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A new method for the determination of sulfobromophthalein in serum
CLINICA CHIMICA ACTA
A NEW
METHOD
PHTHALEIN
FOR
439
THE
DETERMINATION
OF
SULFO~RO~O-
IN SERUM*
H. OTT .+ND D. I’IRRWITZ Sevvic~ de Biologic, (Received
June
27,
Euro$ean Comnzunifies-curator,
Joint Research Centre, Is$va {Ifalyj
Ig68)
Sulfobromophthalein (BSP) is discolored by sodium dithionite (Na,S,O,) in alkaline solution due to a reduction of the chromophore chinoide system in the dye. Application of this reaction for the estimation of BSP in serum and elimination of hemoglobin interference are described.
cator
In several methods for the sulfobromophthalein (BSP) retention test, the indiproperties of the dye are utilized in the spectrophotometric determination in
order to avoid a separate blood sampling for the serum blank. The absorbance of the alkaline sample is measured at $30 nm against a neutral dilution of the test serum. The difference in pH between sample and blank gives rise to interferences due to: (a) turbidity changes resulting from the higher solubility of lipids and lipoproteins in alkaline solution, (b) the pH dependency of bilirubin absorbance, (c) the conversion of oxyhemoglobin (which has a strong absorption band at 577 nm in neutral solution) to alkaline hematin at high pH. The latter has lower absorbance, thus simulating lower BSP readings. Corrections for hemoglobin content by additional readings at different wavelengths have been proposed by several authors ly2. It has also been suggested to keep the pH interval as small as necessary for developing full BSP colour, adjusting sample and reference to pH 10.4 and 7.1, resp., by means of buffers and adding p-toluene sulfonate to liberate BSP from plasma binding at low pH (ref. 3). It was found that BSP can be discolored by sodium dithionite (Na,S,O,) in alkaline solution, due to a reduction of the chromophore chinoide system in the dye. This reaction can be used for the convenient determination of BSP in serum. Interferences are minimized, the sample and blank having the same PH. The method should be also suitable for automated techniques.
* This publication
is contribution
No. qrr of Em-atom Biolofiy
Division.
Clin. Chinz. Ada, 22 (1968) 439-442
440
OTT, PIRRWITZ
APPARATI'S
AND
REAGENTS
A C’nicam SP 600 spectrophotometer was used for the spectrophotometric measurements. Absorption spectra were done on a Perkin-Elmer 350 recording spectrophotometer. mann,
BSP standard. IOO /[g/ml sulfobromophthalein sodium (Etablissements Paris) in distilled water. Alkaline reagent. ro(lb w/v solution of sodium hydroxide. Dithionite reagent. A solution of 1.0 g sodium dithionite
(Na,S,O,)
Kuhl-
(E. Merck A(;,
Darmstadt, “for analytical purposes, not free from chloride”) and 1.0 g sodium hydroxide in a final volume of IO ml was freshly prepared every day. EXPERIMENTA4L
Discoloratiort of BSP by sodium dithionite BSP reacts in alkaline medium with an excess of dithionite forming a leukocompound. The absorption spectra of BSP and reduced BSP are shown in Fig. I. The reduction under the conditions described below is complete within z to 3 min. This reaction is reversible upon access of oxygen, and BSP color reappears after several hours. Therefore the spectrophotometric measurements were done in stoppered or covered cuvettes between 5 and IO min after addition of the dithionitc reagent.
ITi
0
h.
1.
Absorption alkaline
- _.. "--L-
spectra: hematin,
--3-_I0.5 mg/ml;
BSI’, Lo fig/ml; P.-o---_ ,~-I :- hemochromogen,
rctluced BSP, 0.5 mg/ml.
LO ,ug/ml;
Calibration cwz~e Two paired series of varying volumes of the BSP standard were pipetted into reagent tubes, and brought to 4.5 ml with distilled water. By addition of 0.5 ml alkaline reagent and shaking, BSP color in one of the series was developed. For the references 0.5 ml dithionite reagent was used instead of the alkaline reagent. The samples were immediately transferred to stoppered cuvettes (r-cm cell path), and absorbances were read after 5 min at 580 nm, using the discolored sample out of each Clin. Chim.
