| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
RAPID COMMUNICATION |
Department of Endocrinology, William Harvey Research Institute, Queen Mary University of London, London EC1M 6BQ, United Kingdom
Address all correspondence and requests for reprints to: Cecilia Camacho-Hübner, M.D., Department of Endocrinology, 51-53 Bartholomew Close, St. Bartholomew’s Hospital, London EC1A 7BE, United Kingdom. E-mail: c.camacho-hubner{at}qmul.ac.uk.
Abstract
Context: Non-islet cell tumor hypoglycemia (NICTH) results from the hypersecretion of pro-IGF-II by a large, usually mesenchymal tumor. Detection of pro-IGF-II in serum is a potential tumor marker in these patients.
Objective: The aim of this study was to validate a rapid and reliable method for determining serum pro-IGF-II.
Patients: Serum samples from 16 patients with NICTH were studied.
Main Outcome Measure: The main outcome measure was serum concentration of pro-IGF-II determined by immunoblot analysis of pro-IGF-II and mature IGF-II after 16.5% tricine-SDS-PAGE, which was compared with pro-IGF-II measured by standard RIA after size-exclusion acid chromatography.
Results: The analyses of patients’ sera by size-exclusion acid chromatography showed that 68 ± 19% of IGF-II were present in the pro-IGF-II form, whereas only 18 ± 4% corresponded to pro-IGF-II in controls. Scanning densitometry of immunoblots showed 67 ± 16% in the bands corresponding to pro-IGF-II in patients’ sera, compared with 27 ± 9% in controls. The detection sensitivity of tricine-SDS-PAGE method was the same as for size-exclusion chromatography, but the tricine-SDS-PAGE method is quicker and requires smaller amounts of serum.
Conclusion: Tricine-SDS-PAGE followed by IGF-II immunoblot analysis provides a rapid, reproducible, and sensitive method for the separation of serum pro-IGF-II from mature IGF-II and is a useful laboratory evaluation of patients with a clinical diagnosis of NICTH.
NON-ISLET CELL TUMOR hypoglycemia (NICTH) is a well-recognized syndrome associated with overproduction of pro-IGF-II, usually secreted by large mesenchymal tumors (1, 2). IGF-II is synthesized as a preprohormone containing 180 amino acids, including a signal peptide at the amino terminus with 24 amino acids, mature IGF-II of 67 amino acids, and an E-peptide at the carboxy terminus with 89 amino acids (3). Proteolytic cleavage of the signal peptide produces the pro-IGF-II, whereas further proteolytic cleavage of the E-domain produces the 7.5-kDa form corresponding to mature IGF-II (Fig. 1
) (4).
|
Although the cause of hypoglycemia in NICTH remains unclear, the following mechanism has been proposed. The overproduction of pro-IGF-II stimulates glucose uptake into the cells, and this may account for elevated glucose consumption in the tumor and other tissues by an autocrine and/or paracrine mode of action. Hepatic glucose production is also decreased. The overproduction of pro-IGF-II causes inhibition of insulin and GH secretion, which leads to decreased secretion of IGF-I, which depends on GH and insulin, and IGF binding protein (IGFBP)-3 and acid labile subunit (ALS), which are GH dependent (10). However, IGFBP-2 concentrations in these patients are increased, which may be due to the decrease in GH secretion (11). These changes in IGFBPs and ALS in patients with NICTH lead to impaired formation of the 150-kDa ternary complexes (12, 13), resulting in a shift of IGFBP-3 into the 50- to 60-kDa binary complexes, possibly increasing the bioavailability of pro-IGF-II to target tissues and causing hypoglycemia (14). Studies by Guler et al. (15) and Davenport et al. (16) showed the kinetics of IGF-I and IGF-II in the human and rat, respectively. They demonstrated that when radiolabeled IGF-I and IGF-II are bound to IGFBPs in the 50-kDa binary complexes, they have a significantly shorter half-life in plasma and prolonged distribution phase, compared with the 150-kDa ternary complexes. This is consistent with the concept that the binary complexes are transporting IGF-I and IGF-II more readily to target tissues.
