This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miraki-Moud, F. Articles by Camacho-Hübner, C. Search for Related Content PubMed PubMed Citation Articles by Miraki-Moud, F. Articles by Camacho-Hübner, C. Related Collections Endocrine Oncology Metabolism

A Rapid Method for Analyzing Serum Pro-Insulin-Like Growth Factor-II in Patients with Non-Islet Cell Tumor Hypoglycemia

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).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Schematic representation of the structures of pre- and pro-IGF-II. Proteolytic cleavage sites are indicated by arrows, modified from Bell et al. (3 ). aa, Amino acids.

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).

View larger version (15K): [in this window] [in a new window]   FIG. 2. Serum IGF-I (A), IGF-II (B), and IGFBP-2 (C) concentrations in sera from controls and NICTH patients.

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).

View larger version (33K): [in this window] [in a new window]   FIG. 3. A, Size-exclusion acid chromatography of two patients with NICTH (patients 7 and 8) and normal human serum (NHS). Serum samples were analyzed by gel filtration chromatography under acid conditions followed by an IGF-II RIA as described in Subjects and Methods. The profiles show two main immunoreactive peaks (arrows), 15 and 7.5 kDa corresponding to pro-IGF-II and mature IGF-II, respectively. B, The tricine-SDS-PAGE from patients 7 and 8 and normal serum. Samples were separated by 16.5% tricine-SDS-PAGE and detected by immunoblot with IGF-II antibody as described in Subjects and Methods. Molecular-weight (Mw) markers are shown on the right.

View larger version (31K): [in this window] [in a new window]   FIG. 4. A, Size-exclusion acid chromatography of two patients with NICTH (patients 10 and11) and normal serum (NHS). Serum samples were analyzed by gel filtration chromatography under acid conditions followed by an IGF-II RIA as described in Subjects and Methods. The profiles show two main immunoreactive peaks (arrows), 15 and 7.5 kDa corresponding to pro-IGF-II and mature IGF-II, respectively. B, The tricine-SDS-PAGE from patients 10 and 11 and normal serum. Samples were separated by 16.5% tricine-SDS-PAGE and detected by immunoblot with IGF-II antibody as described in Subjects and Methods. Molecular-weight (Mw) markers are shown on the right.

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).

View larger version (11K): [in this window] [in a new window]   FIG. 5. Bland and Altman plot of the difference between the two methods against their mean with 95% limits of agreement (broken lines) and regression line.

The IGF-II E_78–88 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 IIE_89–101 domain weakly detected two bands of 16 and 24 kDa, which did not change after digestion with neuramidase and O-glycosidase. The IIE_138–156 antibody did not recognize any specific band (data not shown).

View larger version (38K): [in this window] [in a new window]   FIG. 6. Representative autoradiograph of a Western immunoblot analysis of serum pro-IGF-II in NICTH patients (patients 7, 8, and 10). Serum samples treated with or without neuramidase and O-glycosidase were separated by tricine-SDS-PAGE and detected by immunoblot using anti-IGF-II antiserum clone S1F2 and anti-pro-IGF-II antibody (IIE78–88) as described in Subjects and Methods. Molecular-weight (MW) markers are shown on the right.

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: IIE_78–88, IIE_89–101, and IIE_138–156. 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 IIE_78–88 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 IIE_89–101 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, IIE_138–156 antibody did not recognize any specific band, suggesting that IGF-IIE_101–156 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

