Studies on the Determination of Bile Pigments - Clinical Chemistry

<strong>Studies</strong> on the Determination of Bile Pigments
VI. Urobilinogen in Urine as Urobilinogen-Aldehyde
Richard J. Henry, Alberto A. Fernandez, and S. Berkman
In an attempt to complete the standardization of the “quantitative” method for
the determination of urobilinogen in urine, recoveries of added urobilin varied
erratically between 50 and 80%. Experiments to demonstrate the cause of the low
recoveries suggest that urobilin is altered in the reduction procedure by the presence
of urine.
THE photometric determination of urobilinogen was proposed by
Terwen (i) and employs the reaction of native urobilinogen, plus that
derived from ferrous hydroxide reduction of urobilin, with Ehrlich’s
reagent, p-dimethylaminobenzaldehyde. This basic approach culminated
in the “quantitative” and “semiquantitative” methods of Watson
(2,3) with minor modifications by various authors (4).
Efforts in this laboratory directed at proper methods of standardization
have provided absorptivities of the best available urobilin
preparations and their respective urobilinogen-aldehydes (5). Phenol
red (PSP) was shown to be superior to the Pontacyl dye mixture or
to phenolphthalein as a secondary standard for the urobilinogen-aldehyde
color. Also, increased stability of urobilinogen was achieved in
the urobilinogen-aldehyde reaction by the addition of ascorbic acid to
the reaction mixture.
It was then our purpose to complete the standardization of the
“quantitative” procedure with recovery experiments for urobilin
added to urine and to redetermine normal values using the proposed
method of standardization (5). The results obtained revealed the apparent
inaccuracy of these technics. The problems encountered incidental
to the purpose at hand will be described in this paper.
From the Bio-Seienee Laboratories, 12330 Santa. Monica Blvd., Los Angeles 25, Calif.
Received for publication Mar. 7, 1963.
440

Vol. 10, No. 5, 1964 DETERMINATION OF UROBILINOGEN 441
Experimental
Preparation of Crystalline Reference Urobilin
The procedure outlined by Watson (6) for the preparation of i-
urobilin hydrochloride was follo’wed. This involves: (1) reduction of
bilirubin with sodium amalgam in alkaline solution; (2) extraction
of the product, mesobilirubinogen, with petroleum ether; (3) oxidation
to i-urobilin with an aqueous solution of iodine, with concomitant
extraction of the urobilin into the aqueous phase; (4) acidification and
extraction with chloroform; and (5) crystallization from chloroformacetone
solution.
Assay of Urines by a Modified “Quantitative” Procedure
In order to ensure best possible reduction to urobilinogen and stability
thereafter, some additions were made to Watson’s procedure:
Twenty milliliters of ferrous hydroxide filtrate was prepared by the
usual means. This was centrifuged and the supernatant solution was
decanted into a tube containing 200 mg. of ascorbic acid. This solution
was then filtered into another tube, 100 mg. of potassium borohydride
was added, and the pH was adjusted to 7-8. Borohydride was found to
aid in the complete reduction of urobilinogen, as evidenced by the more
complete removal of the 490 m peak of urobilin in an acidified aliquot
of the filtrate (Fig. 1). After several minutes, the pH was adjusted to
.500
.400
Fig. 1. Effect of potassium
borohydride treatment on fil- .oo
trate from reduction of urobilin E
A
with ferrous hydroxide: Curve 200
A indicates no borohydride <
treatment; Curve B, borohydride
used. 100
C I I
450 475 525
mu
4.0 and a suitable aliquot was taken for extraction, as per Balikov (4).
Additional precautions of working in subdued artificial light and with
minimal delay at each step were observed.

442 HENRY fT AL. Clinical Chemistry
Direct Condensation of Ehrlich’s Reagent with Reduced Urine
To 1 volume of filtrate, prepared as in the “quantitative” procedure,
was added 1 volume of Ehrlich’s reagent. The solution was
mixed and 2 volumes of saturated sodium acetate was added. Washing
the colored solution with several portions of benzene, followed by 1
portion of petroleum ether removed most nonspecific color. Tlrobilinogen-aldehyde
was then extracted with chloroform.
Results
Preparation
of Urobilin
The following observations were made while preparing the reference
urobilin:
1. Reduction of bilirubin with sodium amalgam gave two fractions
positive to Ehrlich’s reagent, only one of which was extractable into
petroleum ether at pH 4.
2. After iodine oxidation to obtain urobilin from mesobilirubinogen
and extraction into water, it was found that some mesobilirubinogen
remained in the petroleum ether-i.e., complete oxidation had not been
achieved with the theoretical amount of iodine and the iodine was consumed
in side reactions. Subsequent exposure of the solution to diffuse
sunlight gave additional urobilin. The two fractions of pigment were
isolated and gave identical spectra and absorptivities in acid-methanol
and also as urobilinogen-aldehyde after rereduction to the chromogen.
3. If the oxidation in petroleum ether was promoted by sunlight,
the product had to be extracted with water as it formed, since prolonged
exposure to sunlight produced a product which was insoluble
in dilute 1101. llrobilin would be expected to be soluble in HC1. Both
products exhibited spectra in acid-methanol which were identical to
that of urobilin.
4. The “biliviolin” side-products, formed when the oxidation of
mesobilirubinogen was promoted by sunlight or by iodine, were of
different colors, depending on which of these oxidation technics was
used.
5. The “urobilinogen-aldehyde” obtained from our “urobilin”
preparation revealed two fractions. The minor fraction was extractable
into benzene from aqueous solution, whereas the major fraction
(about 90% of total) remained in the aqueous phase and was subsequently
extractable by chloroform.

