Endocrinol Japon
1992, 39 (1), 121-127
NOTE
Western Ligand Blot Assay for Human Growth HormoneDependent Insulin-Like Growth Factor Binding Protein (IGFBP-3): The Serum Levels in Patients with Classical Growth Hormone Deficiency YUKIHIROHASEGAWA, TOMONOBUHASEGAWA, TETSUOYOKOYAMA, SHINOBUKOTOH, YUTAKA TSUCHIYA ANDFUMIHIKOKURIMOTO* DivisionofEndocrinologyand Metabolism,TokyoMetropolitan KiyoseChildren'sHospital,Tokyo204, and *MitsubishiYuka Bio-ClinicalLaboratoriesInc., Tokyo174, Japan
Abstract. vitro,
are
binding
The insulin-like known
to
protein
have
and
at
growth least
six
factors
I and II (IGFs), important
binding
is considered
proteins
(IGFBP-1•`6).
growth
In
to be GH-IGF-I-dependent.
human
We have
factors
both in vivo and in
serum,
IGFBP-3
established
is a major
a Western
Ligand
Blot (WLB) assay for IGFBP-3. The method is a densitometric analysis of IGFBP-3 bands on a film WLB. The IGFBP-3 levels of patients with classical growth hormone deficiency (GHD, 5 isolated and multiple hormone deficiencies with appropriate therapy) were studied. Before puberty there is overlap between control (n=31) and the patients with GHD (n=10). However, IGFBP-3 levels of two five pubertal patients with GHD were within the normal range (n=16). We think that measurement serum
IGFBP-3
Key words:
is a useful
Western
diagnostic
ligand
blot,
marker
for
Insulin-like
GHD,
growth
especially
factor
binding
hormone deficiency.
THE
INSULIN-LIKE
growth
factors
I and
II
bound
include
quenced,
degree IGFs many
insulin,
of structural
similarity
and
share
a high
with proinsulin
[1].
have direct effects on the proliferation of tissues and cell types, both in vivo and in vitro
[2]. The IGF-I is considered
the main mediator
of
growth hormone action on somatic growth, and its measurement in plasma and serum plays a di-
agnostic (GHD) In
role and
plasma,
[3] in growth
hormone
deficiency
IGFs
have
BPs,
been
shown
to
be
proteins
IGFBP-1•`6
Pediatric Kiyose
to:
Endocrinology Children's
204, Japan.
Dr.
Yukihiro
size
of 40-44
and Metabolism,
Hospital,
1-3-1
Tokyo
Umezono,
Division
Metropolitan Kiyose,
Tokyo
of
7,
8].
and
Among
(hIGFBP-3)
se-
these
is the
most
kD
and
well
characterized
in im-
munoprecipitation and deglycosylation studies [9, 11]. The IGFBP-3 doublet, which is recognized immunologically [9, 11], is thought to reflect a down
degree to a single
a
reservoir
availability HASEGAWA,
6,
six bind-
cloned
predominant BP in human serum [9, 10]. Upon western ligand blot (WLB), it is a double having a
be
August 7, 1991 December 20, 1991
Correspondence
Growth
[4]. Recently,
[5,
IGFBP-3
of glycosylation, band
lated [11]. Human Received: Accepted:
(IGFBP-3),
(BP) have been
human
different
in acromegaly. the
protein-3
to binding
(IGFs) belong to a family of peptide hormones that and
puberty.
(EndocrinolJapon 39: 121-127, 1992)
ing proteins
relaxin
before
of 10 no of of
IGFBP-3 of
IGFs
of the free
tial tissue [9]. Human GH-IGF-I-dependent
or increased
because
of 29 kD, when
in patients
IGFs
complex and
in plasma
IGFBP-3 because
to
it goes
deglycosy-
is thought regulate and
is reported
to the
intersti-
to be
its level is decreased
with GHD [10, 12, 13, 14]
122
HASEGAWA
and dwarfism with GH insensitivity [15], or acromegaly [12], although we preliminarily reported some overlap between the values for control adults and patients with GHD [10]. In this study, we have established a WLB assay for hIGFBP-3, by using densitometric analysis of hIGFBP-3 bands after WLB, and measured the hIGFBP-3 levels in patients with classical GHD.
et al.
onto
nitrocellulose
proteins
were
IGF-I
for 2h.
incubated
overnight
exposed
at
to
described
film
Densitometric
meter for
Materials
Subjects was
formed
obtained
consent.
in the
Subjects included
adult male volunteers seven healthy children pubertal
and
female,
old,
of
age;
grade
of
and
1.5•`15 obesity;
and
and
5 pubertal;
years,
7
8
5 isolated
hormonal
of classical
and
GHD
III-V, female,
GHD
and
deficiencies). is as follows;
The all
the
peak GH levels are less than 5ng/ml in two or three different GH provocation tests. Other hormonal deficiencies were treated, if any, with appropriate
therapies
glucocorticoid
control
and
such sex
sera for adult
as thyroid
hormone,
hormones,
periods
etc.
