A spectrophotometric micromethod for determining erythrocyte protoporphyrin-IX in whole blood or erythrocytes.
An increased concentration of erythrocyte protoporphyrin-IX in whole blood or erythrocytes is a valuable diagnostic indicator for acquired porphyrias, e.g., iron deficiency anemia and lead poisoning, and for inherited porphyrias. We developed a spectrophotometric micromethod for determining erythrocyte protoporphyrin-IX. In this method, exhaustive release of erythrocyte porphyrins is achieved using hydroquinone and formic acid. The clean-up procedure for 50 microL of whole blood or erythrocytes covers three steps of liquid/liquid solvent partition: two partitions using diethyl and diisopropyl ether and HCl 2.5 mol/L, and one buffered step using ammonium formate. Determinations of erythrocyte protoporphyrin-IX are possible by: (a) absorption using three wavelengths, Rimington's constant and a millimolar absorptivity coefficient m epsilon(408.8)=294.3 L x mmol(-1) x cm(-1) according to With; and (b) 2nd derivative, which is linked to m epsilon(408.8). Determination of erythrocyte protoporphyrin-IX using a 2nd derivative algorithm showed better spectral resolution and higher sensitivity at a five-fold lower detection limit compared to absorption. Within-run precision of medium and high levels was found for absorption and for 2nd derivative with a coefficient of variation (CV) of 1.4-1.9% (n=10). Total precision evaluated was CV=2.5-8.3% (n=20). Levels of reference intervals could only be measured using the 2nd derivative (CV 2.9%). Linearity was proved to E=1.0. Recoveries of protoporphyrin-IX ranged from 95.3% to 103.0%. Method comparison was carried out using a fluorimetric reference method (Piomelli). Reference intervals for gender groups are discussed.
Kufner
Guenther
G
Occupational Health Service AMD-Linz, Linz, Austria. kufner-ag@aon.at
Schlegel
Helmut
H
Jäger
Reinhard
R
D000465:Algorithms; D004912:Erythrocytes / Q000378:metabolism*; D004986:Ether / Q000737:chemistry; D004987:Ethers / Q000737:chemistry; D005470:Fluorometry; D005561:Formates / Q000737:chemistry; D006801:Humans; D006851:Hydrochloric Acid / Q000737:chemistry; D006873:Hydroquinones / Q000737:chemistry; D011524:Protoporphyrins / Q000097:blood*; D012680:Sensitivity and Specificity; D012997:Solvents / Q000737:chemistry; D013053:Spectrophotometry / Q000379:methods*
D004987:Ethers; D005561:Formates; D006873:Hydroquinones; D011524:Protoporphyrins; D012997:Solvents; D004986:Ether; C030544:formic acid; C028025:protoporphyrin IX; C011779:diisopropyl ether; D006851:Hydrochloric Acid; C031927:hydroquinone