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Original Studies |
Institut National de la Santé et de la Recherche Médicale, Unité 515, Hôpital Saint Antoine, Assistance Publique-Hôpitaux de Paris, Université Paris VI, Paris, France
Address correspondence and requests for reprints to: Dr. Michel Binoux, Institut National de la Santé et de la Recherche Médicale, Unité, 515, Hôpital Saint Antoine, 184, rue du Faubourg Saint Antoine, 75571 Paris Cedex 12, France. E-mail: U515{at}st-antoine.inserm.fr
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Abstract |
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IGFBP-bound IGFs are dissociated and separated by acid pH ultrafiltration, and a monoclonal antibody specific to the first 160 amino acids of IGFBP-3 is used to capture hIGFBP-3 in a solid-phase assay. The complex is then incubated with 125I-IGF-I, which binds to intact IGFBP-3 but not to its proteolytic fragments. Binding specificity was demonstrated in competition experiments with unlabeled IGF. Nonspecific binding was 1.4%. The fragments comprising residues 1–160 and 1–95 of recombinant hIGFBP-3 [corresponding to the major proteolytic fragments of approximately 30 kDa and (glycosylated) 20 or (nonglycosylated) 16 kDa detected in serum by Western immunoblotting, respectively] fail to bind 125I-IGF-I when complexed with the monoclonal antibody. Similarly, no binding of 125I-IGF-I was obtained in the LIFA when applied to plasmas from pregnant women during the final 3 months of pregnancy, where the characteristic 42- to 39-kDa doublet of intact IGFBP-3 is undetectable.
The standard curve was established using a pool of plasmas (EDTA) from healthy adults, for which standardization with glycosylated recombinant hIGFBP-3 yielded an intact IGFBP-3 content of 2 µg/mL. The dynamic range of the LIFA was 0.50–3.75 µL equivalent of the plasma pool in a total volume of 300 µL per assay tube, with a sensitivity threshold of approximately 1 ng intact IGFBP-3. Unknown plasma samples were studied at three concentrations. Intra- and interassay variations were 3.6% and 4%, respectively.
In 31 healthy adults, the mean plasma concentration of intact IGFBP-3 was 2.24 ± 0.08 (SEM) mg/L, and that of total IGFBP-3 measured by immunoradiometric assay was 3.27 ± 0.14 mg/L. The calculated mean proportion of proteolysed IGFBP-3 was 29.4 ± 1.9%. In these subjects, a close correlation was found between intact and total IGFBP-3 (r = 0.71, P = 0.0001).
The LIFA for IGFBP-3, therefore, provides accurate and sensitive measurement of intact IGFBP-3, the form with the functional capacity to sequester IGF-I in the bloodstream by association with the acid-labile subunit in 140-kDa complexes. In combination with total IGFBP-3 and IGF-I assays, the LIFA opens new perspectives in investigating the regulation of IGFBP-3 proteolysis and IGF-I bioavailability in man.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This limited proteolysis was first discovered in pregnant women, where virtually all circulating IGFBP-3 is degraded as from the third month of gestation (2, 6). It occurs to a more moderate extent in the normal physiological state and may be more or less marked in pathological conditions where catabolism in enhanced (1, 7). The proteases and their inhibitors involved and their tissual origins remain obscure. The IGFBP-3 protease activity of normal serum, apart from during pregnancy, is discrete (8), but Western immunoblotting of serum IGFBP-3 consistently reveals proteolytic fragments (8, 9) and indicates variations in their relative abundance in different physiological conditions (7).
