Acid-Labile Subunit of Human Insulin-Like Growth Factor-Binding Protein Complex: Measurement, Molecular, and Clinical Evaluation

Abstract

Although the acid-labile subunit (ALS) of the approximately 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.

Diagnostic interest of acid-labile subunit measurement in relationship to other components of the IGF system in pediatric patients with growth or eating disorders

“…Serum ALS concentrations were decreased in GHD children and normalized after GH replacement. Low ALS concentrations in GHD patients have been reported previously (1,5,6). The patients with short stature and normal GH secretion included in this study had normal serum ALS concentrations; it is also known that GH therapy in patients with GH insensitivity does not change serum ALS concentrations (15).…”

“…In contrast, these effects are not seen in response to IGF-I administration (4). Although there is little information available, ALS concentrations appear to be diminished in GH-deficient (GHD) patients (5), at least in adults suspected of being GHD (6), and increased in acromegalic patients (7); however, there is a high percentage of overlap with control values (1). In addition, ALS is subject to nutritional regulation, decreasing after fasting or caloric restriction.…”

Measurement of the acid-labile subunit of the insulin-like growth factor binding protein complex in human serum: a comparison of four immunoassays

“…Methods 1, 2, and 4 detect denatured (i.e. acidified) ALS poorly, if at all, and are believed to detect complexed ALS as effectively as free ALS, whereas Method 3 relies on the denaturation (by SDS) of ALS to increase detection by 200-fold (Khosravi et al 1997). The close parallel in the values for different patient groups detected by these different methodological approaches indicates that most or all of the ALS in the circulation is in the native conformation, since a significant concentration of denatured ALS would lead to systematically lower values by Method 1 compared with Method 3.…”

“…Method 3 This is a commercial two-site enzyme-linked immunosorbent assay (ELISA) (DSL Inc., Webster, TX, USA) using goat antisera raised against synthetic aminoand carboxy-terminal peptides of human ALS (Khosravi et al 1997). In brief, standards and samples were pretreated Method 4…”

“…in SDS to optimize their immunoreactivity, and aliquots (20 µl of a 1:100 dilution) were incubated in microtiter plates coated with capture antibody for 1 h at 22 C. The standard solution was made in an assay buffer matrix containing partially purified ALS from human serum as previously described (Khosravi et al 1997), calibrated against the same natural (glycosylated) ALS reference preparation as in Methods 1 and 2. Enzyme-labeled detection antibody was added, incubated at 22 C for 30 min, wells were aspirated and washed, chromogen solution was added, and color development stopped after 10 min.…”