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The determination of bromsulphthalein in serum
CLINICA
CHIMICA
ACTA
THE DETERMINATION
A. FLECK
Biochemistry (Received
AND
BARBARA
Department, July
OF BROMSULPHTHALEIN
IN SERUM
A. MORRISON
Glasgow
Royal
Infirmary,
Castle Street, Glasgow,
C.4 (U.K.)
24, Igp)
SUMMARY
The conditions for acceptable accuracy and precision in the determination of bromsulphthalein (BSP) in plasma have been explored. Critical factors include: pH ; the dissociation of the protein-dye complex (by sodium p-toluene sulphonate) ; the variation of colour with time after addition of alkali; and the stability of the dye on storage. It is suggested that acceptable results will be obtained only if care is taken regarding the above conditions and the plasma BSP levels determined on the same day as the specimens are obtained from the patient.
INTRODUCTION
The clearance of BSP from plasma following its intravenous injection has been utilised as a test of liver function in manl. It is usually considered that one advantage of the test is its simplicity, since the determination of the dye is based on the observation that its solutions are colourless in neutral or acid solution and purple in alkaline solution. However, several different procedures have been adopted for the determination of BSP in plasma. For example, simple addition of NaOH has been employed, reading against an acidified serum blank2. Similarly, glycine3 and phosphate4 buffers pH 10.6 have been used. More recently it has been claimed that interference due to haemoglobin, etc. can be eliminated by the use of suitable blanks which are obtained by decolorisation of the dye with Na,S,O, at alkaline pH (ref. 5). Little account seems to have been taken of the observations of Seligson et aL4 that plasma protein causes a shift of the wavelength of maximum absorption of the dye and also changes its specific absorptivity (see, however, Henrya). Seligson et aL4 suggested that these errors could be eliminated by the addition of the complex anion p-toluene sulphonate (PTS).
The existence of such a variety of methods, together with the suspicion that the routine results of BSP tests in this laboratory were not entirely satisfactory, led to the present investigation.
Clin. Chim. Acta,
~CJ(1970) 751-75g
FLECK, MORRISON
752 METHODS
The bromsulphthalein used was obtained in 3-ml ampoules from Hynson, Westcott & Dunning (suitable for intravenous injection). The standard procedure adopted was to add 3 ml of alkali (or alkaline buffer) to I ml plasma. The method of obtaining a suitable blank was the subject of special section of this investigation (see below). In order to determine recoveries, small known amounts of dye were added to samples of serum or plasma. The buffers used were 0.25 M sodium phosphate buffer, and I M glycine buffer. Also 0.05 M NaOH was used in place of a buffer solution. Where pH is quoted this refers to the pH of the final buffer-plasma mixture. The suitability of $-toluene sulphonate (sodium p-toluene sulphonate, 12.8 g/l of buffer) andurea (8.3 M in buffer, giving 5 M in final protein-buffer mixture) as protein-dye dissociating compounds was investigated. Pooled and fresh human serum or plasma was used throughout. l.O-
I
08-
A
550
500
Wavelength
580
600
I
nm
B
Wavelength
nm
Fig. 1. Absorption spectra of BSP in the presence and absence of plasma and pTS. A, 0.25 M phosphate buffer pH 10.5; B, 0.25 M phosphate buffer pH 11.2. I-pure dye; II-dyefplasma; III-dye+plasma$-pTS. Clin. Chim. Acta, 30 (1970) 751-759
BROMSULPHTHALEIN
IN SERUM
753
RESULTS
Protein effect on absorption characteristics of dye E$ect on absorption maximum. The absorption spectra of the pure dye and the same concentration of dye in the presence of serum read against the appropriate blanks in phosphate buffer pH 10.5 and pH 11.2 are shown in Figs. I A and I B respectively. These spectra were obtained with a Unicam SP 800 recording spectrophotometer. Clearly the observations of Seligson et aL4 are confirmed; the presence of protein causes a shift in the absorption maximum of the pure dye approximately IO nm towards the longer wavelengths and an apparent reduction in its absorbance. The presence of sodium p-toluene sulphonate reverses this shift, returning the absorption maximum to nearly that of the pure dye (Fig. IA and B). At both pH 10.5 and 11.2 the addition of p-toluene sulphonate to proteincontaining solutions causes a reversion of the maximum absorption to that of the pure dye. However, at pH 10.5 the absorbance after addition of p-toluene sulphonate is virtually unaltered while at pH 11.2 the absorbance is returned