In serum or culture media conditioned by various cell types, limited
proteolysis of IGFBP-3 generates a major fragment of 30 kDa and smaller
quantities of fragments around 20 and 16 kDa (Refs. 10, 11 ; see Fig. 1
). The 30-kDa fragment, which has only weak affinity for the IGFs and
especially IGF-I (3, 14), corresponds to the first 160
amino acid residues of the intact protein. The 20-kDa fragment arises
from secondary cleavage of the major fragment and corresponds to the
first 95 residues. The 16-kDa fragment, which corresponds to the same
sequence, is the nonglycosylated form of the 20-kDa peptide
(15). These 1–95 fragments have null affinity for IGFs in
Western ligand blotting and in solution assay (14).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human IGF-I and -II (rhIGF-I and -II) were gifts from Ciba-Geigy (Basel, Switzerland). The peptides were labeled with 125I (Amersham Aylesbury, UK) using the chloramine T method and purified by gel filtration. Nonglycosylated rhIGFBP-3 (Escherichia coli) and glycosylated rhIGFBP-3 (CHO) were generous gifts from Celtrix Pharmaceuticals Inc. (Santa Clara, CA).
Limited proteolysis of rhIGFBP-3 (E. coli) by plasmin and isolation of the fragments corresponding to residues 1–160 and 1–95 by reverse-phase chromatography were performed as described previously (14, 15).
The polyclonal anti-hIGF-I antibody used for the IGF-I RIA was a gift from F. Frankenne (Centre Hospitalo-Universitaire, Liège, Belgium). The specific rabbit anti-rhIGFBP-3 (E. coli) antiserum used in Western immunoblotting was raised in our laboratory. The mouse monoclonal anti-hIGFBP-3 antibody [mAb 483.6E8; 2.93 mg/mL phosphate-buffered saline (PBS; pH 7.4)] used in the LIFA for IGFBP-3 was generously provided by Biocode (Sclessin, Belgium).
All other chemical reagents used were of analytical grade purity.
Plasma samples
Plasma samples were obtained from fasting subjects between 0800 and0900 h. Blood was collected in tubes containing an EDTA solution (5 µmol/mL blood), on ice. These were centrifuged at 4C, and plasma samples were stored at -20C.
The subjects, all adults, were: 1) thirty-one healthy volunteers (21 women, none using oral contraceptives, and 10 men) aged 20–39 yr (mean, 29); a plasma pool ("reference plasma") was made up from equal-volume aliquots from 10 of these women and the 10 men; IGF-I levels were all in the normal range (data not shown); 2) twenty-six healthy pregnant women, at 22–39 weeks gestation, from whom samples were taken during routine check-ups in the Obstetrics Department of Saint Antoine Hospital; and 3) untreated patients with idiopathic GH deficiency or acromegaly, whose plasma samples were tested individually for comparison with the reference plasma, in view of the GH dependence of both IGF-I and IGFBP-3 plasma levels.
All blood samples were collected according the the rules of the hospital Ethics Committee, as required by French law.
Dissociation and separation of plasma IGFs and IGFBPs
IGFBP-IGF complexes were dissociated in acid medium, and their components were separated by ultrafiltration as described previously (18). Briefly, 25-µL plasma samples were incubated in 2 mL 0.01 M HCl for 30 min at room temperature, then ultrafiltered on Centricon 30 columns (Amicon, Epernon, France). Following filtration, the columns were washed with 2 mL of the HCl solution to ensure complete passage of ultrafiltered proteins. Close to 100% of 125-I-IGF-I and -II mixed with plasma sample was recovered in the ultrafiltrate. Western ligand blotting and immunoblotting using anti-IGFBP-3 antibody demonstrated that all IGFBPs, including intact IGFBP-3 and its proteolytic fragments, present in the plasma sample remained in the retentate (data not shown).
LIFA for IGFBP-3
The assay was performed in Maxisorp tubes (Nunc, Roskilde, Denmark) coated for 2 days at room temperature with 1.4 µg/tube anti-hIGFBP-3 mAb in 500 µL 0.1 M PBS (pH 7.2) and 1 mg/mL NaN3. After washing, the tubes were saturated overnight at room temperature with 3 mg BSA in 1 mL PBS, then stored at 4C until use for the assay.
For each assay, an aliquot of the reference plasma was subjected to ultrafiltration on Centricon 30 under the same conditions as the unknown sample. An internal standard consisting of a known plasma sample was also run in each assay.