1. Ron D, Powers AC, Pandian MR, Godine JE, Axelrod L 1989 Increased insulin-like growth factor II production and consequent suppression of growth hormone secretion: a dual mechanism for tumor-induced hypoglycemia. J Clin Endocrinol Metab 68:701–706[Abstract/Free Full Text]
2. Zapf J, Futo E, Peter M, Froesch ER 1992 Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia? J Clin Invest 90:2574–2584
3. Bell GI, Merryweather JP, Sanchez-Pescado R, Stempien MM, Priestley L, Scott J, Rall LB 1984 Sequence of a cDNA clone encoding human preproinsulin-like growth factor II. Nature 310:775–777[CrossRef][Medline]
4. Duguay SJ, Jin Y, Stein J, Duguay AN, Gardner P, Steiner DF 1998 Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis. J Biol Chem 273:18443–18451[Abstract/Free Full Text]
5. Daughaday WH, Trivedi B 1992 Measurement of derivatives of proinsulin-like growth factor-II in serum by a radioimmunoassay directed against the E-domain in normal subjects and patients with nonislet cell tumor hypoglycemia. J Clin Endocrinol Metab 75:110–115[Abstract]
6. Hudgins WR, Hampton B, Burgess WH, Perdue JF 1992 The identification of O-glycosylated precursors of insulin-like growth factor II. J Biol Chem 267:8153–8160[Abstract/Free Full Text]
7. Kuenen BC, van Doorn J, Slee PH 1996 Non-islet-cell tumour induced hypoglycaemia: a case report and review of literature. Neth J Med 48:175–179[CrossRef][Medline]
8. Daughaday WH, Emanuele MA, Brooks MH, Barbato AL, Kapadia M, Rotwein P 1988 Synthesis and secretion of insulin-like growth factor II by a leiomyosarcoma with associated hypoglycemia. N Engl J Med 319:1434–1440[Abstract]
9. Daughaday WH, Trivedi B, Baxter RC 1993 Serum "big insulin-like growth factor-II from patients with tumor hypoglycemia lacks normal E-domain O-linked glycosylation, a possible determinant of normal propeptide processing. Proc Natl Acad Sci USA 90:5823–5827[Abstract/Free Full Text]
10. Zapf J 1995 Insulin-like growth factor binding proteins and tumor hypoglycemia. Trends Endocrinol Metab 6:37–42
11. Baxter RC 1996 The role of insulin-like growth factors and their binding proteins in tumor hypoglycemia. Horm Res 46:195–201[Medline]
12. Baxter RC, Daughaday WH 1991 Impaired formation of the ternary insulin-like growth factor-binding protein complex in patients with hypoglycemia due to nonislet cell tumors. J Clin Endocrinol Metab 73:696–702[Abstract/Free Full Text]
13. Bond JJ, Meka S, Baxter RC 2000 Binding characteristics of pro-insulin-like growth factor-II from cancer patients: binary and ternary complex formation with IGF binding proteins-1 to -6. J Endocrinol 165:253–260[Abstract]
14. Baxter RC, Holman SR, Corbould A, Stranks S, Ho PJ, Braund W 1995 Regulation of the insulin-like growth factors and their binding proteins by glucocorticoid and growth hormone in nonislet cell tumor hypoglycemia. J Clin Endocrinol Metab 80:2700–2708[Abstract]
15. Guler HP, Zapf J, Schmid C, Froesch ER 1989 Insulin-like growth factors I and II in healthy man. Estimations of half-lives and production rates. Acta Endocrinol 121:753–758
16. Davenport ML, Clemmons DR, Miles MV, Camacho-Hubner C, D’Ercole AJ, Underwood LE 1990 Regulation of serum insulin-like growth factor-I (IGF-I) and IGF binding proteins during rat pregnancy. Endocrinology 127:1278–1286[Abstract/Free Full Text]
17. Morrell DJ, Dadi H, More J, Taylor AM, Dabestani A, Buchanan CR, Holder AT, Preece MA 1989 A monoclonal antibody to human insulin-like growth factor-I: characterization, use in radioimmunoassay and effect on the biological activities of the growth factor. J Clin Endocrinol Metab 2:201–206
18. Bowsher RR, Lee WH, Apathy JM, O’Brien PJ, Ferguson AL, Henry DP 1991 Measurement of insulin-like growth factor-II in physiological fluids and tissues. I. An improved extraction procedure and radioimmunoassay for human and rat fluids. Endocrinology 128:805–814[Abstract/Free Full Text]
19. Schagger H, von Jagow G 1987 Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379[CrossRef][Medline]
20. Bland JM, Altman DG 1995 Comparing methods of measurement: why plotting difference against standard method is misleading. Lancet 345:1085–1087[CrossRef][Medline]
21. Daughaday WH, Kapadia M 1989 Significance of abnormal serum binding of insulin-like growth factor II in the development of hypoglycemia in patients with non-islet cell tumors. Proc Natl Acad Sci USA 86:6778–6782[Abstract/Free Full Text]
22. Cotterill AM, Holly JMP, Davies SC, Coulson VJ, Price PA, Wass JAH 1991 The insulin-like growth factors and their binding proteins in a case of non-islet-cell tumour-associated hypoglycemia. J Endocrinol 131:303–311[Abstract/Free Full Text]
23. Liu F, Baker BK, Powell DR, Hintz RL 1993 Characterization of proinsulin-like growth factor-II E-region immunoreactivity in serum and other biological fluids. J Clin Endocrinol Metab 76:1095–1100[Abstract]
24. Christofilis MA, Remacle-Bonnet M, Atlan-Gepner C, Garrouste F, Vialettes B, Fuentes P, Guidicelli R, Pommier G 1998 Study of serum big-insulin-like growth factor (IGF)-II and IGF binding proteins in two patients with extrapancreatic tumor hypoglycemia, using a combination of Western blotting methods. Eur J Endocrinol 139:317–322[Abstract]
25. Boulle N, Gicquel C, Logie A, Christol R, Feige JJ, Le Bouc Y 2000 Fibroblast growth factor-2 inhibits the maturation of pro-insulin-like growth factor-II (Pro-IGF-II) and the expression of insulin-like growth factor binding protein-2 (IGFBP-2) in the human adrenocortical tumor cell line NCI-H295R. Endocrinology 141:3127–3136[Abstract/Free Full Text]
26. Khosla S, Ballard FJ, Conover CA 2002 Use of site-specific antibodies to characterize the circulating form of big insulin-like growth factor II in patients with hepatitis C-associated osteosclerosis. J Clin Endocrinol Metab 87:3867–3870[Abstract/Free Full Text]

This article has been cited by other articles:

				P. E. Cryer, L. Axelrod, A. B. Grossman, S. R. Heller, V. M. Montori, E. R. Seaquist, and F. J. Service Evaluation and Management of Adult Hypoglycemic Disorders: An Endocrine Society Clinical Practice Guideline J. Clin. Endocrinol. Metab., March 1, 2009; 94(3): 709 - 728. [Abstract] [Full Text] [PDF]

				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]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miraki-Moud, F. Articles by Camacho-Hübner, C. Search for Related Content PubMed PubMed Citation Articles by Miraki-Moud, F. Articles by Camacho-Hübner, C. Related Collections Endocrine Oncology Metabolism