88 III. PORPHYRIN CHEMISTRY 



Spectrophotometry and photoelectric colorimetry. Photoelectric colorimetry 

 with suitable light filters in the visible range has been used by Dobriner and 

 collaborators {601). For the estimation of the exceedingly small amounts of 

 protoporphyrin in erythrocytes, photoelectric measurement of the strong 

 absorption band at about 410 m^i has been recommended by Grinstein and 

 Watson (1050). While protoporphyrin is rather unstable and easily altered 

 by irradiation, the absorption of the Soret band diminishes much less than 

 the strength of fluorescence. Spectrophotometric methods in the visible and 

 ultraviolet can also be used {1298,2053,2506,2508,2509). 



Fluorimetric method. Measuring the strength of fluorescence has been 

 used extensively for porphyrin estimations {226,365,369,063,757,760,1059, 

 159Jt,1781,1879^2792,2851). Porphyrins are the only substances with a red 

 fluorescence in acid solution which occur in feces or urine. 



Several complicated fluorimeters have been constructed or are on the 

 market. Estimations sufiiciently exact for clinical purposes can be carried 

 out with a very simple apparatus consisting of a box with a source of ultra- 

 violet light in Woods' glass in the upper part and two windows in the front 

 and back. Through the latter the solutions of standard and unknown in 

 nonfluorescent thin-walled test tubes are inserted. They are inspected 

 through the front window, which is closed by a filter, allowing only red light 

 to pass. Standards containing 0.05 to 1 ng. porphyrin per ml. are used. A 

 somewhat more elaborate apparatus on the same principle, constructed by 

 Schuster, is used by Rimington {2519). Rimington's paper should be con- 

 sulted for details of the method {2266). A final acid concentration of 0.25% 

 hydrochloric acid is more suitable for the estimation of copro- and uropor- 

 phyrins (maximal fluorescence) than 5% hydrochloric acid used by earlier 

 investigators. 



It may be advisable to repeat the estimation after repurification of the 

 porphyrin by passage through ether and hydrochloric acid, in order to ensure 

 absence of substances interfering with fluorescence. For this purpose extrac- 

 tion of the hydrochloric acid solutions with chloroform and petroleum ether 

 has been suggested {760,1594.) '■, the former would, of course, also remove proto- 

 porphyrin from fecal porphyrin solutions. Another suggestion was to oxidize 

 the interfering substances {760), but this procedure is not to l)e recommended 

 (76^). 



Boas {298) transforms the porphyrins into hematins with ferrous acetate, 

 and then extracts them with chloroform. He uses the benzidine reaction for 

 the estimation. Schreus and Carrie {406) found this method as reliable as 

 the fluorescence method, but one may doubt whether the very small amounts 

 of porphyrin can be quantitatively converted into hematin; since the por- 

 phyrins will have to be isolated beforehand in any case, the method appears 

 to be unduly complicated. 



Twenty-four hours' specimens of urine are required, and feces should be 

 collected if possible over 2-3 days. Losses of porphyrins may occur during 

 the collection of the urine, even when kept in a dark bottle under toluene, 

 by adsorption to urinary sediments {2S30). For the extraction of fecal 

 porphyrins, hydrochloric acid of no more than 5% strength should be used, 