to almost that of the pure dye. Accordingly we have added p-toluene sulphonate as the sodium salt to all protein-containing solutions of the dye and taken absorbance readings at 580 nm throughout. E_ffect on pH---dissociation cwves of BSP. In the presence of protein the maximum absorption of the dye is attained at higher pH than for the pure dye solution (Fig. 2). Thus pH of IO or greater is acceptable for the determination of the pure dye but pH of at least 1x.0 is required when protein is present. This applies also in the presence of urea or sodium p-toluene sulphonate, both of which, however, raise the absorption of the protein-dye mixture to approximately g5-g7o/o that of the pure dye in buffer. In some cases the absorbance of the dye on the presence of protein was found to decline above pH 11.5, accordingly pH 11.2 was selected as optimum. Stability of the colour The investigation of the stability of the dye in alkaline solution in the presence of serum was prompted by the observation that alkaline solutions of the dye plus serum lost approximately 50% of their colour after standing overnight at room temperature. The absorbance of solutions containing the same amount of the dye, with and without serum, in phosphate or glycine buffer or NaOH at pH 11.2 was read at intervals of time up to 2 h. The effect of temperature (3”, room temperature-approx. 20°, 3o”, and 50”) was observed (Fig. 3). Using the programmed Unicam SP 800 (i.e. SP 825) and Servoscribe external recorder, readings (at least one per minute) were recorded for 20 min at room temperature. With serum present a slight initial fall in absorbance up to 3 min was commonly observed; this was followed by a gradual rise of usually approximately 2% of the total absorbance up to 2 h. The most striking effects were observed at 3’ and 50”. At 3” there was a continuous and gradual increase in colour up to 2 h both for pure dye and dye plus serum (Fig. 3). This was not due to condensation and all reagents were pre-chilled. In contrast, at 50” the absorption of the pure dye remains constant, while in the presence of serum after 40 min there is a rapid decline in absorption (Fig. 3). It was concluded that reagents should be used at room temperature (in the Clin. Chim. Acta, 30 (rgp) 751-759
FLECK, MORRISON
7.54
0.6 -
I
u C---
/
0.5 ~,~,, -
0.4
-III Ip
lf
0.3
/
0.2 / 0.1
/ / / zo
8.0
9.0
10.0
11.0
12.0
Fig. 2. Dissociation of BSP. 0.25 M phosphate buffer was made up to the designated PH. I-pure BSP; II-BSP+plasma+pTS; III-BSP+plasma+R M urea; IV-BSP+plasma. Fig. 3. The effects of time and temperature on absorbance of solutions of BSP. --A--, pure BSP 3?; --o--, pure BSP50”; -A-, BSP+plasma 3O; -a--, BSP+plasma 50”. Solutions in 0.25 M phosphate buffer pH r 1.2 containing 12.8 g/l p-toluene sulphonate.
region of 20') and that absorption of dye solutions should be read between IO and 20 min after mixing. All reagents used (i.e. phosphate, and glycine buffers and NaOH) gave similar results. Stability of the dye on storage Tubes each containing I ml serum and I ml dye solution (3o~g~ml) were stored; some in refrigerators (3”) and some in the deep freeze (-IO”). Three ml phosphate buffer was added to one of each of these daily and the absorption read IO min after mixing. Storage over 3 days at --0’ caused a slight increase in absorbance and at 3” a decrease in absorbance of 5%. Serum to which concentrated dye solution was added (to give an approximate concentration of 40 pglrnl in serum) was also stored at 3” and -IoO. The absorption recorded on addition of buffer to this serum each day showed at --IO* a 10% increase over 3 days and at 3” an initial increase followed by a marked decrease after 3 days. Because of these observations it was concluded that it was unwise to store plasma or serum withdrawn from a patient after injection of BSP and that estimation of the dye should be carried out as soon as possible after withdrawal of blood.
We were unable to confirm the work of Ott and Pirrwitz? who claimed that the use of sodium dithionite as a decolourising agent gave a suitable blank for estimation of BSP. A standard curve prepared using I ml standard, I ml serum and 3 ml phosphate buffer containing 1% dithionite indicated that decolourisation was not complete. Comparison of absorption of different sera with and without dithionite shows that the dithionite lowers the absorption of the serum and the degree of this is dependent on the original colour of the serum (see Table I). Ch.
Chim. Acta,
30 (1970) 75x-759
BROMSIJLPHTHALEIN TABLE
I
THE EFFECT IN SERUM
OF
pH AND DETERMINATION
of SeYUm
Colour
Serum PH
1. 2. 3. 4. 5. 6.
