsuperscript{2}clonidine were given for the clonidine tests.
Blood samples were collected for growth hormone level measurement at 30,
60, 90, and 120 min for both tests, and the fact that sex steroids had
not been administered before the growth hormone stimulation tests was
confirmed from the old records.

Peak growth hormone levels \textless{}10 ng/ml following growth hormone
stimulation tests were regarded as representing insufficient response
(8). Cases with growth hormone levels \textless{}10 ng/dl following the
first test then underwent the other stimulation test. Cases with peak
growth hormone levels \textless{}10 ng/dL after both tests constituted
the GHD group, and those with peak growth hormone levels
\textgreater{}10 ng/dL from either test constituted the ISS group.

Blood specimens were collected at 08.00 following 10-h fasting and were
used for the examination of serum GH levels, IGF-I, and IGFBP--3 levels.
Serum IGF-1 and IGFBP-3 levels were measured using the
solid-phase,~enzyme-labeled~chemiluminescent immunometric method, and
growth hormone levels were determined using the chemiluminescence method
on a Siemens Immulite 2000XPi device, with commercial kits.

The study protocol was approved by the local ethics committee (Ad\selectlanguage{polish}ı\selectlanguage{english}yaman
University Non-Interventional Clinical Research Ethics Committee. No.
2020/3-5, dated 21.04.2020).

\textbf{Statistical Analysis}

Descriptive statistics are presented as frequency and percentage values
for categorical variables and as mean plus standard deviation for
continuous variables. Differences between distributions of different
variables in the groups were examined using chi-square analysis, and
cross tables are presented. The independent samples t test was applied
in the comparison of mean variable values by groups. ROC analysis was
performed to examine the sensitivity and specificity of the tests used
in the research. Data analysis was performed on~SPPS 25 software (IBM
Corp. Released 2017. IBM SPSS Statistics for Windows, Version 25.0.
Armonk, NY, USA). p values~\textless{}0.05 were regarded as significant
for all analyses.

\textbf{RESULTS}

The study population consisted of 68 patients ranging in age between 2.5
and 16.6 years, 35 (51.5\%) girls and 33 (48.5\%) boys. GHD was
diagnosed in 35 (51.5\%) cases and ISS in 33 (48.5\%). Thirty-three
(48.5\%) cases were prepubertal and 35 (51.5\%) were pubertal. Three
separate chi-square analyses were applied in order to determine whether
the two groups were similar in terms of diagnosis, gender, and puberty.
The results showed no significant differences in these three variables
(\emph{p\textgreater{}} 0.05) (Table 1).

Two separate independent samples t test analyses were applied to
determine whether there was any significant variation in terms of age,
bone age, birth weight, height, BMI-SDS, and IGF1, or IGF BP 3 values
between the GHD and ISS groups. The results showed a significant
difference between the groups in terms of BMI-SDS values
(\emph{p}\textless{}0.05), but no significant difference in terms of the
other variables (\emph{p} \textgreater{}0.05) (Table 2).

Peak GH response was insufficient in 27 (79.41\%) patients undergoing
L-dopa was applied as the first test. Clonidine test results were also
low, consistent with the L-dopa test, in 18 (66.7\%) of these. Growth
hormone response was insufficient in 17 (50\%) of the patients
undergoing the clonidine test as the first test. A sufficient growth
response was also achieved at the L-dopa test performed as the second
test in 15 (88.2\%) individuals. Chi-square analysis was performed to
examine whether there was any significant variation in distribution
rates in the groups, and significant variation was determined in
distributions by groups (\emph{p} \textless{}0.001) (Table 3).

The sensitivity and specificity values of the L-dopa and clonidine tests
and appropriate cut-off points for these values were also investigated.
The L-dopa test cut-off point for 50\% sensitivity and 100\% specificity
was 8.93. The cut-off point for 88\% sensitivity and 94\% specificity on
the clonidine test was 9.76 (Table 4).