Acta,
ZL
(1968)
439-.+.+1
SULFOBROMOPHTHALEIN
Fig.
2. Calibration
curve
pair as a reference.
441
IN SERUM
for BSP
determination
In Fig. z absorbance
at 580 nm.
is plotted
against
,crg BSP/ml
in the final
test solution. Determination of BSP in seTurn Paired o.5-ml samples of serum obtained from rats subjected to the BSP test were diluted with 4.0 ml of a 0.9% sodium chloride solution; 0.5 ml alkaline reagent or dithionite reagent, resp., was added, following the described procedure. The serum content of BSP in pg/ml was obtained by multiplying the value from the calibration curve by a factor of IO. No deviations from the calibration curve in water could be detected when known amounts of BSP were added to non-hemolyzed serum. For routine measurements on serum from experimental were reduced by a factor of IO, using microcuvettes
animals the volumes (I-cm cell path).
of liquid
Disturbances from bilkbin, turbidity and hemoglobin Spectra of serum with added bilirubin in alkaline solution were identical in the range from $50 to 600 nm in presence or absence of dithionite. Turbidity changes could not be observed. Disturbances from these factors can thus be excluded. Since the BSP test was applied to small experimental animals, where strong hemolysis is frequently associated with the withdrawal techniques used to obtain the blood samples, hemoglobin interference has been studied in detail. Alkaline hem atin, formed from hemoglobin in strong alkali, is rapidly reduced to hemochromogen by dithionite4. A hemoglobin solution was prepared by hemolysis of washed red cells from rats, and hemoglobin content was determined by the cyanmethemoglobin method5. The spectra were recorded after conversion to alkaline hematin and hemochromogen, resp., (Fig. I) in order to measure the interference of hemoglobin. At 580 nm, I mg/ml hemoglobin simulates a BSP content of 3 pg/ml. This error can practically be neglected in the case of weak hemolysis. As shown in Fig. I, molar absorbances of alkaline hematin and hemochromogen are equal at 570 nm. After application of a correction for the Sq/, lower absorbance of BSP, hemoglobin interference can be eliminated completely when measuring at 570 nm, even in the case of strong hemolysis.
OTT, PIRRWITZ
A NEW
METHOD
PHTHALEIN
FOR
439
THE
DETERMINATION
OF
SULFO~RO~O-
IN SERUM*
H. OTT .+ND D. I’IRRWITZ Sevvic~ de Biologic, (Received
June
27,
Euro$ean Comnzunifies-curator,
Joint Research Centre, Is$va {Ifalyj
Ig68)
Sulfobromophthalein (BSP) is discolored by sodium dithionite (Na,S,O,) in alkaline solution due to a reduction of the chromophore chinoide system in the dye. Application of this reaction for the estimation of BSP in serum and elimination of hemoglobin interference are described.
cator
In several methods for the sulfobromophthalein (BSP) retention test, the indiproperties of the dye are utilized in the spectrophotometric determination in
order to avoid a separate blood sampling for the serum blank. The absorbance of the alkaline sample is measured at $30 nm against a neutral dilution of the test serum. The difference in pH between sample and blank gives rise to interferences due to: (a) turbidity changes resulting from the higher solubility of lipids and lipoproteins in alkaline solution, (b) the pH dependency of bilirubin absorbance, (c) the conversion of oxyhemoglobin (which has a strong absorption band at 577 nm in neutral solution) to alkaline hematin at high pH. The latter has lower absorbance, thus simulating lower BSP readings. Corrections for hemoglobin content by additional readings at different wavelengths have been proposed by several authors ly2. It has also been suggested to keep the pH interval as small as necessary for developing full BSP colour, adjusting sample and reference to pH 10.4 and 7.1, resp., by means of buffers and adding p-toluene sulfonate to liberate BSP from plasma binding at low pH (ref. 3). It was found that BSP can be discolored by sodium dithionite (Na,S,O,) in alkaline solution, due to a reduction of the chromophore chinoide system in the dye. This reaction can be used for the convenient determination of BSP in serum. Interferences are minimized, the sample and blank having the same PH. The method should be also suitable for automated techniques.
* This publication
is contribution
No. qrr of Em-atom Biolofiy
Division.