75.5
IN SERUM
Pale clear Yellow clear Orange clear Deep orange clear Haemolysed clear Turbid clear
ON
THE
BLANK
VALUE
in buffer
I
pH 11.2 dithionite II
0.129
0.087
0.165 0.148 0.170 0.350 0.230
0.126
11.2
o.ogj 0.144 0.270 0.218
plus
pH 7.1
IN
THE
DETERMINATION
OF
BSP
A I-II
A I-III
0.042
0.020
III
0.109 0.145 0.380 0.254
0.039 0.051
0.026 0.080 0.012
Mean A -Co.042
0.003 -0.030 -0.024 -0.008
Dithionite appears to decolourise other substances, thus giving falsely high values of BSP. The effect is most marked in haemolysed serum, and the spectra obtained indicate that this is due to dithionite converting oxyhaemoglobin to haemoglobin. Even slight haemolysis will invalidate the results, and it is recommended that estimations should not be performed on specimens in which any haemolysis is visible. Blanks prepared by adding 3 ml of phosphate buffer of pH 7.1 to I ml serum gave absorption very near to that of serum in buffer of pH 11.2 (see Table I) and this was the method chosen as giving the least likelihood of error. However, turbid or haemolysed sera give high blanks, and these will give falsely low values of BSP (Table I). Precision
and evoY
It is possible to demonstrate that for simple single beam spectrophotometers such as the Unicam SP 600, photometric error is given by the expression
“C_dT C
I
T In T
(where dC is the error in concentration, C; and similarly AT is the error in Transmission, T)738. It follows that plotting the error in concentration (K/C as %) against o/oT scale reading (or Absorbance) yields a U-shaped curve (see Fig. 4). An assessment of the reading error of the photometer (Unicam SP 600) used in the present study was obtained by taking at least IO readings from each of several solutions of different absorbances. The mean results, plotted in Fig. 4 show good agreement with the U-shaped theoretical curve and indicate that the operator precision with the photometer was of the order of 0.3% T. Thus at the anticipated upper limit of the normal range (i.e. 3% retention of dye) with the proposed method employing a I in 4 final dilution of serum, the photometric error alone will be just less than 2% when using a cuvette of I cm light path and assuming that 5% retention of dye corresponds to 5 pg/ml, yielding an absorbance close to 0.100 or 80% T (see Fig. 5). Methods in which greater dilutions of serum are used2p0,for example I/IZ, will lead to very low absorbance at the upper limit of the normal and correspondingly high photometric error, certainly greater than 5%. It is apparent from Fig. 4 that the use of a cuvette of 2 cm light path is desirable in order Clin. Chim. Acta, 30 (1970) 751-759
756
FLECK,MORRISON
5
$
4
0.7 -
k w f
3
05-
2 Effect of protein total proterl 39 gn00ml
7
on
standard
curve
albumm
20 gnCO ml
= 70
90x30
Fig. 4. Photometric error curves. y0 Error (K/C x IOO) plotted against “/;I T; -o-Mean error with Unicam SP 600. The figures 0.1, 0.3, 0.5 refer to the error in o/0 T (i.e. LIT). The hatched areas indicate the approximate y0 T range of specimens taken 45 min after giving 5 mg HP/kg body weight to normal individuals when the BSP is estimated by the present recommended method and the dye absorption read in ~-cm or z-cm cuvettes. Fig. 5. BSP calibration curves in the presence of varying ,ug BSP per ml. Ordinate: Absorbance.
concentration
of protein.
Abscissa:
to reduce error to acceptable levels when assaying specimens close to the normal range. It is possible when the data for a suitable standard curve are available to calculate the error in concentration from the statistics of simple linear regression. Using the general formula of Bowker and Lieberman’O for the error limits in the abscissa and taking t for 99% probability the error was computed and found to be less than 1% over the calibration range (lowest standard approximately 5 pg BSP/ml). These levels of error confirm that the proposed method should give acceptable results down to the generally accepted limit of the normal range at 45 min (i.e. 5% retention of dye). Sqgested procedure for the determination of BSP in scyum Reagents: I. bromsulphthalein stock solution 500 ,ugjml, prepared from the 3-ml vials containing 50 mg/roo ml supplied by Hynson, Westcott and Denning; 2. phosphate buffer, pH 11.2,prepared from 0.25 M di- and tri-sodium phosphate, and containing x2.8 g/l sodium-p-toluene sulphonate; 3. phosphate buffer, pH 7.1 (0.25 M); 4. pooled serum. This must not be turbid. Standards: Prepare standards of 5, 7.5, IO, 12.5, 15, 25 ,ug/ml by weighing into IO-ml volumetric flasks 0.1, 0.15. 0.2, 0.25, 0.3, 0.5 g of stock BSP solution, and calculate the exact concentrations. Make up to the mark with pooled serum, mix well and stand 30 min. Using bulb pipettes throughout, to r ml of each standard, and to I ml of Cl&. Chi%. A&,
30
(1970)
7 jr-759
BROMSULPHTHALEIN IN SERUM
757
pooled serum, add 3 ml phosphate buffer pH 11.2, mix well and stand 10-15 min. Read absorbance against water in SP 600 at 580 nm. Subtract the absorbance of zero standard from absorbance of standards and prepare standard curve. Note-The calculation of the equation of the line from the best least squares fit is useful. Unknowns:
Test: To I ml serum add 3 ml phosphate buffer pH 11.2. Blank : To I ml serum add 3 ml buffer pH 7.1. Mix well, stand 10-15 min and read test and blank against water in SP 600 at 580 nm. Subtract blank from test reading, and calculate the concentration from standard curve. Using the equation of the least squares fit to the standard curve saves time and improves precision. Recoveries Recoveries obtained using this method over a range of sera, containing from 2 to 26 pg/ml BSP gave a mean recovery of 99.56% (N = 33) with S.D. of 2.3 giving 99% confidence limits of 98.9 to 100.2~/~.At the 5 pg/ml level, which is usually taken to be the normal retention of the dye after 45 min, mean recovery was 99.33 (N = 16) S.D. 2.18 with 99% confidence limits 98.3-100.3. In this series of recoveries, however, there was one “rogue” recovery of 125% which we could not account for. Where serum gave a high blank (absorbance > 0.09) recoveries were low, and these also were omitted from the calculations. Normal range
