Preparation of Retinoid Solutions and Stability

Retinoids are insoluble in water, but soluble in organic solvents, such as ethanol and dimethylsulfoxide (DMSO), which are the most commonly used solvents for administration of tRA. Although one always performs control experiments, it is important to be aware of the fact that DMSO acts as a differentiating agent to embryonal carcinoma cell cultures in the same way that tRA does, so perhaps ethanol is a "better" solvent. The maximum solubility of tRA in DMSO is about 50 mg/mL.

Retinoids are stable in powder form for many months at -20°C. Light and oxygen cause isomerization and breakdown, so they are often stored in brown vials in an atmosphere of nitrogen. Solutions of retinoids are unstable, especially at room temperature, and tRA in DMSO begins to isomerize to 13-cis-RA, 11-cis-RA and 9-cis-RA in a few minutes when left on the laboratory bench. This can be very useful for generating standards for HPLC, but a problem when administering retinoids to animals over periods of several hours! The latter tends to negate the emphasis on absolute purity that many retinoid researchers insist on—as soon as one administers these compounds to the embryo, either the light or the embryonic cells will change the nature of thesubstance you have given it. Nevertheless, for experimental purposes it is better to make up one solution and aliquot it as follows;

1. Prepare 1 mL of a l0 mg/mL solution of tRA (Sigma, Dorset, UK) in DMSO (Spectrosol or Analar-Grade, BDH, Poole, UK) or ethanol.

2. Split the 1 mL into 5-pL aliquots in capped tubes.

4. Use one aliquot for each experiment, never refreeze and use again.

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