DETERMINATION OF BROMSULPHALEIN IN NORMAL, TURBID, HEMOLYZED,
OR ICTERIC SERUMS
OLIVER H. GAEBLER,
M.D.
From the Department of Laboratories, Henry Ford Hospital, Detroit, Mich.
In the original method of Rosenthal and White1,
bromsulphalein was determined in serum by adding
alkali and comparing the color with that of known
alkaline solutions of the indicator placed behind an
acidified portion of the same serum. Various photoelectric colorimeters are now recommended for the
comparison. Cantarow and Wirts2 used a photoelectric method for determination of bromsulphalein
in bile. In the present study the necessary conditions
and calculation factors for determination of bromsulphalein in 0.5 or 1.0 ml. of serum, with the Evelyn
photoelectric colorimeter3 (macro type) or Beckman
spectrophotometer4, have been determined. The
sensitivity of the instruments also made it necessary to
establish the range of values hitherto reported "negative" in analyses by the original comparator block
method.
The procedures described below are not quite as
rapid as the original method, the recognized value of
which should not be minimized. Several authors state
however that the bromsulphalein test is inapplicable
in cases of jaundice because the alkaline color of the
indicator in serums containing much bilirubin cannot be
matched correctly. Analysts also have to contend with
small blood samples and with turbid or slightly hemolyzed serums. Devising photoelectric procedures
which surmount these difficulties was facilitated by the
relatively large concentrations provided by the 5 mg.
per kilo dose of bromsulphalein which Macdonald6
introduced for other and more basic reasons.
Each of the following photoelectric procedures
has its advantages. Thefirstone requires two readings
and slightly more calculation than the second, but the
inconvenience of making up a separate blank for each
determination is thereby avoided, and only a single
0.5 ml. sample of serum is required.
PROCEDURES
1. Place 0.5 ml. of serum in an Evelyn colorimeter tube. Add 2.5 ml. of water, and 3.0 ml. of
0.1 N sodium hydroxide. Read against a water
blank, using the 620 and 565 millimicron filters,
and the special setting for 6 ml. volumes. If a
Beckman spectrophotometer is used, the 6 ml. of
solution, prepared in any clean dry tube, suffice
for rinsing and filling the 3 ml. cells with 1 cm.
light path. Readings in this case are preferably
made at 620 and 580 millimicrons.
2. The unknown, prepared as above, may be
read against a blank consisting of 0.5 ml. of
serum, 2.5 ml. of water, and 3.0 ml. of 0.1 N
hydrochloric acid, a single reading being made
with the Evelyn instrument and filter 565.
Under these conditions the following formulas
give the results in milligrams of bromsulphalein
per 100 ml. of serum:
Evelyn macro-colorimeter:
First procedure,
L6(
Second procedure, -
1.3L6i
0.095
0.005
0.111
(1)
(2)
Beckman spectrophotometer:
First procedure only, 1 cm. light path,
E58O — 1.2Ee20
(3)
0.0655
r
Es65 ~~ 1.3E620
6.0488
(4)
! A concentration of 4 mg. per cent of bromsulphalein in serum was defined as 100 per cent
retention of the 2 mg. per kilo dose1. On the
same basis, 10 mg. per cent represents 100 per cent
retention of the 5 mg. per kilo dose, so that per
cent retention in this case is obtained by multiplying milligrams per cent by 10, or simply shifting
the decimal point. Should the 2 mg. per kilo
dose still be used, per cent retention would be
milligrams per cent times 25. Results for the
5 mg. per kilo dose have also been reported in
terms of the standards for the 2 mg. per kilo dose6
because the weaker standards were preferred in
matching, but in a photoelectric method the
reason for this practice is invalid.
In the first procedure, application of the usual
• formulas for determination of two light absorbing
substances in the presence of one another was
452
DETERMINATION OF BROMSULPHALEIN
facilitated by a previous study of the turbidity
factor'. At the indicated wave lengths, which
also correspond with readily available filters, the
extinction ratios for turbidity, alkali hematin,
and bilirubin are sufficiently similar to permit
treating the three interfering substances as one,
and the extinction ratio of bromsulphalein is as
different as possible from that of the interfering
substances. The absorption maximum of bromsulphalein is near 580, and measurement at this
point is preferable when 1 cm. cells are used.