The retentate containing the IGFBPs was taken up in 50 µL 0.1 M acetic acid, then neutralized with 50 µL 0.5 M Tris-HCl (pH 7.4) and made up to 2 mL with 0.1 M phosphate buffer and 1 mg/mL BSA (pH 7.4; PO4BSA). Duplicate aliquots, each corresponding to an initial 3.75 µL plasma, were incubated at three concentrations (four for the reference plasma), dilution factor: 2, in 300 µL (total volume) PO4BSA in the anti-hIGFBP-3 mAb-coated tubes for 3 h at room temperature. Thereafter, the incubation medium was sucked out and the tubes were washed with 1 mL PO4BSA, then incubated with 10,000 cpm 125I-IGF-I in 300 µL PO4BSA for 24 h at 4C. After aspiration of the contents and washing with PO4BSA, the tubes were counted in a gamma spectrometer.
IRMA for IGFBP-3
IRMA was performed using an IGFBP-3 IRMA kit (Immunotech, Marseille, France). The mAb pool used specifically detects IGFBP-3 and its N-terminal proteolytic fragments, as confirmed by Western immunoblotting (data not shown). Each plasma sample was tested at two concentrations (0.25 and 0.50 µL) in a total volume of 450 µL per assay tube. The sensitivity threshold was 0.05 µg/mL plasma. The intra-assay variation was close to 5%, and the interassay variation was 10%.
Western ligand blotting and immunoblotting
IGFBPs were analyzed by methods routinely used in the laboratory and derived from those described previously (12, 13). After separation of the plasma proteins by SDS-PAGE under nonreducing conditions and transfer to nitrocellulose membranes, the various IGFBPs were detected by incubation with their ligands (a mixture of 125I-IGF-I and -II). In immunoblotting, IGFBP-3 was detected using either the specific polyclonal Ab or the mAb used for the LIFA. The complexes were revealed using antibodies coupled to horseradish peroxidase in the Amersham enhanced chemiluminescence Western blotting detection system.
Statistics
Conventional methods were used to determine the means (±SEM), intra- and interassay coefficients of variation, and linear regression analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specificity
The IGFBP-3 mAb used is specific for human IGFBP-3. Figure 1
shows that in Western immunoblotting it
recognizes intact (1–264) IGFBP-3 and its 1–160 and 1–95 fragments
either present in plasma or obtained in vitro from rhIGFBP-3
(E. coli) submitted to the action of plasmin and isolated by
high-performance liquid chromatography (14, 15). This mAb
does not recognize the 161–264 fragment. The lack of cross-reaction of
the IGFBP-3 mAb with either rhIGFBP-1 or rhIGFBP-2 was confirmed by
liquid phase assays; with IGFBP-5, by Western immunoblotting; and with
native human IGFBP-4 and -6, by immunoblot analysis of media
conditioned by human cell lines and cerebrospinal fluid, respectively
(data not shown).
IGFBP-3 binding to the mAb on the test tube walls does not
mask its IGF-I-binding sites. The saturable and reversible nature of
ligand binding was demonstrated by competitive binding experiments
between radiolabeled and unlabeled IGF-I. An example is shown in Fig. 2, where plasma extracts from a healthy
adult and an acromegalic patient were tested in the same experiment. On
average, nonspecific binding of 125I-IGF-I to tubes not
coated with antibody was 1.4%.
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shows the dose-response curve obtained
in the presence of increasing concentrations of rhIGFBP-3. Under the
same conditions, 125I-IGF-I fails to bind to the 1–160 and
1–95 fragments. This loss of affinity was confirmed in studies of
pregnant women where 125I-IGF-I binding measured by the
LIFA was either extremely weak or absent (Fig. 4 and Table 1). This reflects the more or less
complete disappearance of the intact form of IGFBP-3 beyond the fourth
week of gestation owing to intense protease activity
(2).