\textbf{DISCUSSION}

Short stature is defined as a mean height under at least two standard
deviations according to age and gender or a height below the third
percentile according to standard growth curves. Although most short
stature is a variant of normal, it may also be due to an underlying
systemic disease or endocrine disorder\textbf{.\textsuperscript{9}} GHD
represents the most important group among the endocrine causes of short
stature. One study reported an endocrine cause in 26\% of children
presenting due to short stature, with GHD being the most common
endocrine cause (45.2\%).\textsuperscript{10} Growth hormone therapy
contributes to the achievement of normal height in adulthood in children
with GHD. The accurate identification of children with GHD is therefore
highly important.\textsuperscript{11} Growth hormone is released in a
pulsatile manner, and release is higher at night than in the daytime.
Random serum growth hormone values are not a useful guide in the
diagnosis of GHD. Stimulation tests are therefore required in order to
confirm a diagnosis of GHD\textbf{.\textsuperscript{6}} Since false
negative results are obtained in between 10\% and 20\% of growth hormone
stimulation tests, at least two stimulation tests are recommended in
patients with suspected GHD. Peak growth hormone levels \textless{}10
ng/ml with both these different tests are defined as
GHD.\textsuperscript{8} Various growth hormone stimulation tests have
been used to identify GHD in children, including the insulin tolerance
test, clonidine, glucagon, levodopa, arginine, and growth hormone
releasing hormone\textbf{.\textsuperscript{8, 12}} Although the insulin
tolerance test is regarded as the gold standard for GHD in adults, there
is no consensus regarding the first choice test for GHD in children.
Clinical application of the insulin tolerance test is difficult since it
can lead to severe hypoglycemia, and rarely
death\textbf{.\textsuperscript{13, 14}} The L-dopa and clonidine
stimulation tests, which are safer than the insulin tolerance test, were
therefore used to diagnose GHD in the present study. However, various
side-effects can also be seen in children after these two
tests.\textsuperscript{15} Gastrointestinal problems such as nausea and
vomiting can develop with the L-dopa test, and sedation and hypotension
with the clonidine test\textbf{.\textsuperscript{16-19}} Drug-related
side-effects developing following stimulation tests can cause anxiety in
children and adults, which can prevent the performance of a second test.
In addition, the first stimulation test applied in the diagnosis of GHD
has low sensitivity, thus imposing the burden of a second test on the
patient. The selection of the first stimulation test is therefore highly
important.

Petriczko et al. examined the prognostic significance of the L-dopa and
clonidine tests in cases of GHD and reported that the L-dopa test was
superior to the clonidine test in showing pituitary growth hormone
reserves.\textsuperscript{20} However, in contrast to that research, the
purpose of the present study was to determine which of these two tests
should primarily be employed on differentiating GHD and ISS. When the
groups undergoing L-dopa or clonidine as the first stimulation test were
compared, the rate of confirmation the GHD diagnosis of the second test
in the group with a growth hormone level of \textless{}10 ng / mL
detected with clonidine was significantly higher than in the group in
which L-dopa was employed as the first test. However, the clonidine test
exhibited greater sensitivity than the L-dopa test, while specificity
levels were similar between the two. The results of this study show that
the clonidine stimulation test is superior to the L-dopa test in
determining GHD.

In conclusion, growth hormone stimulation tests performed for the
purpose of determining GHD are laborious and time-consuming.
Side-effects also give rise to anxiety in children and their families.
The first test to be employed for differentiating GHD from ISS must
therefore be carefully selected. The application of the clonidine
stimulation test, which has higher sensitivity then and similar
specificity to the L-dopa test in demonstrating GHD, as the first test
may provide a number of advantages for both the child, the family and
the clinician.

DISCLOSURES

The authors declare they have no conflict of interest

AUTHOR CONTRIBUTIONS

Semih Bolu---Study conception and design, Collection of data, analysis
and interpretation of results, drafting the manuscript, study conception
and design, Collection of data, analysis and interpretation of results,
drafting the manuscript, critical revision of manuscript.

Abdulvahit A\selectlanguage{polish}şı\selectlanguage{english}k---Collection of data, study conception and design,
drafting of manuscript, analysis and interpretation of data.

DATA AVAILABILITY STATEMENT

The datasets generated during and/or analysed during the current study
are available from the corresponding author on reasonable request.

