| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Diagnostic Systems Laboratories (Canada), Inc. (M.J.K., A.D.), and the Department of Clinical Biochemistry, University of Toronto (M.J.K.), Toronto, Ontario, Canada; and Diagnostic Systems Laboratories, Inc. (J.M., R.G.K., A.K.), Webster, Texas 77598
Address all correspondence and requests for reprints to: M. J. Khosravi, Ph.D., Diagnostic Systems Laboratories (Canada), Inc., Mount Sinai Hospital, Room 653, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
150-kDa insulin-like
growth factor (IGF)-binding protein (IGFBP) complex was described over
a decade ago, details of ALS physiology have remained largely
uncertain. We evaluated antibodies to synthetic human ALS and
constructed a noncompetitive ALS enzyme-linked immunosorbent assay.
Whereas uncomplexed ALS is directly measured, determination of total
levels required sample pretreatment with SDS, which was found to
optimally dissociate complexed ALS and significantly enhance ALS
immunoreactivity. ALS in random adult sera was approximately 50%
uncomplexed, and samples devoid of complexed ALS by immunoaffinity
separation contained about 54% of the total levels. Serum ALS
fractionated by gel filtration high performance liquid chromatography
eluted in a single peak at approximately 150 kDa with IGF-I and
IGFBP-3, but appeared at about 400–500 kDa after sample acidification
and fractionation under acidic condition. The unexpected shift in ALS
immunoreactivity remained unchanged when acid-neutralized or
SDS-treated samples were fractionated under neutral pH and was
reproducible when the 150-kDa complex was isolated, treated with acid
or SDS, and rechromatographed. ALS in adult sera more tightly
correlated with IGFBP-3 than IGF-I or IGF-II. The total levels
(mean ± SD) were 16.7 ± 3.7 mg/L in 22 normal
subjects, 28.3 ± 8.1 mg/L in 20 acromegalic patients, and
9.5 ± 3.8 in 32 GH-deficient adults. Little or no ALS was
detectable in amniotic fluid, cerebrospinal fluid, seminal plasma, or
milk, whereas high levels were present in synovial fluid. The
development of ALS enzyme-linked immunosorbent assay should
greatly facilitate further investigations of this unique glycoprotein. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In human circulation, IGFs are mostly confined to an approximately
150-kDa GH-dependent ternary protein complex consisting of IGFBP-3, a
molecule of IGF-I or IGF-II, and a unique acid-labile subunit (ALS)
(4, 5, 6, 7, 8, 9). ALS was first described by the observation that acidification
would result in dissociation of the 150-kDa complex into acid-stable
IGF (7.5 kDa) and IGFBP-3 (50 kDa) components, whereas
neutralization of the acidified sample would no longer support
formation of the complex (11, 12). The existence of ALS was later
confirmed by demonstrating that serum-derived ALS could readily convert
binary IGF/IGFBP-3 complexes to the 150-kDa form and that IGFBP-3
occupancy by IGF-I or IGF-II was a prerequisite for the complex
assembly (13, 14). The ALS-dependent increase in the molecular size of
the binary complexes appears to be an important regulator of IGF and/or
IGFBP-3 access to extravascular compartments. However, ALS is
reportedly capable of IGF-independent interaction with IGFBP-3 and may
have the ability to increase the binding capacity of IGFBP-3 for the
IGF peptide (15).
Previous reports have shown that ALS is a glycoprotein that contains 578 amino acid residues and has a molecular mass of 63.3 kDa. It is produced by the liver, and the native molecule appears in serum as a 84/86-kDa glycoprotein doublet (13, 14, 16, 17, 18, 19). The midregion of ALS is largely composed of 18–20 leucine-rich repeats of 24 amino acids. These repeat sequences are also common in a wide variety of proteins that, like ALS, are involved in protein-protein interaction (18). Unique ALS-specific sequences are expressed in the amino- and carboxyl-terminal regions of the molecule (13, 18).
Despite significant progress in biochemical characterization of ALS (13, 14, 16, 17, 18, 19), details of ALS physiology and its potential diagnostic and clinical relevance have remained uncertain. This has been largely due to the unavailability of simple and reliable ALS methods. As ALS circulates in both complexed and uncomplexed (free) forms, methods capable of independent analysis of its subfractions may facilitate investigations in both research and clinical laboratories. Although free ALS levels may be intimately involved in the regulation of ALS physiology, determination of its total levels, including the IGFBP-3-bound ALS subfraction, may prove to be a better diagnostic indicator. Finally, the ability to quantify changes in the ratio of ALS subfractions may allow a more precise definition of physiological ALS and a better determination of its potential diagnostic value.
