Improved ethanol extraction procedure for determining zinc protoporphyrin in whole blood.

We describe a fluorometric method for determining zinc protoporphyrin, in which whole-blood specimens collected in heparin/ethylenediaminetetraacetate are prediluted with water, and zinc protoporphyrin is extracted into ethanol/ water (95/5 by voi). The resulting extract has a low absorbance (0.05) at the excitation wavelength (415 nm) of zinc protoporphyrin, and the extraction efficiency is about 33% greater than Is true for ethyl acetate/acetic acid extraction. When 10 zl of whole blood is used, the ethanol extraction procedure with predilutlon has a detection limit of 40 tg of zinc protoporphyrin per liter of whole blood, linear response to at least 100 mg/lIter, day-to-day precision of better than 5% (CV) over the concentration range 360-2000 tg/llter, and within-run precision of better than 1.5% at concentrations exceedIng 300 zg/llter. Analysis of 143 blood samples by both the ethanol-with-predilution and the ethyl acetate/acetic acid extraction procedures yielded the relationship [erythrocyte porphyrin] = 0.75 (±0.05) [zInc protoporphyrin] 0.95 (± 1.58) at the 95% confidence level, erythrocyte protoporphyrln” being that measured by the ethyl acetate/acetic acid procedure. Comparison of erythrocyte porphyrin data obtained by calculation from zinc protoporphryin values and by direct measurement showed no significant difference between the two procedures (Student’s t-test). The preliminary dilution step is essential for maximum extraction.