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1. 1. Two new procedures are described for the quantitative assay of “free” and zinc-protoporphyrin in red blood cells. 2. 2. Major clinical findings have included the following: (i) An average of about 10% of the total erythrocyte protoporphyrin is “free” in normal humans, but about one-third or more is usually “free” in blood of normal cows, rats and rabbits, (ii) In addition to human and bovine protoporphyria, “free” protoporphyrin was increased in (a) genetic carriers (clinically normal) of bovine protoporphyria, (b) acute Pb poisoning, and (c) some patients undergoing dialysis, (iii) Significant amounts of zinc-coproporphyrin were found in the blood of several dialyzed patients and in some “normal” cows.

The use of porphyrins and metalloporphyrins to modify the radiosensitivity of tumor and nontumor systems has been investigated intermittently in our laboratory since the early 1950s (1, 2)

Direct dilution of freshly passed urine with 19 to 99 volumes of an acid-alcohol solution of chloranil (2, 3, 5, 6-tetrachloro-1, 4-benzoquinone) results in rapid and apparently quantitative conversion of porphyrinogen to fluorescing porphyrin. Dilution must be sufficient to yield an essentially colorless solution. Under these conditions, excitation of fluorescence by 401--405 nm light and fluorescence analysis at 652 nm results in almost complete elimination of quenching and of non-specific fluorescence under the conditions described. Fluorescence intensity is relatively stable for up to several weeks. Alternatively, urine is added to NaHCO3 to permit quantitative conversion of porphyrinogen to porphyrin in 1 to 2 days, and assayed similarly following dilution with the same acid-alcohol diluent (with or without chloranil) or with dilute HCl solution alone. Quantitative results are shown to agree well with those obtained following purification by other, more tedious, procedures. The method has been tested successfully with urines from humans, cows, and rats.