Our thanks to all who replied. Nobody volunteered to offer us the ferrochelatase assay. Some suggested groups that might do it. Others pointed out the technical difficulties. Most pointed out that it was probably not a good idea anyway. We will consider the offer of some groups to do gene studies on our case with a view to family screening.
Some collated and edited replies:
My advice ([log in to unmask]) with regard to ferrochelatase assays is don't bother. We have tried measuring this enzyme in lymphocyte preparations with no success due to blank problems. The enzyme assay is not required for diagnostic purposes. Patients with EPP have extremely high levels of free protoporphyrin in red cells. Some assay unfortunately measure both ZnPP and PP. However, we use the method of Schwartz et al (Int J. Biochem Vol 12, p1053, 1980) which measures both ZNPP and PP using synchronous scanning 2nd derivative fluorescence spectroscopy. This does require a modern fluorimeter capable of performing this type of scanning work. With this method we can pick up carriers in an EPP family as they have slightly raised red cell PP levels of around 2x ULN. If you are still keen on looking at ferochelatase activity then I would suggest reading a paper by Sassa S . et al. (J. Clin Invest. vol 69, p809, 1982). They use cultured lymphocytes and measure the PP produced post incubation with ALA. You can modify this method and use a HCL/DMSO extract of the cells post incubation to look at the porphyrins produced by HPLC. This method does appear to work but is very time consuming.
From ([log in to unmask]): The big problem for ferrochelatase was always the separation of a buffy coat (rich inleukocyte mitochondria) and transport of the preparation frozen to a reference lab. Some of the interesting things that we have done with blood from patients with EPP is to look for natural fluorescence exhibited by RBCs using a fluorescent microscope with blue filter - all cells would exhibit a red orange fluorescence that rapidly fades - we have also used a FACS to show that RBCs from patients with EPP show a increased fluorescence (peak shifted to right) using a non-EPP patient as control. EPP patients still give an elevated "ZnPP" when using an hematofluorimeter - so we have not found this to technique to be useful in distinguishing free from zinc protoporphyrin. EPP would usually be diagnosed in childhood and other kin would be expected to be affected. In theory you would expect the bile and faecal levels of protoporphyrin to be increased - in practice many faecal samples do not show elevations. The other thing to do distinguish EPP from ZnPP elevations is to measure both whole blood porphyrins and plasma porphyrins both should elevated with EPP as FEP leaks out of RBCs while the plasma porphyrins should not be elevated as the ZnPP remained trapped in the RBCs.
The policy at the Cardiff Porphyria Laboratory has been to perform a red
cell scan to detect ZN and free protoporphyrin, then quantitate the plasma
porphyrin. If the rised total porphyrin is all free protoporphyrin, and
there is a plasma peak at 630 (+/-4 nm), then the diagnosis is EPP and there is no need to do ferrochelatase (which we don't). However we do perform mutational analysis, looking for the severe mutation and the low expression allele. We ([log in to unmask]) would be happy to help with this if you wish. The only lab I know that might do a ferrochelatase assay is the Centre Francais des Porphyries, Service de Biochimie et Génétique Moléculaire, CHU Louis Mourier, 178, Rue des Renouillers 92700 Colombes run by Jean-Charles Deybach. I'm afraid I don't have an email address, but they do have a website:<http://www.porphyries.com.fr/>
We have/are developing a mutation screening assay (DHPLC) for the FECH-gene and would happily analyse DNA samples from the family starting with the index-person. (Niels Erik Petersen Odense, Denmark: [log in to unmask])
Thanks again to all
Dr Chris Florkowski
Christchurch, NZ
**********************************************************************
This email and any files transmitted with it are confidential and
intended solely for the use of the individual or entity to whom they
are addressed. If you have received this email in error please notify
the system manager.
This footnote also confirms that this email message has been swept by
MIMEsweeper for the presence of computer viruses.
www.mimesweeper.com
**********************************************************************
------ACB discussion List Information--------
This is an open discussion list for the academic and clinical
community working in clinical biochemistry.
Please note, archived messages are public and can be viewed
via the internet. Views expressed are those of the individual and
they are responsible for all message content.
ACB Web Site
http://www.acb.org.uk
List Archives
http://www.jiscmail.ac.uk/lists/ACB-CLIN-CHEM-GEN.html
List Instructions (How to leave etc.)
http://www.jiscmail.ac.uk/
|