Initially, the confirmation of the elevated levels of serum pro-IGF-II in the diagnosis of NICTH required size-exclusion acid chromatography. This is still considered the gold standard method because it provides good separation of pro-IGF-II from mature IGF-II. However, this method is very time consuming, and a more convenient yet accurate method is needed. Therefore, the aim of this study was to validate a more rapid and reliable method for determining serum pro-IGF-II using SDS-PAGE, which allows reproducible separation of proteins in the 5- to 20-kDa range.
Subjects and Methods
Subjects
Sixteen patients with a clinical diagnosis of NICTH were studied. Serum samples were obtained from healthy sex- and age-matched blood donors, who served as controls. All samples were stored at –20 C until further analysis. Written consent was obtained for all patients.
Immunoassay
Serum IGF-I and IGF-II were measured by RIA after formic acid-acetone extraction of binding proteins as previously described (17, 18). The mean intra- and interassay coefficients of variations were 2.7 and 10.4% for IGF-I and 7.0 and 8.9% for IGF-II, respectively. The assay sensitivity was 45 ng/ml (5.85 nmol/liter) for IGF-I and 125 ng/ml (17 nmol/liter) for IGF-II.
Serum IGFBP-2 was measured using a RIA kit (Diagnostic System Laboratories Inc., Webster, TX). The mean intra and interassay coefficients of variation were 8.55 and 4.9%, respectively.
Size-exclusion acid chromatography
Serum samples from NICTH patients and normal subjects (250 µl) were acidified by incubation with 250 µl of 1% formic acid + 0.2 g/liter protamine sulfate for 30 min at room temperature before chromatography. The total volume (500 µl) was loaded onto a 1.5 x 100 cm column, packed with Sephadex G50 in 0.05 M PBS (pH 7.5). At a pump speed of 6 ml/h, 1-ml fractions were collected every 10 min. The fractions were then neutralized with 145 µl 1 M NaOH, diluted in assay buffer and analyzed using an IGF-II RIA.
Western immunoblot analysis of IGF-II
Tricine-SDS-PAGE (16.5% tricine SDS-PAGE) was used for separating IGF-II peptides according to Schägger and Von Jagow (19). Tricine-SDS-PAGE allows reproducible separation of the proteins in the 5- to 20-kDa ranges. Samples (5 µl) treated with or without neuramidase and O-glycosidase (1 mU neuramidase and 1 mU O-glycosidase overnight at 37 C). Then samples were diluted with sample buffer [0.15 mol/liter Tris-HCl (pH 6.8); 6% (wt/vol) sodium dodecyl sulfate; 22% (wt/vol) glycerol, and 0.02% (wt/vol) bromophenol blue] in a ratio of 1:9, heated at 60 C for 10 min, cooled, and loaded onto 16.5% tricine-SDS-PAGE. The separated proteins were electroblotted onto nitrocellulose membranes and then blocked for 1 h at room temperature with 10% milk and followed by 3% BSA in PBS + 0.1% Tween 20. Membranes were then probed with anti-IGF-II antiserum clone S1F2, which recognizes human and rat IGF-II (Upstate Biotechnology Inc., Lake Placid, NY; 1:1000). For immunoblots using pro-IGF-II antibodies to the E domain, which do not cross-react with mature IGF-II, membranes were blocked for 2 h with 5% milk in Tris-buffered saline + 0.1% Tween 20 and then probed with primary IGF-IIE antibodies (IIE 77–88 1:3000; IIE 89–101 1:1500, or IIE 138–156 1:1500; GroPep Ltd., Adelaide, Australia) at 4 C overnight. After washing, the membranes were incubated with antimouse or antirabbit secondary antibody conjugated to HRP for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence detection system (Amersham International PLC, Buckinghamshire, UK).
Statistical analysis
Results were analyzed using Microsoft analysis for Excel (Redmond, WA) and SPSS for Windows (version 6.1, Chicago, IL). Variables were normally distributed (Kolmogorov-Smirnov test), and thus, parametric tests were used. Mean differences and their 95% confidence interval were calculated. The two methods were compared using the Bland-Altman test (20).
