GENERAL METHODS

All radioimmunoassays and related procedures are conducted with a high standard of quality control procedures. Extraction recoveries are measured by extracting pools in every tenth position containing the radioactive analyte being assayed, and by also extracting one or more controls containing known concentrations of the non-radioactive analyte. Within- and between-extraction means and coefficients of variation are measured. Assay results are corrected for recovery. Within- and between-assay means and coefficients of variation are also measured using pools containing unlabeled analyte in every tenth position, and between-laboratory comparisons of means and coefficients of variation are obtained by assaying commercial controls, when available, in each assay run. Results are not reported to investigators for assays having within- or between-assay pool or control concentrations outside the 95% confidence limits. Instead, the assays are repeated after correcting the cause of the excessive imprecision or inaccuracy.

1. Aldosterone. Plasma is extracted into 7 volumes of dichloromethane, and the extract is evaporated to dryness and reconstituted in buffer. Assay is by radioimmunoassay using standards from Sigma, and tracer and a highly specific liquid-phase antibody from Diagnostic Products.

2. Angiotensin II + III (AII, AIII). Plasma is extracted by C-18 Cartridge (Sep-Pak Plus) and assayed by radioimmunoassay using standards, tracer, and antibody from Beckman, New England Nuclear, and Chemicon, respectively. An AI assay (see "Renin activity and Angiotensin I (AI) concentration" below) of the extract is also done to enable correction for cross-reaction with AI. (Budgeted costs of the AII assay include the cost of the AI assay.) The antibody cross-reacts 100% with AIII, and no correction is done for AIII.

3. Cortisol and Cortitosterone. Cortisol and cortitosterone are measured respectively by radioimmunoassay using an antibody-coated tube kit from Diagnostic Products.

4. Insulin. Insulin is measured by radioimmunoassay. For serum or heparinized samples from most species, the radioimmunoassay uses standards, tracer, and antibody-coated tubes from Diagnostic Products. Because of lot-to-lot variations in cross-reactivity of insulin antibody with human, dog, and rabbit insulin, the Core's assay lab tests, then stockpiles a several-year supply of a satisfactory lot. For samples from rats and mice, the radioimmunoassay employs a kit from Linco. Since the antibody in this kit has very high cross reactivities with human, dog, and rabbit insulins, and is not adversely affected by EDTA, the Linco kit is used as a backup assay for samples from those species which were collected with EDTA.

5. Renin activity and Angiotensin I (AI) concentration. Renin activity in plasma is measured by a modification of the method of Haber and associates (25). This consists of adding buffer and peptidase-inhibitors to the sample and incubating 1 aliquot of the sample for 1 hour at 37° C and pH 7.4 while keeping another aliquot at 0-4° C. The AI concentrations in the two aliquots are then assayed at 2° C by radioimmunoassay using AI standards, tracer, and antibody from the National Bureau of Standards, New England Nuclear, and Arnel, respectively. The AI concentration in the aliquot kept at 0-4° C is then subtracted from that in the 37° C aliquot to yield the renin activity.

6. Leptin. Plasma concentrations of leptin in our rat/mouse samples are measured by radioimmunoassay using a kit from Linco. Plasma concentrations of leptin in human or dog samples are measured by ELISA using a kit from NEOGEN.

7. ACTH. Plasma concentrations of ACTH are measured by radioimmunoassay using a kit from DiaSorin.