The large diameter of the Evelyn tubes makes the
565 filter, which is also useful in other methods,
entirely satisfactory.
CALIBRATION
Since the 5 per cent solution of bromsulphalein
supplied by the manufacturer (Hynson, Westcott,
and Dunning) was found to be uniform, and was
used both for calibration and for liver function
tests, its correctness was assumed. Calibration
of other instruments with any solution to be used
should be very simple, for it will be noted that
formulas 1 and 4, for two very different instruments and cell depths, have the same numerator.
To determine the denominators, one prepares
bromsulphalein solutions containing in 100 ml.
the number of milligrams (conveniently 0.5, 1.0,
2.0, and 4.0) to be added per 100 ml. of serum.
The blank contains 0.5 ml. of bromsulphalein-free
serum, 2.5 ml. of water, and 3.0 ml. of 0.1 N sodium
hydroxide. The standards contain 0.5 ml. of
the same serum, 0.5 ml. of one of the bromsulphalein solutions, 2.0 ml. of water, and 3.0 ml.
of 0.1 N sodium hydroxide. The optical density
or extinction (E), or with filter instruments the L
value, of each standard is determined at 620, and
at 580 or 565 millimicrons, and is divided by the
number of milligrams of bromsulphalein added
per 100 ml. of serum. This result we will designate
as K. With the Evelyn instrument K56o averaged
0.111 in calibrations with many types of serum.
This value appears unchanged in formula 2, in the
numerator of which 0.005 is the average difference
between acidified and alkaline blanks of various
serums .free of bromsulphalein. The denominators
of formulas 1 and 4 are K665 — 1.3 KB8o, and that of
formula 3 is K£8o — 1.2 K620. At the specified
wave lengths the values of K were essentially the
same whether normal, turbid, hemolyzed, or
icteric serums were used.
453
RESULTS
In table 1 are given the results of 34 determinations carried out by procedure 2 with the Evelyn
instrument. The amounts of bromsulphalein
shown in the first column were added to 7 very
different serums (see footnote of table 1) all known
to be free of bromsulphalein. It is evident that
concentrations of bromsulphalein which represent
up to 50 per cent retention when the 5 mg. per kilo
dose is used can be determined accurately.
TABLE 1
DETERMINATIONS OP BROMSULPHALEIN ADDED TO
SERUMS
All values are given in milligrams per cent
BROM- BROMSULPHALEIN FOUND, IN 7 DIFFERENT SERUMS, BY
SULPHA- USING PROCEDURE 2 AND THE EVELYN INSTRUMENT
ADDED 1
2
4
s 6 7 Aver3
age
0.0 -0.04 -0.03 0.01 -0.01 0.00 0.10 0.04
0.45 0.49 0.51 0.51 0.49 0.60 0.51 0.51
0.5
0.98 1.00 0.99 1.04 1.07 1.08 1.06 1.03
1.0
1.90 2.02 2.00 2.00 1.90 2.08 2.02 1.99
2.0
4.84 4.87 4.77 4.61
4.90 4.85 4.81
5.0
The first four serums were normal but varied in
color; the fifth was slightly hemolyzed and icteric,
the sixth extremely hemolyzed, and the seventh
extremely icteric.
Table 2 presents the results obtained in actual
clinical tests. In the comparator block method
the standard representing 100 per cent retention
contained 10 mg. of bromsulphalein per 100 ml.,
and results of the photoelectric methods have
been converted to the same basis by multiplying
milligrams of bromsulphalein found per 100 ml. of
serum by 10. Values obtained by using the four
formulas vary from their average by less than 1 per
cent of retention, regardless of the amount retained.
In the comparator block method the error is
greater, for the standards have 10 per cent intervals. All but a few specimens were however
matched with the nearest standard. Results for
serums considered "negative" show the interesting
fact that bromsulphalein could always be detected,
and that the 60 minute specimen, as one would
expect, always gave the lower value, although the
amounts determined here are near the limit of the
method. It would seem practical to report values
below 2 per cent retention of the 5 mg. per kilo
454
OLIVER H. GAEBLER
TABLE 2
Determinations of bromsulphalein in clinical tests
All values represent per cent retention of the 5 mg. per kilo dose
APPEARANCE OP SERUM
MINUTES AFTER COMPARATOR
BROMSULPHALEIN BLOCK METHOD
45
60
Normal.