Results
Patients’ clinical characteristics and analysis of serum IGF-I and IGF-II
The clinical characteristics of the patients studied are shown in Table 1. All patients had suppressed insulin and C-peptide concentrations in the presence of hypoglycemia and radiological evidence of tumor (data not shown).
|
). No changes in total serum IGF-II concentrations were found in NICTH patients, compared with controls (Fig. 2B). Mean serum IGFBP-2 concentrations were significantly increased in NICTH patients, compared with controls (P < 0.0001, Fig. 2C).
|
Serum samples were analyzed by gel filtration chromatography under acid conditions followed by an IGF-II RIA. Two IGF-II immunoreactive peaks were obtained. Representative IGF-II profiles from four patients and normal serum are shown in Figs. 3A and 4A. The first peak of 10–20 kDa corresponds to pro-IGF-II, whereas the 7.5-kDa peak corresponds to mature IGF-II. In normal serum most of the IGF-II elutes in the second peak (7.5 kDa), whereas most of the IGF-II in patient serum eluted in the 10- to 20-kDa peaks with a small amount in the 7.5-kDa peaks. The results of the size-exclusion acid chromatography from all patients showed that 68 ± 19% of total IGF-II was present in the pro-IGF-II form, whereas only 18 ± 4% corresponded to pro-IGF-II in controls (P < 0.0001).
|
|
and 4B. Bland-Altman plot
The agreement between the size-exclusion acid chromatography (standard method) and tricine-SDS-PAGE (new method) was assessed by the Bland and Altman analysis (Fig. 5). Mean value for the standard method was 68.1 with SD of 20.3 and the new method was 67.8 with SD of 17.2. The mean difference between the two methods was 0.25, and the 95% confidence intervals for the mean difference were –2.5 to +2.96. The correlation between these two methods was 0.98 (95% confidence interval 0.93–0.99, P = 0.01).
|
The IGF-II E78–88 antibody detected a band of approximately 14–16 kDa in NICTH sera when the sera from patients with NICTH were digested with neuramidase and O-glycosidase; the 14- to 16-kDa bands were reduced in size to approximately 9–10 kDa. Similar results were obtained when the primary antibody used in the immunoblot was IGF-II (clone S1F2, Fig. 6). In contrast, the antibody against the IIE89–101 domain weakly detected two bands of 16 and 24 kDa, which did not change after digestion with neuramidase and O-glycosidase. The IIE138–156 antibody did not recognize any specific band (data not shown).
|
Most cases of NICTH result from the hypersecretion of pro-IGF-II by mesenchymal tumors that suppress insulin, C-peptide, and GH, which in turn decrease serum IGF-I, IGFBP-3, and ALS levels; IGF-II levels are usually within the normal range, as confirmed in our study. The suggestion that pro-IGF-II may cause hypoglycemia in NICTH is consistent with the observations previously described that the pro-IGF-II level decreases significantly with concomitant disappearance of hypoglycemia after successful removal of the tumor (8, 21, 22).
Until now, size-exclusion acid chromatography has been considered the gold standard method for detection of pro-IGF-II in NICTH (8, 13, 21, 22). This method provides good separation of pro-IGF-II from mature IGF-II, but the procedure is very time consuming. Usually, the chromatographic analysis requires 3 d per sample including the size-exclusion chromatography and the immunoassay, whereas the IGF-II immunoblotting technique requires 1 d for the analysis of 18 samples.
In recent studies, Western immunoblot analysis has been used to separate pro-IGF-II from mature IGF-II (23, 24, 25, 26), but the two methods have not been previously compared rigorously.