A small number of technicians and patients with no history or evidence of liver disease were subjected to the conventional BSP clearance test. The dose of 5 mg/kg body weight was given i.v. By weighing the syringe before and after injection the amount given was found to deviate by not more than + 2% from the calculated dose. A check of the urine output of BSP during the test indicated that this was never more than 1.8% of the i.v. dose. BSP was determined in a venous blood sample taken at 45 min after injection and the 95% confidence limits for the normal range (n = 7) found to be from 1.4 to 4.6% retention of dye (taking IO mg/roo ml dye in serum as IOO~/~).The recovery of dye in vitro from a “0 time” sample taken from these subjects had a range of from 98.4 to 100.6~/~with a mean of 99.4% recovery. It would seem therefore that the proposed method gives satisfactory recovery in use and the normal range is close to that suggested by Zieve and HillI’. DISCUSSION The interaction of proteins and dyes is widely known and utilised. For example, Nayyar and Glick12 have described a method in which BSP is utilised in the quantitative determination of protein and there is the well known protein error of indicators utilised in “Albustix”. Thus changes in the absorption spectrum of pure BSP in buffer solution caused by the presence of protein, described by Se&on et al.4 and confirmed by us, were to be expected. The extent of the changes is dependent on the amount of protein present and Clin.
Chim.
Acta,
30
(1970)751-759
758
FLECK,
MORRISON
when the total serum protein concentration changes from 5 g/roe ml through 7 g/Ioo ml to IO g/xoo ml changes in the spectrum of the dye were detectable using the Unicam SP 800. These changes can however be virtually though not completely eliminated by the introduction of sodium fi-toluene sulphonate as suggested by Seligson et aL4 (see Fig. I). It was found to be advantageous to use twice the concentration of pTS advocated by Seligson, although the improvement is only slight. This action of pTS is possibly due to protein denaturation, since 5 M urea gives similar results (Fig. Z) as does heat (e.g. raising the temperature to 60”). This last, however, led to difficulties due to the development of turbidity and was regarded as an unsuitable procedure. It may also be anticipated that the presence of protein would alter the pH dissociation curve of the dye. This does not seem to have been taken into account in the dete~ination of BSP in plasma or serum by others, and clearly (see Fig. 2) influences the results. It is therefore suggested that a final pH of x1.2 is more appropriate for the determination of plasma or serum BSP than is the final pH of 10.5 as employed by Seligson et ah4 and Tovey 3. It was also concluded that because of these effects of protein the standard curve should be determined in the presence of serum or plasma. The proposed measures, however, do not completely overcome the effect of protein as is shown by standard curves prepared using sera containing different amounts of protein (Fig. 5). This would suggest that ideally the patient’s own serum should be used for the preparation of the standard curve, but as this is usually not practicable, the protein content should be estimated and taken into consideration when interpreting the results of the BSP test. The error introduced by generally ignoring this effect is small, particularly for levels close to 5% retention of dye (Pig. 5). Yet another possible difficulty could arise from the presence of conjugates of BSP in plasma. We have been unable to detect these in plasma using thin-layer chromatography even in samples from human subjects considered to have some degree of liver dysfunction due to alcoholism. This is in agreement with the conclusion of Higgins et aZ.l3who found that the greater part of the dye in plasma 40 or 60 min after injection in human subjects was unconjugated BSP. It was initially not anticipated that changes due to temperature and time after mixing reagents would be significant and we have no explanation for the observed effects. These are sufficiently large that it is advisable to mix reagents at room temperature, to avoid extremes of temperature and to read the absorption at a specified time after mixing which should not be less than 5 min, nor greater than 30 min and we suggest between IO and 20 min after mixing as ideal. After detecting the changes in colour intensity of dye-plasma-buffer mixtures with temperature and time, changes on storage were anticipated. Storage of the pure dye either in concentrated form or dilute solution in the refrigerator seems to be satisfactory for up to a month or more. This is not the case when protein is present. In the presence of protein storingin thedeep freeze at --IO’ even for a few days leads to marked changes. It follows that for satisfactory results BSP in plasma or serum should be determined on the same day as the blood sample was withdrawn. When care is taken over these various points it is possible to determine BSP with acceptable accuracy and precision in plasma; that is, to obtain over-all precision Clilz. Chiwz.Acta, 30 (1970) 7.j-759
BROMSULPHTHALEIN
IN SERUM
759
better than 5% and usually not greater than 2%, Claims of precision of better than 1% (ref. g) must be viewed with suspicion in view of our findings. In fact, our present results suggest that unless considerable care is taken the error at the level usually accepted as the upper limit of normal for the 45-min test (i.e. 5% retention) may be much greater than 5% and occasionally of the order of 50%. When employed with suitable care (see ref. 14) both with injection of dye and its determination in the laboratory, the “45-min” BSP test would seem to have a place as a sensitive and simple test of liver function. ACKNOWLEDGEMENTS
We wish to thank Mr. C. Bryce and Mrs. Margaret Lockhart for technical assistance. REFERENCES I S. SHERLOCK, A. G. BEAN, B. H. BILLING AND