We here report the development of highly specific and rapid enzyme-linked immunosorbent assays (ELISAs) for direct measurement of free and total ALS levels. The assays incorporates site-specific antibodies raised against unique N- and C-terminal sequences of human ALS. The availability of these assays was instrumental in the development of novel procedures for dissociation of ALS from the 150-kDa ternary complex, further molecular characterization of ALS by gel filtration chromatography, and direct comparison of free and total ALS levels. Preliminary evaluation of ALS in various biological fluids and direct comparison of serum ALS with IGF-I, IGF-II, and IGFBP-3 in normal subjects and in patients with GH deficiency or acromegaly are also presented.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Adult serum samples from patients with GH deficiency (17 women, aged 21–60 yr; 15 men, aged 21–60 yr), patients with acromegaly (10 women, aged 29–75 yr; 10 men, aged 35–66 yr), and normal subjects (4 women, aged 54–66 yr; 4 men, aged 49–66 yr) were provided by Dr. C. Camacho-Hubner (St. Bartholomew’s Hospital, London, UK). Additional serum samples from normal adults (7 women, aged 23–38 yr; 7 men, aged 32–39 yr) were obtained from Diagnostic Systems Laboratories (Webster, TX). Randomly selected serum samples, synovial fluids (SF), and cerebrospinal fluids (CSF) were obtained from the clinical laboratories at Mount Sinai Hospital (Toronto, Canada). The samples were residuals from routine clinical test samples and were from an adult population. Upon collection, blood samples were allowed to clot, then were separated; after clinical testing, the residuals were used for these studies within 48 h after collection. SF and CSF were visually clear of potential blood contamination and were stored at -20 C for less than 4 weeks until use. Amniotic fluid from pregnancies at 14–17 weeks gestation and seminal plasma were provided by Dr. E. P. Diamandis (Mount Sinai Hospital). Human milk was obtained from consenting donors.
Recombinant human IGF-I and IGF-II were obtained from GroPep (Adelaide, Australia), and recombinant nonglycosylated IGFBP-3 was obtained from Celtrix Pharmaceutical (Santa Clara, CA). Recombinant human IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6 were purchased from Austral Biologicals (San Roman, CA). IGFBP-1, purified from human amniotic fluid and calibrated against pure recombinant human IGFBP-1, was obtained from Diagnostic Systems Laboratories. Other materials and chemicals were obtained as previously described (20, 21).
Purified human ALS was obtained from Diagnostic Systems Laboratories.
The purification procedure was similar to a previously reported method
(14), except that human plasma was first precipitated with 15–45%
ammonium sulfate, and after dialyzing the precipitate against 0.05
mol/L NaPO4, pH 7.2, the solution was chromatographed on an
anti-IGFBP-3 affinity column as described below. The ALS component of
the complex eluted in 0.05 mol/L Tris-HCl, pH 8.5, containing 0.6 mol/L
NaCl and ALS positive fractions further purified by ion exchange
chromatography on a Fast Flow Q-Sepharose column (Sigma Chemical Co.,
St. Louis, MO) using a linear 0.2–0.5 mol/L NaCl salt gradient. The
physical purity was established by SDS-PAGE, followed by Coomassie blue
staining and densitometric comparison of the stained band with
different concentrations of BSA, which was similarly treated and
subjected to SDS-PAGE. The preparation showed a major doublet
corresponding to ALS and several minor bands. As judged by gel
densitometry, the preparation appeared as 70% ALS. The purified
preparation, stored frozen at -20 C in 0.5 mL aliquots, was initially
used for ALS standard and control calibration. The stock standards and
controls were subsequently calibrated against a reference preparation
of human serum-derived ALS provided by Dr. R. C. Baxter (New South
Wales, Australia). The immunoreactivity of the new preparation was
lower by 4.8-fold relative to that of the preparation developed by
D. R. Baxter. Both preparations showed closely parallel responses
to serial dilution (data not shown).
Goat antisera to N- and C-terminal regions of ALS were obtained from Diagnostic Systems Laboratories. The antisera were raised against synthetic human ALS peptides ALS-(1–34) and ALS-(551–578), and purified by affinity chromatography. The antibodies specifically detected an ALS doublet of approximately 84 kDa in serum by Western immunoblot analysis (data not shown). Development and characterization of a similar antiserum raised in rabbits to ALS-(1–34) have been recently reported (22).
Total and free ALS ELISA
The principal difference between the two methods is inclusion of SDS in the total ALS ELISA. Both methods incorporate identical components, except for the following. The total ALS ELISA sample pretreatment buffer was 0.05 mol/L sodium borate (pH 8.5), 9 g/L NaCl, 10 g/L BSA, 50 mL/L normal goat serum, 25 mL/L normal mouse serum, 0.5 mL/L Tween-20, 1.0 g/L SDS, and 0.1 g/L thimerosal; the standard matrix buffer and the assay buffer were the same as described above, except that the latter contained 0.25 g SDS/L. The free ALS ELISA assay buffer was 0.05 mol/L Tris-maleate (pH 7.0), 9 g/L NaCl, 20 g/L BSA, 0.5 g/L bovine globulin, 25 mL/L normal goat serum, 25 mL/L normal mouse serum, 0.5 mL Tween-20, and 0.1 g/L thimerosal; the standard matrix buffer was 0.05 mol/L Tris-maleate (pH 7.0), 0.9 g/L NaCl, 60 g/L BSA, 0.05% Tween-20, and 0.1 g/L thimerosal. Other buffers were previously described (20, 21).
