A comparative analysis of methods to measure insulin-like growth factor- I in human serum

T. Grogean, D. Vereault, P. S. Millard, D. Kiel, D. MacLean, Eric Orwoll, S. Greenspan, C. J. Rosen

Research output: Contribution to journalArticle

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Abstract

Objective: To compare two simple methods for extracting insulin-like growth factor-I (IGF-I) in serum from its binding proteins (IGFBPs) with a more complex (but more complete) method of IGFBP removal (size exclusion chromatography) in order to determine if the simpler extraction methods allow for accurate measurement of serum IGF-I. Design: Cross-sectional measurements of serum IGF-I by radioimmunoassay (RIA) after three extraction techniques. Subjects and Measurements: Serum samples from 73 elderly men and women were measured by RIA using a polyclonal antibody to IGF-I after separation of the IGFIBPs by: (1) acid ethanol extraction (AE), (2) acid ethanol cryoprecipitation (AEC), and (3) acid-gel (size exclusion) chromatography using fast protein liquid chromatography (FPLC). Thirty-eight samples were measured by all three methods and 73 samples were determined by at least two extraction techniques. Results: Recovery of IGF-I from serum after extraction was nearly complete and did not differ by separation method. Interassay and intra-assay coefficients of variation ranged from 2.4-5.7% for each method. Serum IGF-I levels did not differ between males and females after adjustment for age. The lowest serum IGF-I concentrations were in elderly white females who had recently suffered a hip fracture (median = 50 ng/ml after AEC). The highest levels of IGF-I were noted in elders receiving rhGH therapy (median = 232 ng/ml after AEC). In general, correlations were high between IGF-I after AE and IGF-I after FPLC (r = 0.98) and between IGF-I after AEC and IGF-I after FPLC (r = 0.96). However, IGF-I levels after AE and AEC both underestimated the serum IGF-I value after FPLC by as much as 82 ng/ml (AE) and 144 ng/ml (AEC), respectively. Conclusions: Although all three methods of extracting IGF-I from serum IGFBPs produced comparable results by RIA, size exclusion chromatography (FPLC) prior to RIA led to the highest serum levels of IGF-I. Linear regression models allowed prediction of serum IGF-I levels from one method to another but substantial residuals suggest that pooling data when more than one method has been employed may lead to bias. It is likely that these differences could be accounted for by remaining IGFBPs or IGFBP fragments that interfere with the measurement of IGF-I by RIA.

Original languageEnglish (US)
Pages (from-to)109-114
Number of pages6
JournalEndocrinology and Metabolism
Volume4
Issue number2
StatePublished - 1997
Externally publishedYes

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Insulin-Like Growth Factor I
Serum
Ethanol
Acids
Liquid Chromatography
Radioimmunoassay
Carrier Proteins
Gel Chromatography
Proteins
Linear Models
Hip Fractures

ASJC Scopus subject areas

  • Endocrinology

Cite this

Grogean, T., Vereault, D., Millard, P. S., Kiel, D., MacLean, D., Orwoll, E., ... Rosen, C. J. (1997). A comparative analysis of methods to measure insulin-like growth factor- I in human serum. Endocrinology and Metabolism, 4(2), 109-114.

A comparative analysis of methods to measure insulin-like growth factor- I in human serum. / Grogean, T.; Vereault, D.; Millard, P. S.; Kiel, D.; MacLean, D.; Orwoll, Eric; Greenspan, S.; Rosen, C. J.

In: Endocrinology and Metabolism, Vol. 4, No. 2, 1997, p. 109-114.

