Four immediate thoughts:

1. your skin isn't producing enough active form of VitD to be detectable. Typically only 1% gets activated in any case. Have you tried UV irradiating them for longer? If the skin you're using is pigmented, then UVB might not have as much effect. Alternatively, not enough 1a hydroxylase around for second activation stage.

2. Active vitamin D is retained and acts in an autocrine fashion, so not enough gets into your medium (it's fat soluble, so solubility in non-lipid rich medium might be low) or it's used up very quickly

3. You haven't left it long enough for diffusion to significantly make a difference to concentrations in your culture medium.

4. Your light source also contains UVA (kills VitD more or less)

Some crude experiments you could try just to see if any of the above are at play: irradiative for longer; leave skin in insets for longer; homogenize the skin after irradiation (early and late after UVB) and collect the clarified supernatant and/or try to elute lipid fraction; Take some RNA and PCR for 1a hydroxylase; rule out contaminating UVA from your light source (unlikely, but best to check).

Hope this helps.

All the best,

B

light Exposition will produce only Vitamin D3 (cholecalciferol). To optimize this step you might add the precursor 7-dehydrocholesterol to your system.

But then, the next step to the storage form (25-hydroxyvitamin D3) is performed in the liver. I doubt that the Enzyme  (25-Hydroxylase) is in Skin.

Furthermore, more complicated, the third step to 1,25(OH)2D3 is in kidney and the 1-Hydroxylase might also not be present in Skin in sufficient activity.

So,  i see the Chance quite low to detect 1,25(OH)2D3

 Hi Henry,

Thanks for the answer, there are several publications indicating that the skin is able to complete the full process from 7-DHC to 1,25(OH)2D3, and in fact is the only organ that is capable of doing so:

Both in-vivo:

Demonstration of UVB-induced synthesis of 1 alpha,25-dihydroxyvitamin D3 (calcitriol) in human skin by microdialysis.

and in-vitro:

UVB‐induced Conversion of 7‐Dehydrocholesterol to 1α,25‐Dihydroxyvitamin D3 (Calcitriol) in the Human Keratinocyte Line HaCaT

1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation.

UVB-Induced Conversion of 7-Dehydrocholesterol to 1α,25-Dihydroxyvitamin D3 in an In Vitro Human Skin Equivalent Model

But the suggestion of adding 7-DHC to increase production is something we will definitely try. 

Hi Reuben

You are quite right, skin does have the capacity to produce active 1,25(OH)2D3, having both hydroxylases.

I suspect your problem is simply that your skin sample is too small. It may also be that the body site you have taken the biopsy from has low 7-DHC content - it is interesting to note that the last paper you cite used a skin equivalent model with exogenous 7-DHC and 1,25(OH)2D3 was produced in femtomolar concentrations, can you detect such small amounts? You might want to consider adding 7-DHC to your system as Henry suggested but this is somewhat artificial in terms of physiological concentrations.

Prolonging the UVB irradiation will not necessarily increase 1,25(OH)2D3. There is more photochemistry going on than just 7-DHC to pre-D, and excessive UVB leads to accumulation of inactive end-products.

Thanks Mark, 

no we can only detect upwards of 1nM which is definitely something I noted. I was optimistically hoping a whole skin biopsy might produce more, what is also interesting is it seems the concentration of 1,25 VitD in cell lysates is actually higher than is released extracellularly, so even if we where inducing enough synthesis perhaps it is being maintained for auto and paracrine signalling. And DBP seems to actually sequester away from T-cells, pergola

Hi Reuben,

your right there are other tissue capable to produce 1,25VD. Without reading your cited Papers: the sensitivity of the Analysis seems challenging. What method are you using?

- UPLC/MS-MS (here may the sensitivity of the method be limiting)

- immuno assays (here may the cross-reaction to 25VD and VD3 be a problem

- radioactive precursors (the old method) and separation by chromatography?