Antibody coating to microtiter wells was performed at a concentration of 2.5–30 mg/L using previously described methods (20). Conjugation of antibody to horseradish peroxidase (HRP) was performed as recently described (21). Standards were prepared by diluting calibrated stock ALS in the appropriate standard matrix buffers to give ALS standards of approximately 0.0075, 0.030, 0.120, 0.240, and 0.480 mg/L (0.75–48.5 mg/L after correction for the sample pretreatment dilution factor of 101-fold), or about 1.09, 4.38, 17.5, 35, and 70 mg/L for the total and free ALS ELISAs, respectively. The total ALS standards are lower because ALS immunoreactivity was enhanced by more than 200-fold after sample pretreatment, and all samples required at least 100-fold dilution to bring them within the measuring range of the assay. Standards were stable for up to 4 days at 4 C and for more than 4 months at -20 C or lower. The quality control samples used in the free ALS ELISA were fresh serum samples, whereas evaluation of the total ALS ELISA involved SDS-treated control sera.
The total ALS ELISA was performed according to previously described procedures (23, 24). Standards or treated samples (0.02 mL after 101-fold dilution, as described bellow) were added in duplicate to precoated wells, followed by addition of the total ALS assay buffer (0.1 mL) and 1-h room temperature incubation with continuous shaking. The wells were washed four times and incubated with 0.1 mL/well of the anti-ALS antibody-HRP conjugate (diluted in the assay buffer to approximately 0.1–0.25 mg/L) for 30 min as described above. The wells were washed, 0.1 mL TMB/H2O2 substrate solution was added, and color development during a 10-min incubation was quantified by dual wavelength measurement at 450 nm with background wavelength correction set at 620 nm. In the free ALS ELISA, standards or untreated serum samples (0.05 mL) were added in duplicate to the precoated wells, followed by addition of the free ALS assay buffer (0.05 mL) and completion of the assay as described above.
ALS ELISA validation procedures
For validation of total and free ALS ELISAs, SDS-treated or untreated serum samples were used, respectively. In both assays, the lower limit of detection (sensitivity) was determined by interpolating the mean plus 2SD of 12 replicate measurements of the zero standard. The intraassay coefficients of variation (CVs) were determined by replicate analysis (n = 12) of 3 samples at ALS levels of 0.048–0.29 mg/L (in total ALS ELISA, 4.8–29.2 mg/L after correction for the dilution factor) and 2.29–12.8 mg/L (in free ALS ELISA) in 1 run; interassay CVs were determined by duplicate measurement of appropriate samples in 8–12 separate runs. The free ALS ELISA recovery was assessed by adding 50 µL ALS in acid-neutralized samples (final treatment dilution, 10-fold) to 450 µL freshly drawn, undiluted serum, followed by analysis. The recovery of the total ALS ELISA was similarly evaluated by adding 50 µL ALS in SDS-treated samples (final treatment dilution, 10-fold) to 450-µL serum samples that had been treated with SDS at the recommended 101-fold dilution as described below. Recovery was determined by comparison of added ALS to the amount measured after subtracting the endogenous levels. Linearity was tested by analyzing serum samples serially diluted (2- to 16-fold) in the corresponding zero standard buffer.
Other assays
IGF-I, IGF-II, and IGFBP-3 were analyzed by immunoassay kits manufactured and marketed by Diagnostic Systems Laboratories. These assays are based on noncompetitive ELISA involving a solid phase capture antibody and a soluble HRP-labeled detection antibody. The performance characteristics of theses assays have been recently described (23, 25, 26). Optical density measurements for all ELISAs were performed with the Labsystems Multiskan Multisoft microplate reader (Labsystems, Helsinki, Finland).
Sample pretreatment procedures for measuring total ALS
Acid-neutralization treatment. Acid treatment of sample followed by neutralization (acid-neutralization) was performed by a modification of a previously described method (23). The basic procedure involves the addition of 0.01 mL serum to 0.5 mL acidification buffer (0.2 mol/L glycine-HCl, pH 2.0) in a 5-mL glass tube, incubation at room temperature for 30 min, and subsequent addition of 0.5 mL neutralization solution (0.85 mol/L Tris base containing 0.05% SDS). The final sample dilution factor is 101-fold.
SDS treatment. The alternative procedure is based on sample treatment with an optimized concentration of SDS dissolved in the total ALS sample pretreatment buffer. The optimized procedure involves addition of 0.01 mL serum to 1.0 mL sample pretreatment buffer and incubation at room temperature for 30 min. The final sample dilution factor is 101-fold.