Clin. Chinz. Ada, 22 (1968) 439-442
440
OTT, PIRRWITZ
APPARATI'S
AND
REAGENTS
A C’nicam SP 600 spectrophotometer was used for the spectrophotometric measurements. Absorption spectra were done on a Perkin-Elmer 350 recording spectrophotometer. mann,
BSP standard. IOO /[g/ml sulfobromophthalein sodium (Etablissements Paris) in distilled water. Alkaline reagent. ro(lb w/v solution of sodium hydroxide. Dithionite reagent. A solution of 1.0 g sodium dithionite
(Na,S,O,)
Kuhl-
(E. Merck A(;,
Darmstadt, “for analytical purposes, not free from chloride”) and 1.0 g sodium hydroxide in a final volume of IO ml was freshly prepared every day. EXPERIMENTA4L
Discoloratiort of BSP by sodium dithionite BSP reacts in alkaline medium with an excess of dithionite forming a leukocompound. The absorption spectra of BSP and reduced BSP are shown in Fig. I. The reduction under the conditions described below is complete within z to 3 min. This reaction is reversible upon access of oxygen, and BSP color reappears after several hours. Therefore the spectrophotometric measurements were done in stoppered or covered cuvettes between 5 and IO min after addition of the dithionitc reagent.
ITi
0
h.
1.
Absorption alkaline
- _.. "--L-
spectra: hematin,
--3-_I0.5 mg/ml;
BSI’, Lo fig/ml; P.-o---_ ,~-I :- hemochromogen,
rctluced BSP, 0.5 mg/ml.
LO ,ug/ml;
Calibration cwz~e Two paired series of varying volumes of the BSP standard were pipetted into reagent tubes, and brought to 4.5 ml with distilled water. By addition of 0.5 ml alkaline reagent and shaking, BSP color in one of the series was developed. For the references 0.5 ml dithionite reagent was used instead of the alkaline reagent. The samples were immediately transferred to stoppered cuvettes (r-cm cell path), and absorbances were read after 5 min at 580 nm, using the discolored sample out of each Clin. Chim.
Acta,
ZL
(1968)
439-.+.+1
SULFOBROMOPHTHALEIN
Fig.
2. Calibration
curve
pair as a reference.
441
IN SERUM
for BSP
determination
In Fig. z absorbance
at 580 nm.
is plotted
against
,crg BSP/ml
in the final
test solution. Determination of BSP in seTurn Paired o.5-ml samples of serum obtained from rats subjected to the BSP test were diluted with 4.0 ml of a 0.9% sodium chloride solution; 0.5 ml alkaline reagent or dithionite reagent, resp., was added, following the described procedure. The serum content of BSP in pg/ml was obtained by multiplying the value from the calibration curve by a factor of IO. No deviations from the calibration curve in water could be detected when known amounts of BSP were added to non-hemolyzed serum. For routine measurements on serum from experimental were reduced by a factor of IO, using microcuvettes
animals the volumes (I-cm cell path).
of liquid
Disturbances from bilkbin, turbidity and hemoglobin Spectra of serum with added bilirubin in alkaline solution were identical in the range from $50 to 600 nm in presence or absence of dithionite. Turbidity changes could not be observed. Disturbances from these factors can thus be excluded. Since the BSP test was applied to small experimental animals, where strong hemolysis is frequently associated with the withdrawal techniques used to obtain the blood samples, hemoglobin interference has been studied in detail. Alkaline hem atin, formed from hemoglobin in strong alkali, is rapidly reduced to hemochromogen by dithionite4. A hemoglobin solution was prepared by hemolysis of washed red cells from rats, and hemoglobin content was determined by the cyanmethemoglobin method5. The spectra were recorded after conversion to alkaline hematin and hemochromogen, resp., (Fig. I) in order to measure the interference of hemoglobin. At 580 nm, I mg/ml hemoglobin simulates a BSP content of 3 pg/ml. This error can practically be neglected in the case of weak hemolysis. As shown in Fig. I, molar absorbances of alkaline hematin and hemochromogen are equal at 570 nm. After application of a correction for the Sq/, lower absorbance of BSP, hemoglobin interference can be eliminated completely when measuring at 570 nm, even in the case of strong hemolysis.
OTT, PIRRWITZ