ORCID

Semih Bolu: 0000-0002-8183-2188

\textbf{REFERENCES}

\begin{enumerate}
\tightlist
\item
  Esen \selectlanguage{polish}İ, \selectlanguage{english}Demirel F, Tepe D, Demir B. Character\selectlanguage{polish}ı\selectlanguage{english}st\selectlanguage{polish}ı\selectlanguage{english}cs Of Pat\selectlanguage{polish}ı\selectlanguage{english}ents Who
  Referred To A Ped\selectlanguage{polish}ı\selectlanguage{english}atr\selectlanguage{polish}ı\selectlanguage{english}c Endocr\selectlanguage{polish}ı\selectlanguage{english}nology Outpat\selectlanguage{polish}ı\selectlanguage{english}ent Cl\selectlanguage{polish}ı\selectlanguage{english}n\selectlanguage{polish}ı\selectlanguage{english}c. Turkish J.
  Pediatr. Dis. 2011;5:133-8
\item
  Ranke MB. Towards a consensus on the definition of idiopathic short
  stature. Horm Res 1996;45:64-6.
\item
  Wit JM, Ranke MB, Kelnar CJH. ESPE classification of paediatric
  endocrine diagnosis. Horm Res 2007;68:1-120
\item
  Murray PG, Dattani MT, Clayton PE. Controversies in the diagnosis and
  management of growth hormone deficiency in childhood and
  adolescence.~Arch Dis Child.~2016;101(1):96-100.
\item
  Antoniazzi F, Cavarzere P, Gaudino R. Growth hormone and early
  treatment.~Minerva Endocrinol.~2015;40:129-143
\item
  Ghigo E, Bellone J, Aimaretti G, et al. Reliability of provocative
  tests to assess growth hormone secretory status. Study in 472 normally
  growing children.~J Clin Endocrinol Metab.~1996;81:3323-3327
\item
  Tanner JM, Whitehouse RH. Clinical longitudinal standards for height,
  weight, height velocity, weight velocity, and stages of puberty. Arch
  Dis Child 1976;51:170-79
\item
  Hilczer M, Smyczynska J, Lewinski A. Limitations of clinical utility
  of growth hormone stimulating tests in diagnosing children with short
  stature. Endocr Regul 2006;40:69-75
\item
  Grimberg A, Lifshitz F. Worrisome growth. In: Lifshitz F, ed.
  Pediatric Endocrinology. 5th ed. New York: Infroma Heathcare USA;
  2007:1-50
\item
  Hussein A, Farghaly H, Askar E, et al. Etiological factors of short
  stature in children and adolescents: experience at a tertiary care
  hospital in Egypt. Ther Adv Endocrinol Metab. 2017;8(5):75-80
\item
  Growth Hormone Research S. Consensus guidelines for the diagnosis and
  treatment of growth hormone (GH) deficiency in childhood and
  adolescence: summary statement of the GH Research Society. GH Research
  Society. J Clin Endocrinol Metab 2000;85:3990-3
\item
  Hanew K, Utsumi A. The role of endogenous GHRH in arginine-, insulin-,
  clonidine- and l-dopa-induced GH release in normal subjects.~Eur J
  Endocrinol.~2002;146:197-202.
\item
  Biller BM, Samuels MH, Zagar A, et al. Sensitivity and specificity of
  six tests for the diagnosis of adult GH deficiency.~J Clin Endocrinol
  Metab.\emph{~} 2002;87:2067-2079
\item
  Shalet SM, Toogood A, Rahim A, Brennan BM. The diagnosis of growth
  hormone deficiency in children and adults.~Endocr Rev.\emph{~}
  1998;19:203-223
\item
  Moon JS, Lee SY, Nam CM, et al. 2007 Korean National Growth Charts:
  review of developmental process and an outlook.~Korean J
  Pediatr.~2008;51:1-25
\item
  Richmond EJ, Rogol AD. Growth hormone deficiency in children.
  Pituitary 2008;11:115-120
\item
  Lanes R, Recker B, Fort P, Lifshitz F. Low-dose oral clonidine. A
  simple and reliable growth hormone screening test for children.
  American Journal of Diseases of Children 1985;139:87-88.
\item
  Al-Ruhaily AD, Malabu UH. Growth hormon deficiency sort stature in a
  third world adult endocrine clinic: usefulness of clonidine test in
  its diagnosis Indian J Med Sci. 2008;62(4):149-56.
\item
  Collu R, Leboeuf G, Letarte J, Ducharme JR. Stimulation of Growth
  Hormone Secretion by Levodopa-Peopranolol in Children and Adolescents.
  Pediatrics 1975;56:262-266
\item
  Petriczko E, Szmit-Domagalska J, Horodnicka-J\selectlanguage{ngerman}ózwa A, Walczak M.
  Prognostic value of the L-Dopa and clonidine GH stimulation tests in
  patients with endogenous growth hormone deficiency - retrospective
  analysis after discontinuation of treatment - own experience. Pediatr
  Endocrinol Diabetes Metab. 2009;15(1):13-9.
\end{enumerate}

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