Molecular sieve chromatography
To study distribution of ALS immunoreactivity, aliquots of fresh
serum samples (0.2–0.5 mL) were subjected to size-exclusion
chromatography on a precalibrated 600 x 7.5-mm TSK-Gel G-3000 SW
high performance liquid chromatography (HPLC) column (Tosohaas,
Montgomeryville, PA) as previously described (21). The column was
preequilibrated and eluted with 0.05 mol/L Tris-HCl (pH 7.2) and 9.0
g/L NaCl at 0.5 mL/min, with collection of 0.5-mL fractions. Gel
filtration molecular mass markers (Bio-Rad Laboratories, Richmond, CA)
included thyroglobulin (60 kDa), 
-globulin (158 kDa), ovalbumin (44
kDa), myoglobin (17 kDa), and vitamin B12 (1.4 kDa). The ALS
immunoreactivity of the fractionated serum was evaluated by the free
ALS ELISA. IGFBP-3 immunoreactivity was measured with IGFBP-3 ELISA. In
the same fractions, total ALS immunoreactivity was measured after
aliquot of each fraction was acid-neutralized [i.e. 1:4
(vol/vol) 0.2 mol/L glycine, pH 2.0, 30 min; then neutralized with 1:1
(vol/vol) 0.85 mol/L Tris base; final dilution factor, 8]. Where
indicated, IGF-I in the acid-neutralized samples was measured using
IGF-I ELISA.
To determine the potential effect of various treatments on molecular distribution of ALS immunoreactivity, aliquots of pretreated serum samples (0.2 or 0.5 mL) were fractionated on size-exclusion HPLC as described. Acid-neutralization was performed using similar treatment ratios as those described above, with an 8-fold final sample dilution. SDS treatment was performed by mixing samples with the total ALS sample pretreatment buffer [1:8 (vol/vol), 30 min; final dilution factor, 8]. Where indicated, detergents such as Tween-20, Triton X-100 (1.0 or 2.5 mL/L), or SDS (1.0 mL/L) were also added to the neutralization buffer. Fractions were analyzed for total ALS, IGFBP-3, and IGF-I.
Acid-gel filtration chromatography
Size-exclusion chromatography of acidified serum samples was performed using a Bio-Sil SEC-250 HPLC column (600 x 7.5, Bio-Rad) as described (23). Briefly, the column was preequilibrated and eluted with 1.0 mol/L acetic acid containing 0.1 mol/L NaCl. Sample (0.1 mL) was preincubated with 0.4 mL 1.25 mol/L acetic acid containing 0.125 mol/L NaCl at room temperature for 15 min. The column was loaded with 0.2 mL of the acidified sample and eluted at 0.8 mL/min. Each fraction was immediately neutralized with 0.8 mL 1.25 mol/L Tris base as described. Fractions were then assayed for total ALS, IGFBP-3, IGF-I, and IGF-II.
Affinity chromatography on anti-IGFBP-3 column
A polyclonal anti-IGFBP-3 antibody (5 mg; Diagnostic Systems
Laboratories) was coupled to approximately 3 g cyanogen
bromide-activated Sepharose-4B (Pharmacia, Piscataway, NJ) according to
the manufacturer’s instructions and packed into a minicolumn
(0.6 x 3-cm packed bed volume; 2-mL void volume). More than
90% of the antibody was coupled, as determined by absorbance
monitoring at 280 nm. Fresh serum samples (1.0–2 mL; n = 7)
gently layered onto the column at 4 C and effluent fractions containing
free ALS were collected at approximately 0.05 mL/min and assayed for
ALS, IGFBP-3, and absorbance at 280 nm. In all applications, IGFBP-3
analysis, used to monitor column efficiency in removing the 150-kDa
complex, showed little or no detectable IGFBP-3 in the effluent
fractions, and absorbance at 280 was generally less than 0.05 after
washing the column with 4 column vol buffer (0.05 mol/L
NaPO4, pH 6.5); washing was generally performed with 6–8
column vol. The column was regenerated by sequential washing with 4–6
column vol 0.2 mol/L glycine-HCl, pH 2.0, and 6–8 column vol phosphate
buffer before use or storage at 4 C.
Data analysis
ELISA data were analyzed using data reduction packages included in the Labsystems microplate reader with cubic spline (smoothed) curve fit. Other statistical analyses were performed using the Statworks statistical software package (Starlight Network, Mountain View, CA) on an Apple Macintosh SE computer. Descriptive data are presented as the mean and SD unless otherwise specified. Linear regression analysis was performed by the least squares method, and correlation coefficients were determined by the Pearson method.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An ELISA was developed using a two-step (sequential)
noncompetitive immunoreaction format in which the anti-ALS-(1–34) and
ALS-(551–578) antibodies were used for detection and coating,
respectively. The optimized protocols were established by evaluating
effects of various technical manipulations on the analytical
performance of the assay, particularly the detection limit, dynamic
range, precision and delayed sample addition (21, 23). The addition of
IGF-I (up to 300 µg/L); IGF-II (up to 3000 µg/L); IGFBP-1,
IGFBP-2, and IGFBP-4–6 (up to 500 µg/L); and IGFBP-3 (up to 4.2
mg/L) to the zero standard or a standard preparation of approximately
0.14 mg/L ALS did not show any cross-reactivity or interferences. The
total and free ALS ELISA validation data are summarized in Table 1
.
