I am going to extract protoporphyrin from human cells. I followed two extraction methods from publised articles but the results are not satisfactory. Can anyone suggest a proper extraction protocol?
Dear Ripon,
The attached protocol is Laboratory Procedure Manual for Erythrocyte Protoporphyrin from Whole Blood which was developed by New York State Department of Health Extraction Method
Method No.: 0164A Revised: as performed by: New York State Department of Health Wadsworth Center Lead/Poisoning Trace Elements Laboratory
The following text is the summary of the method, the full method is contained in the attached file.
1. SUMMARY OF TEST PRINCIPLE AND CLINICAL RELEVANCE
Porphyrins and heme components are extracted from whole blood into a 4:1 mixture of ethyl acetate-acetic acid. Porphyrins are then separated from heme by back-extraction into a 1.5 M hydrochloric acid solution, and quantitatively determined by molecular fluorometry using a spectrofluorometer calibrated with
protoporphyrin IX (PPIX) standard solutions; however, the exact concentration of the standards must first be established using molecular absorbance, Beer’s Law, and the millimolar absorptivity of PPIX.
The analytical method for erythrocyte protoporphyrin (EP) routinely employed by the EP Lab is based largely on those originally described by Sassa et al. (1973) and Chisolm and Brown (1975). New York State’s extraction method owes much to contributions from other public health labs, including the CDC, and closely follows the key elements of the consensus method for EP as published by the National Committee for Clinical Laboratory Standards (NCCLS C42-A*, 2001). At the invitation of Dr. Sassa, the EP Laboratory’s routine method for EP was published as Unit 8.8 in Current Protocols in Toxicology, 1999 by J. Wiley & Sons, Inc. Elements of this protocol are reproduced below, but a reprint of the original publication is available from the EP lab director.
Hoping this will be helpful,
Rafik
Dear Rafik Karaman
Thank you for you suggestion. method is very helpful for quantification of PPIX. I am not working with whole blood. I want to extract cellular or mitochondrial PPIX from epithelial cell line. Can you suggest me any method for it, or this method is also applicable for cellular PPIX extraction.
Ripon
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