Studies on the Determination of Bile Pigments - Clinical Chemistry

Vol. 10, No. 5, 1964 DETERMINATION OF UROBILINOGEN 445
exposure of chromogeii extracts even to diffuse sunlight can produce
pigments other than urobilin (“biliviolins”). Doubt has thus been
cast upon the fundamental assumption of the test-viz., that urobilinogen
which has undergone oxidation in the urine can again be reduced
to urobiliriogen; indeed, this suspicion has been raised by Watson
(7) and by Heilmeyer (8).
The data in Table 1 suggest that it is the urine itself which interferes
with the reduction of urobilin. The good recovery of urobilinogen
added to urine lends support to this hypothesis. Spectrophotometric
data presented also support this possibility; Fig. 2 shows that the reduction
of urobilin in the presence of urine can produce another substance
different from urobilinogen (Curve C). In addition, recovery
experiments using direct Ehrlich’s aldehyde reaction in urine suggest
the concept of a negative interference: In these experiments, the observation
that the apparent urobilinogn level was somewhat increased
when samples had stood for 24 hr. suggests that the interfering factor
itself changes with time and that low recoveries from urine are due
primarily to such interference rather than to an irreversible oxidation
of
urobilinogen.
To be sure, elevated levels of urobilinogen in disease states such as
infectious hepatitis would not go undetected, despite the analytical
problems ; however, in obstructive jaundice, where diminished amounts
of urobilinogen are excreted because of the absence of intestinal re-
Table 2. RECOVERY OF UROBILINOGEN FROM URINE BY DIRECT REACTION WITH EHRLICH’S
REAGENT
Sample preparation (nil.) Am, tm. CHClii* --
20 mg./
100 ml.
Sample Water Urine URGt
Initial teat 24-hr. teat
Urine 5 45 - 0.006 0.006
*Readings represent “urobilinogen-aldebyde” extracted in 24 ml. chloroform from reduction
of 7.5 ml. sample. Samples were preserved overnight at 30#{176} with 0.1 gm. Na,00a per 10 ml.
sample and overlayed with 3 ml. mineral oil.
tA 20-mg./100 ml. solution of urobilinogen was prepared by borohydride reduction of urobilin.
The solution was then acidified with HCI to decompose the excess potassium horohydride.
0.005 0.006
Urohilinogen 45 - 5 0.500 0.380
0.470 0.360
Urine plus - 45 5 0.210 0.275
urohilinogen 0.200 0.300