|
As an alternative to acid-neutralization, the effects of
variations in pH, ionic strength, and detergents on ALS
immunoreactivity were examined. As shown in Table 2, serum ALS remained relatively constant
when analyzed untreated or after 2-fold dilutions in various buffers,
pH 6.5–9.5. Increases of up to about 3-fold in ALS levels were
observed for samples diluted 10-fold in alkaline buffers (pH 
8.5) in
the absence or presence of a relatively high salt concentration (1.0
mol/L). The addition of SDS was highly effective in enhancing ALS
immunoreactivity. ALS immunoreactivity increased by more than 200-fold
after sample predilution in buffers containing 1 g/L SDS (Table 2). The
SDS effect was concentration dependent and appeared most effective at
alkaline pH, reaching a plateau within about 30 min of incubation (data
not shown). Compared with acid-neutralization, the enhancing effect of
SDS on ALS immunoreactivity was highly independent of the dilution
ratios introduced by the sample pretreatment method. The mean ALS
responses (absorbance ± SD) obtained for a 40-fold
diluted serum sample after the initial 1:10, 1:20, or 1:40 sample
pretreatment dilution ratios were 1.17 ± 0.08, 1.30 ± 0.05,
and 1.92 ± 0.04 U for the acid-neutralized samples and 1.90
± 0.05, 1.95 ± 0.09, and 1.95 ± 0.08 U for the SDS-treated
samples, respectively. However, under optimal conditions, described in
Materials and Methods, total ALS immunoreactivity measured
in acid-neutralized (y) and SDS-treated (x)
samples showed excellent correlation: y = -0.82 +
0.91x (r = 0.97), Sy,x = 0.035
(P < 0.001; n = 42).
|
Randomly selected serum samples from an adult population (n =
41) were assayed. Regression analysis showed a high correlation between
total and free ALS levels and indicated that uncomplexed ALS may, on
the average, constitute approximately 50% of the total levels (Fig. 1); the total and free ALS levels
(mean ± SD) were 13.0 ± 4.4 and 6.7 ± 4.0
mg/L, respectively. For confirmation, ALS was measured in selected
serum samples (n = 7) before and after removal of the 150-kDa
complex by anti-IGFBP-3 immunoaffinity chromatography. As shown in
Table 3, the effluent fractions contained
less than 0.5% of the immunoreactive IGFBP-3, but about 54% of the
total ALS immunoreactivity; the total and free ALS levels measured in
this manner were 19.7 ± 4.9 and 10.7 ± 2.5 mg/L,
respectively. The finding of comparable free ALS levels in the whole
sera (12.0 ± 2.2 mg/L) and their corresponding effluent fractions
(13.6 ± 2.9) is consistent with the inaccessibility of complexed
ALS for measurement by the free ALS ELISA. The apparent increase in
free ALS in the effluent fractions by approximately 14% is most likely
due to increased ALS immunoreactivity (accessibility) in response to
dilution as described (Table 2
).
|
|
To evaluate the molecular profile of immunoreactivity detected,
fresh serum samples (n = 5) were fractionated by size-exclusion
HPLC. As represented in Fig. 2, IGFBP-3
eluted in a single sharp peak at about 150 kDa, whereas ALS
immunoreactivity was completely undetectable. The latter is primarily
due to the inaccessibility of the complexed form of ALS for antibody
binding as well as the considerably lower immunoreactivity of
uncomplexed ALS (Table 2). When an aliquot of each fraction was
acid-neutralized and reassayed, a single peak of ALS immunoreactivity
appeared within that of the IGFBP-3 profile, indicating nearly
identical gel filtration migration for both complexed and uncomplexed
ALS. The IGF-I component of the ternary complex peaked in the same
fractions.
|
). The unexpected shift in the ALS
filtration profile did not change when acidified samples were
neutralized, incubated for 2 h at room temperature, and
fractionated under neutral conditions (Fig. 4). Addition of detergents such as
Tween-20 (1 mL/L), Triton X-100 (1 mL/L), or SDS (1 g/L) to the
neutralization buffer had no effect on the ALS filtration profile (data
not shown). ALS behaved in the manner described above when SDS-treated
samples were fractionated. In both SDS-treated and acid-neutralized
samples, IGFBP-3 peaks appeared at about 40 kDa (Fig. 4).