In this study, we validated the usefulness and reproducibility of the immunoblot technique in a group of patients in which the diagnosis of excess pro-IGF-II secretion has been confirmed by size-exclusion acid chromatography. In addition, we optimized the tricine-SDS-PAGE using anti-IGF-II (clone S1F2) antibody, which recognizes both mature and pro-IGF-II forms. Using the standard method and tricine-SDS-PAGE, we observed that in normal subjects most of serum IGF-II was detected at 7.5 kDa and a small amount in 10- to 18-kDa ranges. In contrast, in NICTH most of the circulating IGF-II migrated in 10–18 kDa, and a small amount in 7.5 kDa. This study has shown strong correlation and identity of values between the standard chromatographic method and the immunoblot technique. Our results also confirmed that the detection sensitivity of tricine-SDS-PAGE method was the same as for size-exclusion acid chromatography, but the tricine-SDS-PAGE method is quicker and requires a smaller amount of serum.
The specific antibodies recognizing various forms of pro-IGF-II E-domain were also used for immunoblotting. Based on the cleavage sites at the E-domain (Fig. 1) these antibodies recognize pro-IGF-II peptides as follows: IIE78–88, IIE89–101, and IIE138–156. Our results showed that the predominant form of pro-IGF-II in NICTH migrated as a doublet of approximately 14–16 kDa and reacted with IIE78–88 antibody. This 14- to 16-kDa band was reduced in size after treatment of samples with neuramidase and O-glycosidase, which suggests that the most abundant form of pro-IGF-II in patients with NICTH is O-glycosylated at the position E-21. We also found IIE89–101 antibody weakly detected two immunoreactive bands of 16 and 24 kDa, which are nonglycosylated or resistant to the standard method of deglycosylation using O-glycosidase and neuramidase.
Finally, IIE138–156 antibody did not recognize any specific band, suggesting that IGF-IIE101–156 isoform is not present in serum samples from patients with NICTH. Our findings using these specific antibodies are slightly different from a previous study comparing pro-IGF-II forms present in patients with NICTH and those found in patients with hepatitis C-associated osteosclerosis (26). The same antibodies used in that study to characterize the circulating forms of pro-IGF-II were used in our study. We found that sera of patients with NICTH have similar forms of pro-IGF-II to those previously described in patients with hepatitis C-associated osteosclerosis who do not present with hypoglycemia. Thus, the clinical presentation of hypoglycemia is associated with differential pro-IGF-II processing and in particular with which is the predominant form of circulating pro-IGF-II. In addition, these patients have elevated IGFBP-2 as previously described (7, 10, 14, 22, 26).
In summary, we propose the use of tricine-SDS-PAGE as a rapid and reliable method for determining serum pro-IGF-II in clinical studies. Tricine-SDS-PAGE analysis provides a useful, reproducible, and sensitive method for the separation of the pro-IGF-II from mature IGF-II in NICTH patients. The uses of specific antibodies to IGF-II and various forms of IGF-IIE are useful in identifying the predominant forms of pro-IGF-II found in patients with NICTH.
Acknowledgments
We are grateful to Professor P. Sönksen, Professor J. A. Wass, Dr. T. Mak, Dr. F. Kelestimur, and Dr. A. Cotterill for providing patient samples. We are also grateful to Pfizer for their support.
Footnotes
This work was supported in part by a Cancer Research Committee Grant (to C.C.-H.).
First Published Online April 19, 2005
Abbreviations: ALS, Acid labile subunit; IGFBP, IGF binding protein; NICTH, non-islet cell tumor hypoglycemia.
Received October 22, 2004.
Accepted April 11, 2005.
References
This article has been cited by other articles:
 |
|
 |
|
  J. W. B de Groot, B. Rikhof, J. van Doorn, H. J G Bilo, M. A Alleman, A. H Honkoop, and W. T A van der Graaf Non-islet cell tumour-induced hypoglycaemia: a review of the literature including two new cases Endocr. Relat. Cancer, December 1, 2007; 14(4): 979 - 993. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Qiu, J.-Y. Jiang, M. Bell, B. K. Tsang, and A. Gruslin Activation of Endoproteolytic Processing of Insulin-Like Growth Factor-II in Fetal, Early Postnatal, and Pregnant Rats and Persistence of Circulating Levels in Postnatal Life Endocrinology, October 1, 2007; 148(10): 4803 - 4811. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
