Assays
Samples for Radiometer ABL 800 Flex blood gas analyzer (Radiometer Medical, Brønshøj, Denmark) were analyzed every hour throughout the experiment to monitor electrolytes (especially potassium), BG levels, and hematocrit. In addition, BG levels were measured with glucometer (HemoCue Glucose 201 DM, HemoCue AB. Ängelholm, Sweden) to obtain BG measurements every 30 min. In the post-HD period, BG levels were measured every 10–30 min to avoid hypoglycaemia.
Plasma concentrations of albumin, fibrinogen, and 25-hydroxy vitamin D were determined with routine automated methods at the Department of Clinical Biochemistry, Aarhus University Hospital. For measurements of hormones and inflammatory biomarkers serum and plasma were separated by centrifugation at 3500 g for 10 min at 4°C and aliquots were stored within an hour at −80°C until further processing.
Serum levels of immunoreactive (total) IGF-I (final dilution 1:1000) were determined in acid ethanol serum extracts using a validated, in-house time-resolved immunofluorometric assay (TR-IFMA) as previously described [
34] with slight modifications: first, the IGF-I assay had been calibrated against the international IGF-I reference preparation WHO 02/254. Second, the detection antibody now consists of a biotinylated goat polyclonal antibody (Sigma-Aldrich, catalogue No. I-8773; Brøndby, Denmark). The biotinylated antibody was incubated with streptavidin europium obtained from Perkin Elmer Life Sciences (Turku, Finland).
Bioactive IGF-I was analysed by a cell-based kinase-receptor activation (KIRA) assay based on human embryonic cells transfected with cDNA of the human IGF-I receptor (IGF-IR) gene, as previously detailed [
35], with slight modifications. The assay measures the ability of serum IGF to phosphorylate (i.e. activate) the IGF-IR
in vitro, and takes into account the presence of the IGFBPs and their ability to modify IGF-IR activation by IGF-I or IGF-II [
36]. In brief, transfected cells were stimulated with serum diluted 1:10 in Krebs-Ringer buffer for 15 min at 37°C, after which cells were aspirated and the cells lysed. The crude cell lysates were then transferred to a TR-IFMA assay specific for phosphorylated (i.e. ligand-activated) IGF-IRs, based on a monoclonal capture antibody against the extracellular IGF-IR domain and an europium-labelled monoclonal anti-phosphotyrosine antibody as tracer. Since the original description [
35], the KIRA assay has been slightly modified. The assay has now been calibrated against the international IGF-I reference preparation WHO 02/254. Furthermore, the antibodies of the phosphorylated IGF-IR TR-IFMA have been changed. Coating is now performed using the monoclonal antibody (part 841431, 4 mg/l) contained in the human phospho-IGF-IR kit from R&D Systems (catalogue no DYC 1770E). For detection we use the biotinylated monoclonal phosphotyrosine antibody BAM 1676 (2 mg/l; R&D Systems). The latter is co-incubated with streptavidin europium.
In the KIRA assay, the ability of serum to phosphorylate the IGF-IR in vitro is compared of a serial dilution of rh-IGF-I (WHO 02/254), and accordingly, it is possible to express the serum signal in IGF-I concentrations. The KIRA also detects IGF-II and pro-IGF-II activation of the IGF-IR with a cross-reactivity of 12% and 2%, respectively, of that of IGF-I, whereas proinsulin, insulin and insulin analogues have a cross reactivity <1%. The KIRA has a detection limit <0.08 μg/l, and intra- and inter-assay CVs averaging <7% and <15%, respectively.
IGFBP-1 was assayed by an in-house TR-IFMA, which represents a modification of our previously described RIA [
37]. In brief, microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 μL per well of MAB 6303 (Medix Biochemica, Kauniainen, Finland, 1 mg/L dissolved in phosphate buffer, 40 mM, pH 8.0) and incubated overnight at 5°C. MAB 6303 recognizes all phosphorylated isoforms of human IGFBP-1 [
38]. Next day, all wells were washed once (PBS, pH 7.4 added 0.5% (vol/vol) Tween 20 and 0.05% (wt/vol) NaN3) before they were blocked (40 mM phosphate buffer, pH 8.0, added 5% (vol/vol) Tween 20 and 25 μM EDTA). After 2 hrs of blocking at room temperature, the wells were washed once and added 100 μL antigen. Purified human amniotic IGFBP-1 (catalogue no 8IGB1, HyTest, Turku, Finland) served as standard (range: 1.56-50 μg/L). Serum was diluted 1:10. All antigens were dissolved in assay buffer (40 mm phosphate, pH 8.0, added 25 μM EDTA, 0.1% (wt/vol) bovine serum albumin, 0.05% (wt/vol) NaN
3, 0.9% (wt/vol) NaCl and 1% (vol/vol) Tween 20)) and assayed in duplicates, whereas non-specific binding (NSB) was assayed in quadruplicate. After an overnight incubation at 5°C, the wells were washed and added 100 μL (400 μg/L) of polyclonal rabbit antibody directed against human IGFBP-1 (catalogue no sc-13097, Santa Cruz Biotechnology, Heidelberg, Germany) dissolved in assay buffer. After two hours of incubation at room temperature all wells were washed and added 100 μL biotinylated goat anti-rabbit IgG (catalogue no. BAF008, R&D Systems) together with Europium-labeled streptavidin added in a final dilution of 1:1000. After two hours incubation at room temperature all wells were washed 6 times, added enhancement solution (200 μL/well, Perkin Elmer Life Sciences, Turku, Finland) and read using time-resolved fluorometry. The coating antibody MAB 6303 has previously been shown to neglect rhIGF-I and -II as well as rhIGFBP-2, -3, -4, and −5 in concentrations up to 10,000 μg/L [
37]. The sensitivity of the assay is well below the lowest standard (the signal to NSB ratio corresponding to the lowest and highest standards averaged approximately 7 and 70, respectively). The intra-assay CV of samples assayed in duplicates averaged less than 5%, the inter-assay CV of an internal control (IGFBP-1 standard) and a control serum sample averaged 8.1 and 7.0%, respectively (22 plates).
IGFBP-2 was assayed by an in-house TR-IFMA as previously detailed with an intra- and inter-assay CV of < 5% and < 12% respectively [
37].
Serum insulin was measured by a commercially available TR-IFMA (AutoDELFIA insulin; Perkin Elmer Life Sciences). Insulin aspart (NovoRapid®) measurements were generously performed at the laboratories of Novo Nordisk A/S (Måløv, Denmark) using a homogenous immunoassay with analogue-specific antibody for insulin aspart determination.
Plasma cytokines (IL-1β, IL-6, and TNF-α) were measured in a magnetic Bio-Plex Pro Assay (Bio-Rad, Hercules, CA, USA), as described by the manufacturer. Detection limits were between 0.5 and 1 ng/L. A Luminex100 using the BioPlex Manager 6.0 software (BioRad) was used for analysis.
High-sensitivity CRP (hsCRP) was determined by an in-house TR-IFMA assay based on two commercial monoclonal antibodies (catalogue no. MAB17071 and BAM 17072 obtained from R&D Systems). The assay was performed essentially as described for the IGFBP-1 assay. As standard we used a preparation from NIBSC (1st international standard, NBISC code 85/506, Potters Bar Hertforshire, UK). The intra- and inter-assay CVs averaged less than 5 and 11%, respectively.