|
|
(fraction 31–36; pooled and
concentrated 7-fold) were acid-neutralized or SDS-treated and then
refractionated by HPLC, ALS and IGFBP-3 components of the complex
peaked at the same positions as those shown in Fig. 4 for the treated
whole serum. ALS and IGFBP-3 eluted at about 400–500 and 40 kDa,
respectively (data not shown). Relationship of ALS with IGF-I, IGF-II, and IGFBP-3
Randomly selected serum samples (n = 38) were assayed for
total ALS, IGF-I, IGF-II, and IGFBP-3. Regression analysis of data
showed a high degree of correlation between ALS and IGFBP-3 (Fig. 5). Significant correlations were also
obtained between ALS and IGF-I or IGF-II levels (Fig. 6). Interrun comparison of ALS values was
excellent (Fig. 5).
|
|
Clinical assessment
Serum samples from adults with GH deficiency (n = 32),
patients with acromegaly (n = 20), and age-matched normal subjects
(n = 22) were simultaneously analyzed for IGF-I, IGFBP-3, and
total or free ALS. As shown in Fig. 7, the total ALS immunoreactivity patterns obtained for the various sample
groups were similar to those detected for IGF-I and IGFBP-3. Compared
to those in the normal subjects (16.7 ± 3.7 mg/L), the total
levels were generally higher in acromegaly (28.3 ± 8.1 mg/L) and
lower in GH deficiency (9.5 ± 3.8 mg/L). A similar picture
emerged when samples were assayed for free ALS (Fig. 7), although there
appeared to be relatively more overlaps among the three sample groups.
The mean ± SD for acromegalic, normal, and
GH-deficient subjects were 10.6 ± 3.6, 6.9 ± 2.1, and
3.6 ± 2.0 mg/L, respectively.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although the existence of ALS has been known for over a decade (11, 12), methods for direct quantification of ALS have not been fully explored. Approaches to ALS immunoassay may have been complicated by antibody cross-reactivity due to ALS midregion homology with other serum proteins (18) or by poor detectability of ALS in its native conformations, as demonstrated in the present report. The fact that circulating ALS is expressed in both complexed and uncomplexed forms with inherently different degrees of immunoreactivity may have further complicated ALS analysis. Previous reports have shown that active ALS molecules could be quantified by its ability to bind and convert binary IGFBP-3/IGF complexes to the 150-kDa complex or by RIA before or after chromatographic removal of the ternary complex (13, 14, 16, 17). Although differential antibody recognition of the complexed vs. uncomplexed ALS has not been investigated, chromatographic separation of the two forms of ALS appears mandatory for determination of their relative concentrations by RIA (17). The present alternative approach is based on sample pretreatment with procedures that would result in unfolding of both complexed and uncomplexed ALS, thus allowing direct measurement of its total levels. Because the assay does not recognize the complexed form of ALS, direct determination of uncomplexed ALS by the free ALS ELISA is also possible.
We here demonstrated that the immunoreactivity of serum ALS is enhanced more than 200-fold in response to SDS treatment. This suggests exposure of epitopes that are not readily accessible to antibody binding, particularly when ALS is associated with the ternary complex. This observation may have important bearings on ALS determinations in untreated serum samples, as measured concentrations may be influenced by changes in the three-dimensional presentation and, thus, immunoreactivity of ALS. The significant scattering of values observed in comparison of free and total ALS may be primarily due to variable immunoreactivity of uncomplexed ALS rather than true variations in its absolute levels. Accordingly, procedures for ALS determination should include a sample pretreatment method that would confer consistent immunoreactivity to both uncomplexed as well as complexed ALS, unless equivalent and consistent immunoreactivity of the two forms could be demonstrated.
The novel SDS-based sample pretreatment method appeared highly efficient in enhancing ALS immunoreactivity, and its effect was relatively independent of variations in the initial sample pretreatment dilution ratios. Direct determinations of ALS by the total and free ALS ELISAs indicated that, on the average, approximately 50% of serum ALS is uncomplexed. This was further supported by the finding that serum samples devoid of the 150-kDa complex by immunoaffinity chromatography contained approximately 54% of the total ALS immunoreactivity. Collectively, our results support the existence of excess amounts of uncomplexed ALS in circulation, but demonstrate the level to be somewhat higher than about 30% of the total levels previously reported (17). Variations in assay design and specificity for the complexed and uncomplexed ALS as well as expected differences in sample population may be among the contributing factors. These variations may well be responsible for the differences in total ALS measured by different methods. The mean serum ALS in randomly selected adults by the present method was 13.0 ± 4.4 mg/L, whereas mean RIA value for a group of normal adults was 24.2 ± mg/L.
ALS measured by the present method eluted in a sharp peak at about 150 kDa in association with IGFBP-3 and IGF-I. The fact that similar to previous observations (17), both forms of ALS exhibited identical gel filtration profiles, appearing in a single superimposable peak with that of the 150-kDa complex, clearly indicates the inadequacy of size exclusion chromatography as a means of ALS separation. Surprisingly, acidified or SDS-treated ALS migrated with a molecular mass of about 400–500 kDa. The unexpected shift in ALS immunoreactivity was not altered after neutralization of acidified samples or addition of substances in the neutralization buffer that could potentially affect protein solubility and, thus, chromatographic migration. Indeed, migration at approximately 400–500 kDa was characteristic of functionally inactive ALS molecules, as identical gel filtration profiles were obtained when isolated 150-kDa ternary complex was subjected to acid-neutralization or SDS treatment and rechromatographed. Whether the observed increase in the apparent molecular size of ALS is due to unfolding and linearalization of the molecule or, less likely, the possibility of ALS aggregation into multimeric forms remains to be investigated. Similar to the effect of acidification (11, 12, 13, 14), SDS-treated ALS was functionally inactive and could not support formation of the 150-kDa complex in reconstitution experiments.
