Storage stability of hormone solutions - How would you prepare your working solution if your boss thinks that n (May/10/2007 )
We treat monolayered cells with hormones (cortisone, cortisol, estradiol, testosterone and similar).
We prepare stock solutions mainly in ethanol, considering that most of these molecules easily dissolve in it. We weigh an amount of powder and prepare the mother solution from which we obtain less concentrated solutions by serial dilutions.
My question is: how can we consider stable a molecule in its stock solution? How many times would you prepare a new mother solution?
I think that the more concentrated is the solution, the more the molecule is stable. So if we open very few times the mother solution (in order to lessen the probabilities that ethanol evaporates and the concentration changes) would it be considered stable if kept at -20°C.
My boss thinks that we should prepare new mother solutions each working day. I think this is a little bit too expensive in terms of time and money. But how can we get informations about storage and stability in solution of these compounds? Is there a sort of Bible of reference and, if any, would you trust in it ?
Ehehehe my boss never trusts in anything...
Hope to have your suggestions soon,
Silvia
The half life of cortisol in serum is 100mins. In this environment cortisol binds to and is protected by transcortin (aka cortisol binding globulin, CBG). Aldosterone, on the other hand, isn't bound by CBG and has a half life of 30mins. Metabolism is by enzymatic degredation.
This wont apply to your mother solution so if the chemical literature can't answer your steriod stability in EtOH issue, why not set up a series of experiments. When you split your cells, take an extra flask of them. Make up your mother solution. Every day test the mother solution stability on some of the cells (split them again). Keep a flask of the cells as a control that you don't give the hormones to. I suspect the cells will live for a few days in hormone depleted media so you may have to lengthen the interval between splitting. At some point the cells you feed hormones will survive and those you starve...will not. Then you can prove to your boss (or he prove to you) that stability is/isnot an issue.
Alternatively, you can analyse a cell marker. Calcium works for cell surface receptors (e.g. acetylcholine, endothelin etc). I'm not sure what nuclear marker's are best used nowadays.
Thank you ! How thorough is your answer!
I will try during our research dead times!!
Where did you find these informations
My one and only hope is on the Merck index....
thank you again
Silvia
I will try during our research dead times!!
Where did you find these informations
My one and only hope is on the Merck index....
thank you again
Silvia
Merk Index should have the info. Otherwise, the "Data for Biochemical Research" is a gold mine. Someone in the lab will have a copy. As far as the physiology of the cortisol etc goes, any medical biochemistry text will help. The best if probably Tietz "Textbook of Clinical Chemnistry." Thisi san American text so uses American units (mass rather than moles).
Just get in touch if I can help, though.