Research output: Contribution to journalArticle

Grogean, T, Vereault, D, Millard, PS, Kiel, D, MacLean, D, Orwoll, E, Greenspan, S & Rosen, CJ 1997, 'A comparative analysis of methods to measure insulin-like growth factor- I in human serum', Endocrinology and Metabolism, vol. 4, no. 2, pp. 109-114.
Grogean, T. ; Vereault, D. ; Millard, P. S. ; Kiel, D. ; MacLean, D. ; Orwoll, Eric ; Greenspan, S. ; Rosen, C. J. / A comparative analysis of methods to measure insulin-like growth factor- I in human serum. In: Endocrinology and Metabolism. 1997 ; Vol. 4, No. 2. pp. 109-114.
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abstract = "Objective: To compare two simple methods for extracting insulin-like growth factor-I (IGF-I) in serum from its binding proteins (IGFBPs) with a more complex (but more complete) method of IGFBP removal (size exclusion chromatography) in order to determine if the simpler extraction methods allow for accurate measurement of serum IGF-I. Design: Cross-sectional measurements of serum IGF-I by radioimmunoassay (RIA) after three extraction techniques. Subjects and Measurements: Serum samples from 73 elderly men and women were measured by RIA using a polyclonal antibody to IGF-I after separation of the IGFIBPs by: (1) acid ethanol extraction (AE), (2) acid ethanol cryoprecipitation (AEC), and (3) acid-gel (size exclusion) chromatography using fast protein liquid chromatography (FPLC). Thirty-eight samples were measured by all three methods and 73 samples were determined by at least two extraction techniques. Results: Recovery of IGF-I from serum after extraction was nearly complete and did not differ by separation method. Interassay and intra-assay coefficients of variation ranged from 2.4-5.7{\%} for each method. Serum IGF-I levels did not differ between males and females after adjustment for age. The lowest serum IGF-I concentrations were in elderly white females who had recently suffered a hip fracture (median = 50 ng/ml after AEC). The highest levels of IGF-I were noted in elders receiving rhGH therapy (median = 232 ng/ml after AEC). In general, correlations were high between IGF-I after AE and IGF-I after FPLC (r = 0.98) and between IGF-I after AEC and IGF-I after FPLC (r = 0.96). However, IGF-I levels after AE and AEC both underestimated the serum IGF-I value after FPLC by as much as 82 ng/ml (AE) and 144 ng/ml (AEC), respectively. Conclusions: Although all three methods of extracting IGF-I from serum IGFBPs produced comparable results by RIA, size exclusion chromatography (FPLC) prior to RIA led to the highest serum levels of IGF-I. Linear regression models allowed prediction of serum IGF-I levels from one method to another but substantial residuals suggest that pooling data when more than one method has been employed may lead to bias. It is likely that these differences could be accounted for by remaining IGFBPs or IGFBP fragments that interfere with the measurement of IGF-I by RIA.",
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T1 - A comparative analysis of methods to measure insulin-like growth factor- I in human serum

AU - Grogean, T.

AU - Vereault, D.

AU - Millard, P. S.

AU - Kiel, D.

AU - MacLean, D.

AU - Orwoll, Eric

AU - Greenspan, S.

AU - Rosen, C. J.

PY - 1997

Y1 - 1997

N2 - Objective: To compare two simple methods for extracting insulin-like growth factor-I (IGF-I) in serum from its binding proteins (IGFBPs) with a more complex (but more complete) method of IGFBP removal (size exclusion chromatography) in order to determine if the simpler extraction methods allow for accurate measurement of serum IGF-I. Design: Cross-sectional measurements of serum IGF-I by radioimmunoassay (RIA) after three extraction techniques. Subjects and Measurements: Serum samples from 73 elderly men and women were measured by RIA using a polyclonal antibody to IGF-I after separation of the IGFIBPs by: (1) acid ethanol extraction (AE), (2) acid ethanol cryoprecipitation (AEC), and (3) acid-gel (size exclusion) chromatography using fast protein liquid chromatography (FPLC). Thirty-eight samples were measured by all three methods and 73 samples were determined by at least two extraction techniques. Results: Recovery of IGF-I from serum after extraction was nearly complete and did not differ by separation method. Interassay and intra-assay coefficients of variation ranged from 2.4-5.7% for each method. Serum IGF-I levels did not differ between males and females after adjustment for age. The lowest serum IGF-I concentrations were in elderly white females who had recently suffered a hip fracture (median = 50 ng/ml after AEC). The highest levels of IGF-I were noted in elders receiving rhGH therapy (median = 232 ng/ml after AEC). In general, correlations were high between IGF-I after AE and IGF-I after FPLC (r = 0.98) and between IGF-I after AEC and IGF-I after FPLC (r = 0.96). However, IGF-I levels after AE and AEC both underestimated the serum IGF-I value after FPLC by as much as 82 ng/ml (AE) and 144 ng/ml (AEC), respectively. Conclusions: Although all three methods of extracting IGF-I from serum IGFBPs produced comparable results by RIA, size exclusion chromatography (FPLC) prior to RIA led to the highest serum levels of IGF-I. Linear regression models allowed prediction of serum IGF-I levels from one method to another but substantial residuals suggest that pooling data when more than one method has been employed may lead to bias. It is likely that these differences could be accounted for by remaining IGFBPs or IGFBP fragments that interfere with the measurement of IGF-I by RIA.

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