In several experiments, total ALS levels in randomly selected serum samples were more closely related to IGFBP-3 levels than to those of IGF-I or IGF-II. Whether the highly parallel association between ALS and IGFBP-3 is suggestive of coregulation of their production or is in some way reflective of association of all of the circulating IGFBP-3 with ALS through IGF-dependent (17) or independent (15) mechanisms remains to be clarified. The results of the present study confirm the GH dependency of ALS and demonstrated that the total ALS levels in GH deficiency are generally lower than those in normal controls, whereas levels are elevated in acromegalic subjects. Although difficulties in the proper diagnosis of GH deficiency (27) may be partly responsible for the observed overlap in ALS levels, the free ALS ELISA showed a more overlapping classification of the various patient groups. As noted earlier, this may be expected, as significant increases in ALS immunoreactivity and, thus, its apparent concentration could be induced by changes in its native molecular conformation. We postulate that conditions that could minimally alter native conformation of ALS could lead to significant alteration of its immunoreactivity and potentially mask the free ALS ELISA response to true changes in ALS levels. Because of this unfavorable characteristic, the free ALS levels recognized by the present method may not totally reflect the true diagnostic potential of free ALS measurements. In the present study, frozen samples undergoing at least two freeze/thaw cycles were employed. Freeze/thawing of samples may potentially alter ALS conformation and, thus, its apparent immunoreactivity. This is in contrast to the total ALS analysis in which a novel sample pretreatment procedure ensures optimal dissociation and unfolding of ALS. In the preliminary comparison to IGF-I and IGFBP-3, the total ALS analysis appeared at least equally effective in classifying the various sample groups. These findings strongly support the usefulness of total ALS analysis in the clinical assessment of GH abnormalities and indicate the need for further evaluation of its diagnostic potentials.
The findings of relatively low ALS levels in amniotic fluid and seminal plasma are in agreement with previous reports that described either low levels of immunoreactive ALS (17) or undetectable levels of the 150-kDa complex (28, 29) in amniotic fluid and seminal plasma. Consistent with the reported presence of the 150-kDa complex in interstitial (30) and peritoneal (31) fluid, synovial fluid contained relatively high levels of ALS. In contrast, little or no ALS immunoreactivity was detectable in cerebrospinal fluid and human milk. Although the origin and potential role of extravascular ALS have not been investigated, it is most likely derived from vascular sources, where uncomplexed ALS, on the average, constitutes about 50% of the total circulating levels.
In conclusion, we demonstrated the development of a simple ALS ELISA that incorporates site-specific antibodies and a new approach for total ALS analysis. We have shown that circulating ALS is about 50% uncomplexed and appears at more than 400 kDa on gel filtration in response to SDS or acid treatment. Circulating ALS is more tightly associated with IGFBP-3 than IGF-I or IGF-II, and its determinations may be of significant value in the differential diagnosis of GH-related disorders. Except for synovial fluid, most biological fluids evaluated contained little or no ALS immunoreactivity, suggesting the circulation to be the primary source of extravascular ALS. Development of ALS ELISA should facilitate further clinical and research investigations of this unique binding protein.
Received May 19, 1997.
Revised August 13, 1997.
