Quantitative fluorometric screening test for fecal porphyrins.

Department of Pathology, Vancouver General Hospital, University of British Columbia, Canada.
Clinical Chemistry (Impact Factor: 7.91). 07/1991; 37(6):826-31.
Source: PubMed


We describe a fluorometric method for screening and quantifying porphyrins in stool. A small sample of stool is extracted with concentrated HCI and is diluted 200-fold in 3 mol/L HCI before analysis. An excitation scan is done from 350 to 450 nm, monitoring emission at 603 nm. Total porphyrin is estimated at the isosbestic point for coproporphyrin and protoporphyrin (402.5 nm). Monitoring emission at 603 nm eliminates interference from chlorophyll, obviating the need for extraction with ether. The position of the excitation peak gives some indication of the nature of the porphyrins in the stool. The acid extract can be injected directly into an HPLC system for fractionation studies. Our method correlates well with the spectrophotometric method developed by Lockwood et al. (Clin Chem 1985;31:1163-7). However, in our method, the sample is easier to process and the assay has higher sensitivity than their assay. The reference interval for porphyrin in healthy individuals by the fluorometric method is less than 300 nmol/g dry weight. We can detect as little as 1 nmol of porphyrin per gram (dry weight) of stool. Results of the method vary linearly with stool porphyrin concentrations as great as 4000 nmol/g dry weight. The within-run imprecision of the method is 3%.

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    • "Fluorescence signals were recorded at the Soret band (i.e. absorption band in the blue region of all porphyrins) peak L exc = 400 nm (Martinez & Mills 1971, Schwartz et al. 1976, Westerlund et al. 1988, Pudek et al. 1991) and corrected for background uorescence by subtracting the corresponding values determined for the blank measurements. e detection limit was determined as the mean of three blank measurements plus three standard deviations. "
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