LDH measurement - can be done in frozen samples? (Nov/13/2007 )

hi!

I´d like to know if I can apply my treatment to the cells (primary rat neurons and astrocytes), then take the medium (where LDH is suppose to be due to releasing from damaged cells) and froze it in aliquots until I perform the LDH measurement (1 week maximum). Is this possible or LDH will degrade?
Thanks

-Pumuki-

LDH is indeed a (non-specific) marker of cell damage. It actually consists of 5 isoenzymes which have different responses to storeage conditions. LDH4 and 5 are sensitive to cold and all activity of these is lost after overnight storeage at -20C. A way round this is to add NAD or glutathione. In serum samples, albumin actually helps protect the LDH but you wont have serum, will you? Serum samples can be stored for 2-3 days with no loss of activity at room temp but need NAD or glutathione and storeage at 4C for longer periods.

One other consideration is which LDH assay you'll use. Will you measure mass or activity? Will you use NADH spectroscopy (LDH catalyses pyr to lact conversion). Will you use the forward or reverse reaction (probably makes no difference to storeage conditions). Will you use a kinetic or end point assay. All that said, there is a decent electrophoresis kit on the market if you needed to look at the isoenzymes (doubt it, that's usually used to differentiate muscle from cardiac damage).

This used to be a hot debate topic in clinical chemistry so the literature will present many opinions. If it were me, I'd try both immediate analysis and post-storeage in the same sample. What controls will you use?

-paraboxa-

Thanks for your answer!

QUOTE (paraboxa @ Nov 13 2007, 11:55 AM)

In serum samples, albumin actually helps protect the LDH but you wont have serum, will you? Serum samples can be stored for 2-3 days with no loss of activity at room temp but need NAD or glutathione and storeage at 4C for longer periods.

My cells grow on DMEM + horse serum (10%) but during the treatment I substitute it for PBS.

QUOTE (paraboxa @ Nov 13 2007, 11:55 AM)

One other consideration is which LDH assay you'll use. Will you measure mass or activity? Will you use NADH spectroscopy (LDH catalyses pyr to lact conversion). Will you use the forward or reverse reaction (probably makes no difference to storeage conditions). Will you use a kinetic or end point assay. All that said, there is a decent electrophoresis kit on the market if you needed to look at the isoenzymes (doubt it, that's usually used to differentiate muscle from cardiac damage).

I have LDH assay kit (Sigma) to measure amount of LDH released to the medium, based on the reduction of NAD by the action of LDH. Then NADH is used in the conversion of a tetrazolium dye which is measured at 490 nm.

QUOTE (paraboxa @ Nov 13 2007, 11:55 AM)

This used to be a hot debate topic in clinical chemistry so the literature will present many opinions. If it were me, I'd try both immediate analysis and post-storeage in the same sample. What controls will you use?

My controls are non-treated cells in PBS.

-Pumuki-

QUOTE (Pumuki @ Nov 13 2007, 02:22 AM)

Thanks

yes, but shock freeze medium; some lost of activity is to expect

-The Bearer-