J. C. S. PATERSON, J. Lab. Clin. Med., 35 (1950)
923. H. J. D. H. R. H. D. 8 G. g 0. IO A.
VARLEY, Practical Clinical Biochemistry, 4th ed., Heinemann, London, 1967, p. 383. E. TOVEY, C&z. Chim. Acta, 15 (1967) 149. SELIGSON, J. MARION AND E. DODSON, Clin. Chem., 3 (1957) 638. OTT AND D. PIRRWITZ, Clin. Chim. Acta, 22 (1968) 439. J. HENRY, Clinical Chemistry, Harper and Row, New York, 1964, p. 551. H. WILLARD, L. L. MERRITT AND 1. A. DEAN, Instrumental Methods of _ Analysis, _.. 4th ed., Van Nostrand, Princeton, N. J., 1966. F. LOTHIAN, Absorption Spectrophotometry, Adam Hilger Ltd., London, 1969. H. GAEBLER, Am. J. Clin. Pathol., 15 (1945) 452. H. BOWKER AND G. J. LIEBERMAN, Engineering Statistics, Prentice-Hall, Englewood Cliffs,
II 12 13 14
ZIEVE AND E. HILL, Gastroenterology, 28 (1955) 766. N. NAYYAR AND D. GLICK, J. Histochem. Cytochem., 2 (1954) 282. E. HIGGINS, W. T. FOULK AND J. L. BALLMAN. J. Clin. Invest., 38 (1960) 194. W. ASTIN, Bit. Med. J., ii (1965) 408.
2 3 4 5 6 7
N.J., 1959.
L. S. F. T.
Clin. Chim. Acta, 30 (1970) 751-759
CHIMICA
ACTA
THE DETERMINATION
A. FLECK
Biochemistry (Received
AND
BARBARA
Department, July
OF BROMSULPHTHALEIN
IN SERUM
A. MORRISON
Glasgow
Royal
Infirmary,
Castle Street, Glasgow,
C.4 (U.K.)
24, Igp)
SUMMARY
The conditions for acceptable accuracy and precision in the determination of bromsulphthalein (BSP) in plasma have been explored. Critical factors include: pH ; the dissociation of the protein-dye complex (by sodium p-toluene sulphonate) ; the variation of colour with time after addition of alkali; and the stability of the dye on storage. It is suggested that acceptable results will be obtained only if care is taken regarding the above conditions and the plasma BSP levels determined on the same day as the specimens are obtained from the patient.
INTRODUCTION
The clearance of BSP from plasma following its intravenous injection has been utilised as a test of liver function in manl. It is usually considered that one advantage of the test is its simplicity, since the determination of the dye is based on the observation that its solutions are colourless in neutral or acid solution and purple in alkaline solution. However, several different procedures have been adopted for the determination of BSP in plasma. For example, simple addition of NaOH has been employed, reading against an acidified serum blank2. Similarly, glycine3 and phosphate4 buffers pH 10.6 have been used. More recently it has been claimed that interference due to haemoglobin, etc. can be eliminated by the use of suitable blanks which are obtained by decolorisation of the dye with Na,S,O, at alkaline pH (ref. 5). Little account seems to have been taken of the observations of Seligson et aL4 that plasma protein causes a shift of the wavelength of maximum absorption of the dye and also changes its specific absorptivity (see, however, Henrya). Seligson et aL4 suggested that these errors could be eliminated by the addition of the complex anion p-toluene sulphonate (PTS).
The existence of such a variety of methods, together with the suspicion that the routine results of BSP tests in this laboratory were not entirely satisfactory, led to the present investigation.