Accepted August 26, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  P. F. Collett-Solberg, M. Misra, and on behalf of the Drug and Therapeutics Committee o The Role of Recombinant Human Insulin-Like Growth Factor-I in Treating Children with Short Stature J. Clin. Endocrinol. Metab., January 1, 2008; 93(1): 10 - 18. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. Domene, P. A. Scaglia, A. Lteif, F. H. Mahmud, S. Kirmani, J. Frystyk, P. Bedecarras, M. Gutierrez, and H. G. Jasper Phenotypic Effects of Null and Haploinsufficiency of Acid-Labile Subunit in a Family with Two Novel IGFALS Gene Mutations J. Clin. Endocrinol. Metab., November 1, 2007; 92(11): 4444 - 4450. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C. Nindl, J. A. Alemany, M. D. Kellogg, J. Rood, S. A. Allison, A. J. Young, and S. J. Montain Utility of circulating IGF-I as a biomarker for assessing body composition changes in men during periods of high physical activity superimposed upon energy and sleep restriction J Appl Physiol, July 1, 2007; 103(1): 340 - 346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M Morrison, M. Bidlingmaier, S. Stadler, Z. Wu, L. Skriver, and C. J Strasburger Sample pre-treatment determines the clinical usefulness of acid-labile subunit immunoassays in the diagnosis of growth hormone deficiency and acromegaly Eur. J. Endocrinol., March 1, 2007; 156(3): 331 - 339. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Pazaitou-Panayiotou, T. Kelesidis, I. Kelesidis, A. Kaprara, J. Blakeman, I. Vainas, A. Mpousoulegas, C. J Williams, and C. Mantzoros Growth hormone-binding protein is directly and IGFBP-3 is inversely associated with risk of female breast cancer Eur. J. Endocrinol., February 1, 2007; 156(2): 187 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Rasmussen, A. Juul, L. L. Kjems, and J. Hilsted Effects of short-term caloric restriction on circulating free IGF-I, acid-labile subunit, IGF-binding proteins (IGFBPs)-1-4, and IGFBPs-1-3 protease activity in obese subjects. Eur. J. Endocrinol., October 1, 2006; 155(4): 575 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Feelders, M. Bidlingmaier, C. J. Strasburger, J. A. M. J. L. Janssen, P. Uitterlinden, L. J. Hofland, S. W. J. Lamberts, A. J. van der Lely, and W. W. de Herder Postoperative Evaluation of Patients with Acromegaly: Clinical Significance and Timing of Oral Glucose Tolerance Testing and Measurement of (Free) Insulin-Like Growth Factor I, Acid-Labile Subunit, and Growth Hormone-Binding Protein Levels J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6480 - 6489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. M. Domene, S. V. Bengolea, A. S. Martinez, M. G. Ropelato, P. Pennisi, P. Scaglia, J. J. Heinrich, and H. G. Jasper Deficiency of the Circulating Insulin-like Growth Factor System Associated with Inactivation of the Acid-Labile Subunit Gene N. Engl. J. Med., February 5, 2004; 350(6): 570 - 577. [Full Text] [PDF]
|
|
|
|
|
|
B. C. Nindl, J. W. Castellani, A. J. Young, J. F. Patton, M. J. Khosravi, A. Diamandi, and S. J. Montain Differential responses of IGF-I molecular complexes to military operational field training J Appl Physiol, September 1, 2003; 95(3): 1083 - 1089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. H. W. Lange, J. L. Andersen, N. Beyer, F. Isaksson, B. Larsson, M. H. Rasmussen, A. Juul, J. Bulow, and M. Kjar GH Administration Changes Myosin Heavy Chain Isoforms in Skeletal Muscle But Does Not Augment Muscle Strength or Hypertrophy, Either Alone or Combined with Resistance Exercise Training in Healthy Elderly Men J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 513 - 523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Marzullo, C. Di Somma, K. L. Pratt, J. Khosravi, A. Diamandis, G. Lombardi, A. Colao, and R. G. Rosenfeld Usefulness of Different Biochemical Markers of the Insulin-Like Growth Factor (IGF) Family in Diagnosing Growth Hormone Excess and Deficiency in Adults J. Clin. Endocrinol. Metab., July 1, 2001; 86(7): 3001 - 3008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Nindl, W. J. Kraemer, J. O. Marx, P. J. Arciero, K. Dohi, M. D. Kellogg, and G. A. Loomis Overnight responses of the circulating IGF-I system after acute, heavy-resistance exercise J Appl Physiol, April 1, 2001; 90(4): 1319 - 1326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Arosio, S. Garrone, P. Bruzzi, G. Faglia, F. Minuto, and A. Barreca Diagnostic Value of the Acid-Labile Subunit in Acromegaly: Evaluation in Comparison with Insulin-Like Growth Factor (IGF) I, and IGF-Binding Protein-1, -2, and -3 J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1091 - 1098. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. Yu and T. Rohan Role of the Insulin-Like Growth Factor Family in Cancer Development and Progression J Natl Cancer Inst, September 20, 2000; 92(18): 1472 - 1489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Diamandi, J. Mistry, R. G. Krishna, and J. Khosravi Immunoassay of Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3): New Means to Quantifying IGFBP-3 Proteolysis J. Clin. Endocrinol. Metab., June 1, 2000; 85(6): 2327 - 2333. [Abstract] [Full Text]
|
|
|
|
|
|
M. E. Wilson Insulin-Like Growth Factor I (IGF-I) Replacement during Growth Hormone Receptor Antagonism Normalizes Serum IGF-Binding Protein-3 and Markers of Bone Formation in Ovariectomized Rhesus Monkeys J. Clin. Endocrinol. Metab., April 1, 2000; 85(4): 1557 - 1562. [Abstract] [Full Text]
|
|
) and ALS (
). IGF-I
(
) and ALS (
) in an acid-neutralized aliquot of each fraction
were also measured. The column was calibrated with gel filtration
molecular mass standards eluting at fractions 23.5 (660 kDa), 31.9 (158
kDa), 37.2 (44 kDa), 44.3 (17 kDa), and 52.6 (1.4 kDa). Values for
IGF-I and IGFBP-3 were divided by 50 to bring them within the graph
scale.
