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An improved method for measuring the concentration of bromsulphthalein in blood
503
CLINICA CHIMICA ACTA
CCA Report CCA 5286
improved in blood
An
method
for measuring
the concentration
of bromsulphthalein
M.G. CUTLER Departmenr of CIinicafPharmacology, Medical School, Birmingham (U.K.) (Received June 10,1972)
INTRODUCTION
The rate of bromsulphthalein clearance in man is a sensitive index of functional impairment of the liver’, but this liver function test suffers from the disadvantage that none of the existing methods for estimating the bromsu~phthalein content of serum is free from errors due to turbidity
and to interfering
haem and serum pigments.
A method has been developed for eliminating such sources of error. Pigments and proteins are precipitated from serum containing bromsulphthalein by a mixture of acetone, saturated magnesium sulphate and sodium hydroxide. Inclusion of ammonia solution with the sodium hydroxide severs the protein-binding of bromsulphthalein in serum and liberates the bromsulphthalein
from the precipitate
into a clear solution.
METHODS
A mixture of 1 mf 80% acetone in water and 0.1 ml saturated magnesium sulphate was added either to 0.5 ml serum or to 0.25 ml blood mixed with 0.25 ml oxalated normal saline. The solution was mixed thoroughly, centrifuged, and 1 ml of the supernatant solution was added to 2 ml of a mixture of ammonia and sodium hydroxide solutions (50 ml of 20% 0.880 ammonia solution plus 25 ml N/l sodium hydroxide solution). If whole blood had been used, 0.05 ml saturated magnesium sulphate solution was added to ensure that all haemoglobin had been precipitated. After 10 min the tube was centrifuged and the optical density of the supernatant solution recorded at 580 nm. Reproducibility
ofthe
method
Standard solutions of bromsulphthalein in normal saline were incubated at 37” with fresh blood for 20 min and their bromsu~phtha~ein content estimated by the method Ciin. Chim Acta, 40 (1972) 503-504
504
CCA REPORTS
TABLE I REPRODUCIBILITY OF THE METHOD FOR ANALYSIS OF THE BROMSULPHTHALEIN CONTENT IN BLOOD
Optical density of b&msuiphthaiein solution in blood
Concentration of bromsulphthalein 4 mg/I 00 ml
IO mg/lOO ml
0.175 2 0.001
0.419 It 0.001
0.179 + 0.001
0.422 ?I 0.002
(r + SEMI Optical density of brornsul~h~h~e~ soiution in alkali (X f SEMI
described and compared with the optical density at 580 nm of standard solutions of bromsulphthalein in alkali. Table I shows that the method was reproducible values similar to those of the standard solution in alkali ~FERENCES 1 L. Zieve and E. Hi& Gffstroentero~og~,
28 11955) 785.
Clin. Chim. Acta, 40 (1972) 503-504
The Author and Subject Index of this volume is gwen on pp. 489-498. .^^-
and gave
CLINICA CHIMICA ACTA
CCA Report CCA 5286
improved in blood
An
method
for measuring
the concentration
of bromsulphthalein
M.G. CUTLER Departmenr of CIinicafPharmacology, Medical School, Birmingham (U.K.) (Received June 10,1972)
INTRODUCTION
The rate of bromsulphthalein clearance in man is a sensitive index of functional impairment of the liver’, but this liver function test suffers from the disadvantage that none of the existing methods for estimating the bromsu~phthalein content of serum is free from errors due to turbidity
and to interfering
haem and serum pigments.
A method has been developed for eliminating such sources of error. Pigments and proteins are precipitated from serum containing bromsulphthalein by a mixture of acetone, saturated magnesium sulphate and sodium hydroxide. Inclusion of ammonia solution with the sodium hydroxide severs the protein-binding of bromsulphthalein in serum and liberates the bromsulphthalein
from the precipitate
into a clear solution.
METHODS
A mixture of 1 mf 80% acetone in water and 0.1 ml saturated magnesium sulphate was added either to 0.5 ml serum or to 0.25 ml blood mixed with 0.25 ml oxalated normal saline. The solution was mixed thoroughly, centrifuged, and 1 ml of the supernatant solution was added to 2 ml of a mixture of ammonia and sodium hydroxide solutions (50 ml of 20% 0.880 ammonia solution plus 25 ml N/l sodium hydroxide solution). If whole blood had been used, 0.05 ml saturated magnesium sulphate solution was added to ensure that all haemoglobin had been precipitated. After 10 min the tube was centrifuged and the optical density of the supernatant solution recorded at 580 nm. Reproducibility
ofthe
method
Standard solutions of bromsulphthalein in normal saline were incubated at 37” with fresh blood for 20 min and their bromsu~phtha~ein content estimated by the method Ciin. Chim Acta, 40 (1972) 503-504
504
CCA REPORTS
TABLE I REPRODUCIBILITY OF THE METHOD FOR ANALYSIS OF THE BROMSULPHTHALEIN CONTENT IN BLOOD
Optical density of b&msuiphthaiein solution in blood
Concentration of bromsulphthalein 4 mg/I 00 ml
IO mg/lOO ml
0.175 2 0.001
0.419 It 0.001
0.179 + 0.001
0.422 ?I 0.002
(r + SEMI Optical density of brornsul~h~h~e~ soiution in alkali (X f SEMI
described and compared with the optical density at 580 nm of standard solutions of bromsulphthalein in alkali. Table I shows that the method was reproducible values similar to those of the standard solution in alkali ~FERENCES 1 L. Zieve and E. Hi& Gffstroentero~og~,
28 11955) 785.
Clin. Chim. Acta, 40 (1972) 503-504
The Author and Subject Index of this volume is gwen on pp. 489-498. .^^-
and gave