Clin. Chim. Acta,
~CJ(1970) 751-75g
FLECK, MORRISON
752 METHODS
The bromsulphthalein used was obtained in 3-ml ampoules from Hynson, Westcott & Dunning (suitable for intravenous injection). The standard procedure adopted was to add 3 ml of alkali (or alkaline buffer) to I ml plasma. The method of obtaining a suitable blank was the subject of special section of this investigation (see below). In order to determine recoveries, small known amounts of dye were added to samples of serum or plasma. The buffers used were 0.25 M sodium phosphate buffer, and I M glycine buffer. Also 0.05 M NaOH was used in place of a buffer solution. Where pH is quoted this refers to the pH of the final buffer-plasma mixture. The suitability of $-toluene sulphonate (sodium p-toluene sulphonate, 12.8 g/l of buffer) andurea (8.3 M in buffer, giving 5 M in final protein-buffer mixture) as protein-dye dissociating compounds was investigated. Pooled and fresh human serum or plasma was used throughout. l.O-
I
08-
A
550
500
Wavelength
580
600
I
nm
B
Wavelength
nm
Fig. 1. Absorption spectra of BSP in the presence and absence of plasma and pTS. A, 0.25 M phosphate buffer pH 10.5; B, 0.25 M phosphate buffer pH 11.2. I-pure dye; II-dyefplasma; III-dye+plasma$-pTS. Clin. Chim. Acta, 30 (1970) 751-759
BROMSULPHTHALEIN
IN SERUM
753
RESULTS
Protein effect on absorption characteristics of dye E$ect on absorption maximum. The absorption spectra of the pure dye and the same concentration of dye in the presence of serum read against the appropriate blanks in phosphate buffer pH 10.5 and pH 11.2 are shown in Figs. I A and I B respectively. These spectra were obtained with a Unicam SP 800 recording spectrophotometer. Clearly the observations of Seligson et aL4 are confirmed; the presence of protein causes a shift in the absorption maximum of the pure dye approximately IO nm towards the longer wavelengths and an apparent reduction in its absorbance. The presence of sodium p-toluene sulphonate reverses this shift, returning the absorption maximum to nearly that of the pure dye (Fig. IA and B). At both pH 10.5 and 11.2 the addition of p-toluene sulphonate to proteincontaining solutions causes a reversion of the maximum absorption to that of the pure dye. However, at pH 10.5 the absorbance after addition of p-toluene sulphonate is virtually unaltered while at pH 11.2 the absorbance is returned to almost that of the pure dye. Accordingly we have added p-toluene sulphonate as the sodium salt to all protein-containing solutions of the dye and taken absorbance readings at 580 nm throughout. E_ffect on pH---dissociation cwves of BSP. In the presence of protein the maximum absorption of the dye is attained at higher pH than for the pure dye solution (Fig. 2). Thus pH of IO or greater is acceptable for the determination of the pure dye but pH of at least 1x.0 is required when protein is present. This applies also in the presence of urea or sodium p-toluene sulphonate, both of which, however, raise the absorption of the protein-dye mixture to approximately g5-g7o/o that of the pure dye in buffer. In some cases the absorbance of the dye on the presence of protein was found to decline above pH 11.5, accordingly pH 11.2 was selected as optimum. Stability of the colour The investigation of the stability of the dye in alkaline solution in the presence of serum was prompted by the observation that alkaline solutions of the dye plus serum lost approximately 50% of their colour after standing overnight at room temperature. The absorbance of solutions containing the same amount of the dye, with and without serum, in phosphate or glycine buffer or NaOH at pH 11.2 was read at intervals of time up to 2 h. The effect of temperature (3”, room temperature-approx. 20°, 3o”, and 50”) was observed (Fig. 3). Using the programmed Unicam SP 800 (i.e. SP 825) and Servoscribe external recorder, readings (at least one per minute) were recorded for 20 min at room temperature. With serum present a slight initial fall in absorbance up to 3 min was commonly observed; this was followed by a gradual rise of usually approximately 2% of the total absorbance up to 2 h. The most striking effects were observed at 3’ and 50”. At 3” there was a continuous and gradual increase in colour up to 2 h both for pure dye and dye plus serum (Fig. 3). This was not due to condensation and all reagents were pre-chilled. In contrast, at 50” the absorption of the pure dye remains constant, while in the presence of serum after 40 min there is a rapid decline in absorption (Fig. 3). It was concluded that reagents should be used at room temperature (in the Clin. Chim. Acta, 30 (rgp) 751-759
FLECK, MORRISON
7.54
0.6 -
I
u C---
/
0.5 ~,~,, -
0.4
-III Ip
lf
0.3
/
0.2 / 0.1
/ / / zo
8.0
9.0
10.0
11.0
12.0
Fig. 2. Dissociation of BSP. 0.25 M phosphate buffer was made up to the designated PH. I-pure BSP; II-BSP+plasma+pTS; III-BSP+plasma+R M urea; IV-BSP+plasma. Fig. 3. The effects of time and temperature on absorbance of solutions of BSP. --A--, pure BSP 3?; --o--, pure BSP50”; -A-, BSP+plasma 3O; -a--, BSP+plasma 50”. Solutions in 0.25 M phosphate buffer pH r 1.2 containing 12.8 g/l p-toluene sulphonate.