(n=6,
was
previously
were scanDensito-
with GHD
for
were
healthy
expressed
of the value of adult pooled D%).
values
controls
and
as a percentage
control
serum
(BP-3-
Statistics All
data
Statistical unpaired
were
expressed
comparisons and paired P
1992, 39 (1), 121-127
NOTE
Western Ligand Blot Assay for Human Growth HormoneDependent Insulin-Like Growth Factor Binding Protein (IGFBP-3): The Serum Levels in Patients with Classical Growth Hormone Deficiency YUKIHIROHASEGAWA, TOMONOBUHASEGAWA, TETSUOYOKOYAMA, SHINOBUKOTOH, YUTAKA TSUCHIYA ANDFUMIHIKOKURIMOTO* DivisionofEndocrinologyand Metabolism,TokyoMetropolitan KiyoseChildren'sHospital,Tokyo204, and *MitsubishiYuka Bio-ClinicalLaboratoriesInc., Tokyo174, Japan
Abstract. vitro,
are
binding
The insulin-like known
to
protein
have
and
at
growth least
six
factors
I and II (IGFs), important
binding
is considered
proteins
(IGFBP-1•`6).
growth
In
to be GH-IGF-I-dependent.
human
We have
factors
both in vivo and in
serum,
IGFBP-3
established
is a major
a Western
Ligand
Blot (WLB) assay for IGFBP-3. The method is a densitometric analysis of IGFBP-3 bands on a film WLB. The IGFBP-3 levels of patients with classical growth hormone deficiency (GHD, 5 isolated and multiple hormone deficiencies with appropriate therapy) were studied. Before puberty there is overlap between control (n=31) and the patients with GHD (n=10). However, IGFBP-3 levels of two five pubertal patients with GHD were within the normal range (n=16). We think that measurement serum
IGFBP-3
Key words:
is a useful
Western
diagnostic
ligand
blot,
marker
for
Insulin-like
GHD,
growth
especially
factor
binding
hormone deficiency.
THE
INSULIN-LIKE
growth
factors
I and
II
bound
include
quenced,
degree IGFs many
insulin,
of structural
similarity
and
share
a high
with proinsulin
[1].
have direct effects on the proliferation of tissues and cell types, both in vivo and in vitro
[2]. The IGF-I is considered
the main mediator
of
growth hormone action on somatic growth, and its measurement in plasma and serum plays a di-
agnostic (GHD) In
role and
plasma,
[3] in growth
hormone
deficiency
IGFs
have
BPs,
been
shown
to
be
proteins
IGFBP-1•`6
Pediatric Kiyose
to:
Endocrinology Children's
204, Japan.
Dr.
Yukihiro
size
of 40-44
and Metabolism,
Hospital,
1-3-1
Tokyo
Umezono,
Division
Metropolitan Kiyose,
Tokyo
of
7,
8].
and
Among
(hIGFBP-3)
se-
these
is the
most
kD
and
well
characterized
in im-
munoprecipitation and deglycosylation studies [9, 11]. The IGFBP-3 doublet, which is recognized immunologically [9, 11], is thought to reflect a down
degree to a single
a
reservoir
availability HASEGAWA,
6,
six bind-
cloned
predominant BP in human serum [9, 10]. Upon western ligand blot (WLB), it is a double having a
be
August 7, 1991 December 20, 1991
Correspondence
Growth
[4]. Recently,
[5,
IGFBP-3
of glycosylation, band
lated [11]. Human Received: Accepted:
(IGFBP-3),
(BP) have been
human
different
in acromegaly. the
protein-3
to binding
(IGFs) belong to a family of peptide hormones that and
puberty.
(EndocrinolJapon 39: 121-127, 1992)
ing proteins
relaxin
before
of 10 no of of
IGFBP-3 of
IGFs
of the free
tial tissue [9]. Human GH-IGF-I-dependent
or increased
because
of 29 kD, when
in patients
IGFs
complex and
in plasma
IGFBP-3 because
to
it goes
deglycosy-
is thought regulate and
is reported
to the
intersti-
to be
its level is decreased
with GHD [10, 12, 13, 14]
122
HASEGAWA
and dwarfism with GH insensitivity [15], or acromegaly [12], although we preliminarily reported some overlap between the values for control adults and patients with GHD [10]. In this study, we have established a WLB assay for hIGFBP-3, by using densitometric analysis of hIGFBP-3 bands after WLB, and measured the hIGFBP-3 levels in patients with classical GHD.
et al.
onto
nitrocellulose
proteins
were
IGF-I
for 2h.
incubated
overnight
exposed
at
to
described
film
Densitometric
meter for
Materials
Subjects was
formed
obtained
consent.
in the
Subjects included
adult male volunteers seven healthy children pubertal
and
female,
old,
of
age;
grade
of
and
1.5•`15 obesity;
and
and
5 pubertal;
years,
7
8
5 isolated
hormonal
of classical
and
GHD
III-V, female,
GHD
and
deficiencies). is as follows;
The all
the
peak GH levels are less than 5ng/ml in two or three different GH provocation tests. Other hormonal deficiencies were treated, if any, with appropriate
therapies
glucocorticoid
control
and
such sex
sera for adult
as thyroid
hormone,
hormones,
periods
etc.
(n=6,
was
previously
were scanDensito-
with GHD
for
were
healthy
expressed
of the value of adult pooled D%).
values
controls
and
as a percentage
control
serum
(BP-3-
Statistics All
data
Statistical unpaired
were
expressed
comparisons and paired P