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The standard curve used for the IGFBP-3 LIFA was established using
the reference preparation comprising the pool of plasmas from healthy
adults and containing 2.9 µg/mL total IGFBP-3 as measured by IRMA
(see Materials and Methods). Measurements of bound
125I-IGF-I (percentage of total counts) as a function of
the log of the intact IGFBP-3 concentration yielded approximately
linear responses for quantities between 0.47- and 3.75-µL equivalent
of plasma in a total volume of 300 µL per assay tube. Figure 5, which shows a representative assay,
also shows the dose-response curves for plasma samples from an
acromegalic patient (5.7 µg/mL total IGFBP-3) and a GH-deficient
patient (1.4 µg/mL total IGFBP-3). The maximum quantities used per
assay tube were 1.9- and 5- µL equivalent, respectively. Parallelism
with reference curve is quite evident.
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Expression of the results
The intact IGFBP-3 concentration of the reference plasma was calculated on the basis of a standard curve obtained with glycosylated rhIGFBP-3 (CHO). It was estimated at 2 µg/mL. Intact IGFBP-3 concentrations of unknown samples were calculated as percentages of that of the reference plasma, then as mg/L on the basis of the estimation above.
In the 31 healthy adults sampled for this study (see Materials
and Methods), good correlation was found between intact and total
IGFBP-3 concentrations (Fig. 6): r =
0.71, P = 0.0001. The mean concentrations of intact and
total IGFBP-3 were 2.24 ± 0.08 (SEM) and 3.27 ±
0.14 mg/L, respectively. The mean proportion of proteolysed IGFBP-3
[(total IGFBP-3- intact IGFBP-3/total IGFBP-3) x 100] for
these subjects was 29.4 ± 1.9%.
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Recovery was determined using known concentrations of rhIGFBP-3 incubated in triplicate in mAb-coated tubes and measurement on the standard curves. These concentrations were 5, 10, 20, and 40 ng per 300 µL, the highest corresponding to twice the concentration obtained from 2.5-µL equivalent of a plasma containing 8 mg/L total IGFBP-3 (the maximum level in an acromegalic patient). Recovery rates were 81%, 83%, 83%, and 84%, respectively.
Accuracy
The intra-assay variation, determined using the same plasma sample ultrafiltered on five different Centricon columns then measured by the LIFA, was 3.6%.
The interassay variation, determined using four different plasma pools, each sampled seven times, fell between 2% and 8% (mean, 4%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this solid phase assay, binding of IGFBP-3 (whether native glycosylated or recombinant glycosylated or nonglycosylated) to the mAb adsorbed to the test tube wall does not mask its IGF-I-binding sites. IGF binding was specific and reversible. Nonspecific binding was below 2%.
For analysis of plasma samples, the indispensable first step of dissociating the IGFBP-IGF-I or -II complexes in acid medium and separating IGFs and IGFBPs by ultrafiltration provides the advantage of simultaneously measuring IGF-I by RIA (18) and intact IGFBP-3 by LIFA. The 1–160 and 1–95 fragments obtained by plasmin-induced proteolysis of rhIGFBP-3 do not bind 125I-IGF-I when coupled with the mAb on the test tube wall. In Western ligand blotting and liquid phase binding experiments, neither IGF-I nor IGF-II binds to the 1–95 fragment (14). This agrees with recent results from biosensor analyses indicating a 1000-fold reduced affinity compared with intact IGFBP-3 (19). The 1–160 fragment retains residual affinity for IGF-I, estimated at one tenth (plasma IGFBP-3) and one fiftieth (rhIGFBP-3) of that of intact IGFBP-3 (3, 14). The total loss of affinity of the 1–160 peptide in the solid phase assay may reflect conformation changes. The lack of affinity for IGF-I of the N-terminal IGFBP-3 fragments was confirmed by analysis of pregnancy plasmas where there was consistently no or extremely weak binding to 125I-IGF-I at times when intact IGFBP-3 is not detectable by Western ligand and immunoblotting.
Recovery determined using known quantities of rhIGFBP-3 was close to 80% and independent of the concentration tested. This was not taken into account in the calculations of intact IGFBP-3 measured, because the concentrations were estimated in comparison with the reference plasma preparation treated in the same way as the unknown samples in each assay and calibrated on the basis of glycosylated rhIGFBP-3.
