ローダミンBを標準物質とした血中プロトポルフィリンの蛍光微量定量法

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Free erythrocyte protoporphyrin (FEP) level is elevated as the blood lead concentration increases in lead poisoning, and it has been recognized as a critical indicator reflecting the biological effects of lead on the bone marrow. However the well-established spectrophotometric methods (classical methods) for FEP determination are not popular in clinical diagnosis because of the complicated and time-consuming procedure in which repeated partitions between hydrochloric acid and organic solvents are involved. Although several rapid micro-methods for FEP-test have been reported, they are still inadequate for the purpose of the routine test. For this reason, a simple and rapid micro-fluorometric method for FEP-test is developed in our laboratory. This method is basically to determine total erythrocyte porphyrins (EP) after conversion of the pigments to the free form by extraction with acidic media. The outline of the method is as follows; 0.05ml of whole blood is pipetted into the test tube containing 0.25ml of 5% (w/v) celite suspension in the physiological saline solution. Add 4ml of ethyl acetate-acetic acid mixture (3:1 v/v), and agitate on a Vortex mixer for 30sec. After centrifugation at 3,000rpm for 5min, all the supernatant is decanted into a small measuring cylinder. From the solvent mixture, porphyrins are extracted with 4ml of 1.5 N-HCl on a Vortex mixer for 30sec. Read volume of the final HCl extract (V ml), and transfer an aliquot of the final extract into a cuvette for fluorometry. Rhodamine B-ethylene glycol solution (3.0×10^<-3>μg/ml) is used as the standard fluorescence. The emission fluorescent spectra, ranging from 560 to 620nm in wave length, of the final acidic extract and of Rhodamine B standard are recorded under the excitation wave length of 403nm with Shimadzu RF-500 spectrofluorometer equipped with Hamamatsu R-446 U red-sensitive photomultiplier tube. A blank solution is prepared in parallel through the whole procedure by replacing the blood to saline, but its fluororescence is almost negligible. Since the maximum fluorescence intensity of the standard (F_R:at577nm) is equivalent to that of the 2.0×10^<-3>μg/ml protoporphyrin in the blank solution (F_s:at605nm), the concentration of FEP is calculated from the formula, EP μg/dl of blood=(F_S/F_R)×0.002×(V/0.05)×100, in which 0.002 is the conversion constant. The EP and FEP levels of 48 lead workers were determined by both the present and the classical methods. A close correlation (r=0.96,n=48) was found between the values determined by both methods. The present method is an adequate one for our purpose.
1979-01-20

ローダミンBを標準物質とした血中プロトポルフィリンの蛍光微量定量法

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