region of 20') and that absorption of dye solutions should be read between IO and 20 min after mixing. All reagents used (i.e. phosphate, and glycine buffers and NaOH) gave similar results. Stability of the dye on storage Tubes each containing I ml serum and I ml dye solution (3o~g~ml) were stored; some in refrigerators (3”) and some in the deep freeze (-IO”). Three ml phosphate buffer was added to one of each of these daily and the absorption read IO min after mixing. Storage over 3 days at --0’ caused a slight increase in absorbance and at 3” a decrease in absorbance of 5%. Serum to which concentrated dye solution was added (to give an approximate concentration of 40 pglrnl in serum) was also stored at 3” and -IoO. The absorption recorded on addition of buffer to this serum each day showed at --IO* a 10% increase over 3 days and at 3” an initial increase followed by a marked decrease after 3 days. Because of these observations it was concluded that it was unwise to store plasma or serum withdrawn from a patient after injection of BSP and that estimation of the dye should be carried out as soon as possible after withdrawal of blood.
We were unable to confirm the work of Ott and Pirrwitz? who claimed that the use of sodium dithionite as a decolourising agent gave a suitable blank for estimation of BSP. A standard curve prepared using I ml standard, I ml serum and 3 ml phosphate buffer containing 1% dithionite indicated that decolourisation was not complete. Comparison of absorption of different sera with and without dithionite shows that the dithionite lowers the absorption of the serum and the degree of this is dependent on the original colour of the serum (see Table I). Ch.
Chim. Acta,
30 (1970) 75x-759
BROMSIJLPHTHALEIN TABLE
I
THE EFFECT IN SERUM
OF
pH AND DETERMINATION
of SeYUm
Colour
Serum PH
1. 2. 3. 4. 5. 6.
75.5
IN SERUM
Pale clear Yellow clear Orange clear Deep orange clear Haemolysed clear Turbid clear
ON
THE
BLANK
VALUE
in buffer
I
pH 11.2 dithionite II
0.129
0.087
0.165 0.148 0.170 0.350 0.230
0.126
11.2
o.ogj 0.144 0.270 0.218
plus
pH 7.1
IN
THE
DETERMINATION
OF
BSP
A I-II
A I-III
0.042
0.020
III
0.109 0.145 0.380 0.254
0.039 0.051
0.026 0.080 0.012
Mean A -Co.042
0.003 -0.030 -0.024 -0.008
Dithionite appears to decolourise other substances, thus giving falsely high values of BSP. The effect is most marked in haemolysed serum, and the spectra obtained indicate that this is due to dithionite converting oxyhaemoglobin to haemoglobin. Even slight haemolysis will invalidate the results, and it is recommended that estimations should not be performed on specimens in which any haemolysis is visible. Blanks prepared by adding 3 ml of phosphate buffer of pH 7.1 to I ml serum gave absorption very near to that of serum in buffer of pH 11.2 (see Table I) and this was the method chosen as giving the least likelihood of error. However, turbid or haemolysed sera give high blanks, and these will give falsely low values of BSP (Table I). Precision
and evoY
It is possible to demonstrate that for simple single beam spectrophotometers such as the Unicam SP 600, photometric error is given by the expression
“C_dT C
I
T In T
(where dC is the error in concentration, C; and similarly AT is the error in Transmission, T)738. It follows that plotting the error in concentration (K/C as %) against o/oT scale reading (or Absorbance) yields a U-shaped curve (see Fig. 4). An assessment of the reading error of the photometer (Unicam SP 600) used in the present study was obtained by taking at least IO readings from each of several solutions of different absorbances. The mean results, plotted in Fig. 4 show good agreement with the U-shaped theoretical curve and indicate that the operator precision with the photometer was of the order of 0.3% T. Thus at the anticipated upper limit of the normal range (i.e. 3% retention of dye) with the proposed method employing a I in 4 final dilution of serum, the photometric error alone will be just less than 2% when using a cuvette of I cm light path and assuming that 5% retention of dye corresponds to 5 pg/ml, yielding an absorbance close to 0.100 or 80% T (see Fig. 5). Methods in which greater dilutions of serum are used2p0,for example I/IZ, will lead to very low absorbance at the upper limit of the normal and correspondingly high photometric error, certainly greater than 5%. It is apparent from Fig. 4 that the use of a cuvette of 2 cm light path is desirable in order Clin. Chim. Acta, 30 (1970) 751-759
756
FLECK,MORRISON
5
$
4
0.7 -
k w f
3
05-
2 Effect of protein total proterl 39 gn00ml
7
on
standard
curve
albumm
20 gnCO ml
= 70
90x30
Fig. 4. Photometric error curves. y0 Error (K/C x IOO) plotted against “/;I T; -o-Mean error with Unicam SP 600. The figures 0.1, 0.3, 0.5 refer to the error in o/0 T (i.e. LIT). The hatched areas indicate the approximate y0 T range of specimens taken 45 min after giving 5 mg HP/kg body weight to normal individuals when the BSP is estimated by the present recommended method and the dye absorption read in ~-cm or z-cm cuvettes. Fig. 5. BSP calibration curves in the presence of varying ,ug BSP per ml. Ordinate: Absorbance.