Vol. 10, No. 5, 1964 DETERMINATION OF UROBILINOGEN 443
Assay of Iirines by the Modified “Quantitative” Procedure
“Pure urobilin” solutions taken through the procedure as described
for urine yielded a mean recovery of 90%. The final colored solutions
were quantitated by comparison with standards not taken through
the extraction procedure and which have been previously described
(5). However, recoveries of urobilin from urine varied erratically
from 50 to 80% of the added urobilin (Table 1).
Precision on normal urines containing low levels of urobilinogen
was poor, with duplicates differing as much as 65%.
Examination of absorption spectra of some reduction mixtures of
urobilin with urine revealed a peak in the 512 m region which was
obtained neither from the reduced urine nor from reduced urobilin
alone (Fig. 2).
Table 1. REcOVERY OF UltomLIN AS UROBILINOGEN FROM PURE SOLUTIONS AND FROM URINE
Added in filtrate Found in filtrate
Urobilin extracted (mg.) extracted (mg.) Recovery (%)
In pure aq. aol.
A 0.065 0.064 99
B 0.060 0.057 95
C 0.065 0.053 82
D 0.063 0.052 83
E 0.089 0.082 92
Added to urine
A 0.105 0.068 65
B 0.095 0.074 78
C 0.095 0.064 67
D 0.091 0.045 49
Urobiinogen
Added to urine 0.150 0.155 103
Fig. 2. Reduction mixtures of
urine (A), urobilin (B), and
urine plus urobilin (C).
a
0
f .100
0
4
450 475
mp

444 HENRY fT AL. Clinical Chemistry
The effect of time of standing with Ehrlich’s reagent prior to sodium
acetate addition on the urobilinogen aldehyde color is shown
in Fig. 3.
:1
E
.500
.430
0
4
Fig. 3. Effect of prolonged
standing of urobilinogen with
Ehrlieh’s reagent in HC1.
.460
0 2 4 6 6 10
Mnutes before a4iton of NoOAc
Direct Condensation of Ehrlich’s Reagent with Reduced Urine
In order to eliminate the problems of incomplete extraction and
losses arising from oxidatioti of urobilinogen or urobilin in the “quantitative”
procedure, a method was devised wherein the color reaction
was performed directly on a urine filtrate in the same manner that
Watson’s “semiquantitative” method is applied to urine. A subsequent
extraction was made to separate the urobilinogen-aldehyde
from other Ehrlich-positive reaction products. Details of the method
have been given above. Table 2 shows that urobilinogen added to urine
could not be quantitatively recovered in this procedure. The same solutions,
analyzed 24 hr. later, showed a loss of urobilinogen in the aqueous
solution; the urine, however, exhibited an apparent increase in
urobilinogen.
Discussion
Recognition that difficulties are encountered ill the testing of urine
for urobilin and urobilinogen is not new. Tn 1936, Watson (2) stated
that “. . . the reduction of urobilin to urobilinogen is in some degree
hindered in the urine” and that “because urobilinogen is a labile
chromogen, it is certain that no quantitative method can aspire to more
than approximate values.” The present data seem to substantiate this
and seem also to indicate that the oxidation of urohilinogen goes iii
part to products other than urohilin. According to our observations,

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

446 HENRY ET AL. Clinical Chemistry
duction of bilirubin, or in other states with diminished bile formation,
it would seem unwise to place much faith in these methods.
It would appear that improvements in the urobilinogen-aldehyde
method for the determination of urobilin and urobilinogen in urine
will depend upon separation of these substances from the interfering
materials prior to chemical manipulation. Convenient methods for this
purpose are not presently available. Until such improvements are
made in the “quantitative” technic, the urobilinogen concentrations
obtained by it are of very questionable value, and it would seem that it
is sufficient for clinical purposes to use the “semiquantitative” technic,
even though it is subject to serious problems of specificity and may be
affected by the same inhibiting substances.
References
1. Terwen, A. .1. L., Deut. Arch. kim. Med. 149, 72 (1925).
2. Watson, C. J., Am. J. Clin. Pathol. 6,458 (1936).
3. Watson, C. J., Schwartz, S., Sborov, V., and Bertie, E., Am. J. CUn. Paihol. 14, 605 (1944).
4. Balikov, B., Standard Methods of Clinical Chemistry, (Vol. 2), Acad. Press, 1958, p. 192.
5. Henry, R. J., Jacobs, S. L. and Berkman, S., Clin. Chem. 7, 231 (1961).
6. Watson, C. 3., J. Bid. Chem. 200, 691 (1953)
7. Watson, C. J., and Hawkinson, V., Am. J. Clin. Pathol. 17, 108 (1947).
8. Heilmeyer, L., Z. Ges. exptl. Med. 76, 220 (1931).