The transition of hemoglobin to bile pigment, at least under normal conditions, is believed to occur via an intermediate biliverdin-globin-iron (verdohemoglobin) and not over the stages of hematin and protoporphyrin. It is probable that the next step is a reduction to bilirubin with splitting off of iron. There is much reason to believe that the globin remains attached until the bilirubin passes through the liver cell, bilirubinglobin exhibiting a delayed or indirect van den Bergh reaction and not being excreted in the urine; the sodium bilirubinate of the bile exhibiting a prompt (1') van den Bergh reaction and being readily excreted in the urine. The former type is characteristic of retention, the latter of regurgitation jaundice.
The appearance of bilirubin in the urine is believed to be related to the concentration in the blood of the 1' or prompt bilirubin, rather than that of the total bilirubin. It is evident that the threshold may be considerably lower at the onset of jaundice, as, for example, in hepatitis, than during its defervescence. This undoubtedly accounts for the appearance of bilirubinuria prior to recognizable jaundice in certain instances, likewise for its presence in the cases of so-called "hepatitis without jaundice." In retention jaundice marked elevation of the total serum bilirubin is unassociated with bilirubinuria; in these cases the increase of bilirubin is mainly of the delayed or indirect reacting type. Further evidence is presented of the essential difference between the 1' or prompt, and the T minus 1', or delayed and indirect reacting bilirubins. This consists of a change of the order of reaction at one minute after adding the diazonium salt. The normal upper limit of the 1' bilirubin has been shown to be in the neighborhood of 0.2. mg. per 100 cc.; figures well below this value are usually obtained.
Further experience with the erythrocyte protoporphyrin in the anemias has revealed that this determination, quite apart from its fundamental interest, is at times of diagnostic value. Thus in several instances a significant elevation of the erythrocyte protoporphyrin has indicated that the initial impression of pernicious anemia was incorrect, and has led to the search for other information. Conversely, a low normal value in the presence of anemia has often correctly indicated or confirmed the diagnosis of pernicious anemia. Marked elevations have aided in confirming the presence of iron deficiency and have given some insight into the degree of its severity and chronicity. In certain cases, high values for the erythrocyte protoporphyrin have suggested the possibility of heavy metal toxicity, the existence of which has then been borne out by subsequent study.