The intra-assay variability (3.6%) and interassay variability (below 10%) were of the order of magnitude required for an immunoassay, in particular that for total IGFBP-3. The accuracy of the LIFA for intact IGFBP-3 is well above that of the semiquantitative estimations obtained from densitometry scanning of immunoblots. In our hands, the (quantitative) reproducibility of immunoblots is disappointing, yielding 20–50% variations even with analysis of samples in duplicate or at two concentrations.
The sensitivity of the LIFA (0.4 µL of the normal plasma pool corresponding to 1 ng intact IGFBP-3) is also much greater than that of Western immunoblotting (approximately 2 µL of the same pool on a standard gel). This means that the LIFA will be applicable to measurement of intact IGFBP-3 in other biological fluids and/or cell culture media without prior concentration.
The physiological relevance of the IGFBP-3 LIFA is that it detects only the intact form that has the functional ability to bind IGF-I. Used in combination with the classical RIA or IRMA for total IGFBP-3, which indiscriminately measures the intact and proteolyzed forms, the proportions of proteolyzed IGFBP-3 can be quantified. This also provides a means of studying the regulation of IGFBP-3 proteolysis under normal and pathological conditions.
The correlation observed between intact and total IGFBP-3 concentrations in plasma samples from healthy adults shows that IGFBP-3 proteolysis is tightly regulated. Our results indicate that in the normal state approximately 30% of the IGFBP-3 analyzed in the bloodstream has undergone proteolysis. This is within the order of magnitude of estimations obtained from densitometry scanning of Western immunoblots (20). However, it does not concord with data from protease activity measurements using 125I-IGFBP-3 as substrate (21), where in the normal state (apart from during pregnancy) there is virtually no IGFBP-3 proteolytic activity in serum (8). Nevertheless, our occasional observation of increased proportions of proteolyzed IGFBP-3 in serum samples stored at -20C for several months points toward residual proteolytic activity in serum. For this reason, all plasmas tested in the present study were obtained from blood samples collected in tubes containing EDTA and on ice.
From studies of a variety of cell models (22), it seems likely that IGFBP-3 proteolysis occurs either in the environment of IGFBP-3-producing cells, or on contact with the vascular endothelium and/or via the action of tissue proteases that are rapidly inactivated in the blood. Our observation that proteolytic activity in lymph is almost eight times that in serum (8) would support the notion of tissual proteases. The proportion of proteolyzed IGFBP-3 is markedly larger in catabolic states, such as in patients with severe illness (23) or after surgery (24). The virtual disappearance of intact IGFBP-3 during the last three months of pregnancy reflects extreme physiological conditions, when the extensive proteolysis of IGFBP-3 associated with elevated IGF-I levels leads to enhanced IGF-I availability to the tissues (3, 4, 5) in response to increased metabolic needs and relative insulin resistance (25). The enhanced IGF-I bioavailability could account for the decrease in insulin requirement that is not uncommon in well-controlled type1 diabetic women (26). It could also explain the increased incidence of hypoglycemic episodes in these patients during the first half of pregnancy (27), interpreted as being partly related to transitionally increased insulin sensitivity (28). A recent study suggests that the main IGFBP-3 protease activity in pregnancy serum is due to a trophoblast-derived protease (29).
The IGF-I that dissociates from the 140-kDa complexes when IGFBP-3 is cleaved either associates with other IGFBPs in the 40-kDa complexes or circulates in free forms (3, 4). Calculation of the ratio of total IGF-I concentration to intact IGFBP-3 would, therefore, provide an index of the exchangeable fraction of IGF-I bound to IGFBP-3. This may have clinical significance considering the results of epidemiological studies revealing a link between IGF-I levels and prostate or breast cancer risk (30, 31). Determination of this ratio would constitute a different but complementary approach vis-à-vis the measurement of free IGF-I that is already in use in clinical investigation (32, 33). In our next study, we describe possible application of the IGFBP-3 LIFA to study of the regulation of IGFBP-3 proteolysis and IGF-I bioavailability in an analysis of physiological and pathological conditions comprising abnormalities of GH secretion or insulin deficiency and/or resistance.
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Footnotes |
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Received July 21, 2000.
Revised November 14, 2000.
Accepted December 6, 2000.
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References |
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