Very turbid
<f it
Hemolyzed, turbid.
Normal
Slightly hemolyzed.
" turbid
((
U
" hemolyzed.
turbid
hemolyzed.
Normal.
Hemolyzed, icteric.
Icteric
Extremely icteric..
45
60
45
60
45
60
45
60
45
60
45
60
45
60
45
45
60
45
60
45
60
45
60
45
60
45
60
45
60
neg.
It
trace
neg.
trace
neg.
4
trace
neg.
10
5
10
5
15
15
40
35
?
?
?
?
dose, or below 5 per cent retention of the 2 mg.
per kilo dose, as negative, and to report values
equal to or greater than these to the nearest full
per cent of retention.
SUMMARY
Procedures are described for determination of
bromsulphalein in 0.5 ml. or 1.0 ml. of serum by
the use of a photoelectric colorimeter having
sufficiently selective filters, or a spectrophotometer. The procedures are applicable to turbid,
hemolyzed, and icteric serums. In one of the
procedures the wave lengths at which measurements are made are chosen so that turbidity,
bilirubin, or alkali hematin formed from hemo-
Evelyn
(1)
1.3
0.7
2.9
1.3
2.8
1.7
1.
1.
0.
0.
1.8
1.0
1.2
1.0
1.0
0.9
4.9
11.9
9.4
12.0
7.2
18.9
15.4
22.8
21.4
36.1
34.4
38.8
34.2
39.6
37.1
INSTRUMENT AND FORMULA EMPLOYED
Beckman
Evelyn
Beckman
(4)
(2)
(3)
1.2
0.8
2.6
2.7
1.5
1.4
2.4
2.2
1.5
1.4
1.8
1.9
0.9
0.7
1.5
0.
2.
1.
0.7
1.4
1.
0.5
0.5
0.
0.6
1.0
1.
0.2
0.6
0.
4.0
4.5
4.
12.1
11.
11.9
9.0
8.8
7.
11.
12.4
11.7
7.3
7.2
7.
18.8
15.7
23.1
23.8
22.6
21.8
22.6
21.2
36.2
35.8
33.6
32.8
33.6
40.6
39.5
40.3
34.8
34.8
34.4
41.2
41.3
39.7
38.1
38.4
37.1
globin, all have similar effects and can be treated
as one interfering substance. This also makes
possible the convenience of using a water blank.
Results are reproducible to within less than 0.1
mg. of bromsulphalein per 100 ml. of serum. This
amount represents 1 per cent retention of the
5 mg. per kilo dose of bromsulphalein.
REFERENCES
(1) ROSENTHAL, S. M., AND WHITE, E. C: Clinical
application of bromsulphalein test for hepatic
function. J. A. M. A., 84: 1112, 1925.
(2) CANTAROW, A., AND WIRTS, C. W.: Excretion, of
bromsulphalein in the bile. Proc. Soc. Exper.
Biol. & Med., 47: 252, 1941.
DETERMINATION OF BROMSULPHALEIN
(3) EVELYN, K. A.: A stabilized photoelectric colorimeter with light filters. J. Biol. Chem., 115:
63, 1936.
(4) CARY, H. H., AND BECKMAN, A. O.: A quartz
photoelectric spectrophotometer. J. Optical
Soc. Am., 31: 682, 1941.
(5) MACDONALD, D.: Practical and clinical test for
liver function. Surg. Gynec. & Obst., 69: 70,
1939.
455
(6) DRILL, V. A., AND IVY, A. C: Comparative value
of bromsulphalein, serum phosphatase, prothrombin time, and intravenous galactose
tolerance tests in detecting hepatic damage
produced by carbon tetrachloride. J. Clin.
Investigation, 23: 209, 1944.
(7) GAEBLER, O. H.: The relationship of turbidity to
wave length in turbid sera and other suspensions. J. Biol. Chem., 149: 251, 1943.