In rat studies, I have found that blood plasma from animals given a certain treatment has a particular effect on cells in culture compared with plasma from untreated rats. I am sure that it is a hormone in the plasma that is affected by the treatment and causes the effect, but I have no idea what it is. Apart from fractionating the plasma and semi-purifying the hormone on the basis of fraction bioactivity in cell cultures, is there any other method of getting to the hormone identity in a more efficient way?
I thing in blood clinical analysis they have all the techniques and equipement to find the hormones you are looking for.
I have microarray data from the cells, so one approach has been to work backwards by gene ontology and pathway analysis to find the receptor. However, it's not specific enough and I can't identify a particular pathway.
I would agree with the clinical lab route. Find a lab in the area that will run samples for you. Let them know what you are looking for so they can tailor the test to your samples.
You can use liquid chromatography-mass spectrometry using blood plasma from animals given your treatment vs plasma from untreated rats and search for differences in peaks that appear.
Thanks for the suggestions.
Clinical lab: we have screened for all the usual suspects using Luminex without success, but most assays for low abundance hormones are immunoassays, and a negative result from a clinical lab might just mean no cross-reactivity with the rat protein (if it's a protein).
LC-MS: Yes, this might be a way forward using tandem mass spectrometry - it will be a complex mix so some preliminary fractionation might be necessary. Sensitivity may also be an issue but this technique would pick up structural changes (e.g. phosphorylation) as well as concentration changes.
Anybody have suggestions for a sensing approach in the bioassay cells?
Without further explanation about the treatment and the effect observed on the cells it is hard to give an opinion. What you are assuming is an hormone may very well be a growth factor or a cytokine. It would be important at least to clarify what is specifically that you observed in the cell culture.
No, it's not a drug or any kind of chemical/physical treatment that could result in the appearance of xenobiotic metabolic products in the plasma. Hence, I'm fairly sure it's a hormone. Yes, it could be any class of hormone including a steroid, and my fractionation should give me some information on molecular weight and whether it's bound to another plasma component (as steroids tend to be).
May be going back to old physiology : a hormone is a messenger produced by an organ and affecting an other organ .You could compare the effect and the proteomic profile of hypophysectomized (or pancreatectomized or ....ed) animal sera mixed with drug treated animal serum to the only drug treated animal serum. It could give clues from what organ is coming your hormone since the serum from the endocrine ablated organ will decrease your effect if the hormone is produced by this organ. Conversely if you have serum from a model animal of hypersecreting endocrine adenoma, you will increase your effect. You can also piked the serum with various hormones.
I am sure there is an elegant and efficient way of identifying the hormone, and going back to the physiology in vivo might be a good approach.
My thanks to all contributers.
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