Step Procedure accomplished Equipment Data Collected 1 Terminal blood drawn and processed for serum - - 2 Serum samples pooled (2 mice) - - 3 Serum samples assayed for vitamin D using radioimmunoassay (RIA) RIA kit, Gamma counter serum Vitamin D
- Balance scale
- Blood collection kits (serum separation tubes, needles, syringes etc.)
- Microcentrifuge tubes
- -80°C freezer
- Gamma counter
- Heartland Assays, Inc. (Iowa State University Research Park, Ames, IA)
- RIA kit with antibody that is co-specific for 25(OH)D2 and 25(OH)D3
- Radioactive tracer, 125I-labeled tracer
- Goat antibody for vitamin D 25(OH)D3 (primary antibody)
- Donkey anti-goat antibody (secondary antibody)
- Secondary antibody-precipitating complex
Blood collection and serum preparation
a. Blood is drawn from 11 wks old male mice.
b. Blood samples are allowed to clot at room temperature for at least 1h.
c. Serum is separated from blood cells using serum separation tubes and a table top centrifuge at a speed of 13,000 x g for 20 min.
d. The serum layer is pipetted carefully without disturbing the packed blood cell layer below and then transferred into a new pre-labeled tube.
e. An aliquot of serum from two individual mice are pooled and stored at -80°C until analysis. Note that a pooled sample is given a unique identification number and does not represent individual animals.
f. Frozen serum samples are sealed in plastic bags and shipped on dry ice for transport.
Measurement of serum Vitamin D concentration
Samples were shipped to Heartland Assays, Inc. for quantification of vitamin D (25(OH)D) by an equilibrium radioimmunoassay (RIA) using a radioactive (125I) tracer, primary goat antibody to 25(OH)D, and donkey anti-goat as secondary antibody (see Wagner et. al. 2009 ). To monitor assay performance each assay includes one in-house control treated as an unknown specimen and multiple determinations are made. Assay variation is determined and acceptable performance limits are defined as ≤15 CV.
a. Vitamin D and other hydroxylated metabolites are rapidly extracted from serum samples with acetonitrile organic solvent.
b. Following extraction, the acetonitrile treated samples are then assayed using an equilibrium radioimmunoassay (RIA) step based on an antibody that is co-specific for 25(OH)D2 and 25(OH)D3.
c. Each sample is mixed with a cocktail of primary antibody and tracer and incubated for 120 min at room temperature (20-25°C).
d. After a 20 min incubation at 20-25°C, phase separation step is accomplished with the addition of a secondary antibody-precipitating complex.
e. To reduce non-specific binding, buffer is added after incubation of each cocktail and before the centrifugation step.
f. Following phase separation, samples are centrifuged to remove unbound complexes and metabolites.
g. 25(OH)D-equivalent values are calculated directly by the γ-radiation counting system with use of a smooth-spline method.
h. Samples with radioactive tracers are disposed of in specifically marked and designated bins.
25(OH)D: 25-hydroxyvitamin D exists in two forms as (1) cholecalciferol (vitamin D3), which is derived from synthesis in the epidermis, and (2) ergocalciferol (vitamin D2), which is derived solely from plant sources.
• serum vitamin D concentration