concentration
of protein.
Abscissa:
to reduce error to acceptable levels when assaying specimens close to the normal range. It is possible when the data for a suitable standard curve are available to calculate the error in concentration from the statistics of simple linear regression. Using the general formula of Bowker and Lieberman’O for the error limits in the abscissa and taking t for 99% probability the error was computed and found to be less than 1% over the calibration range (lowest standard approximately 5 pg BSP/ml). These levels of error confirm that the proposed method should give acceptable results down to the generally accepted limit of the normal range at 45 min (i.e. 5% retention of dye). Sqgested procedure for the determination of BSP in scyum Reagents: I. bromsulphthalein stock solution 500 ,ugjml, prepared from the 3-ml vials containing 50 mg/roo ml supplied by Hynson, Westcott and Denning; 2. phosphate buffer, pH 11.2,prepared from 0.25 M di- and tri-sodium phosphate, and containing x2.8 g/l sodium-p-toluene sulphonate; 3. phosphate buffer, pH 7.1 (0.25 M); 4. pooled serum. This must not be turbid. Standards: Prepare standards of 5, 7.5, IO, 12.5, 15, 25 ,ug/ml by weighing into IO-ml volumetric flasks 0.1, 0.15. 0.2, 0.25, 0.3, 0.5 g of stock BSP solution, and calculate the exact concentrations. Make up to the mark with pooled serum, mix well and stand 30 min. Using bulb pipettes throughout, to r ml of each standard, and to I ml of Cl&. Chi%. A&,
30
(1970)
7 jr-759
BROMSULPHTHALEIN IN SERUM
757
pooled serum, add 3 ml phosphate buffer pH 11.2, mix well and stand 10-15 min. Read absorbance against water in SP 600 at 580 nm. Subtract the absorbance of zero standard from absorbance of standards and prepare standard curve. Note-The calculation of the equation of the line from the best least squares fit is useful. Unknowns:
Test: To I ml serum add 3 ml phosphate buffer pH 11.2. Blank : To I ml serum add 3 ml buffer pH 7.1. Mix well, stand 10-15 min and read test and blank against water in SP 600 at 580 nm. Subtract blank from test reading, and calculate the concentration from standard curve. Using the equation of the least squares fit to the standard curve saves time and improves precision. Recoveries Recoveries obtained using this method over a range of sera, containing from 2 to 26 pg/ml BSP gave a mean recovery of 99.56% (N = 33) with S.D. of 2.3 giving 99% confidence limits of 98.9 to 100.2~/~.At the 5 pg/ml level, which is usually taken to be the normal retention of the dye after 45 min, mean recovery was 99.33 (N = 16) S.D. 2.18 with 99% confidence limits 98.3-100.3. In this series of recoveries, however, there was one “rogue” recovery of 125% which we could not account for. Where serum gave a high blank (absorbance > 0.09) recoveries were low, and these also were omitted from the calculations. Normal range
A small number of technicians and patients with no history or evidence of liver disease were subjected to the conventional BSP clearance test. The dose of 5 mg/kg body weight was given i.v. By weighing the syringe before and after injection the amount given was found to deviate by not more than + 2% from the calculated dose. A check of the urine output of BSP during the test indicated that this was never more than 1.8% of the i.v. dose. BSP was determined in a venous blood sample taken at 45 min after injection and the 95% confidence limits for the normal range (n = 7) found to be from 1.4 to 4.6% retention of dye (taking IO mg/roo ml dye in serum as IOO~/~).The recovery of dye in vitro from a “0 time” sample taken from these subjects had a range of from 98.4 to 100.6~/~with a mean of 99.4% recovery. It would seem therefore that the proposed method gives satisfactory recovery in use and the normal range is close to that suggested by Zieve and HillI’. DISCUSSION The interaction of proteins and dyes is widely known and utilised. For example, Nayyar and Glick12 have described a method in which BSP is utilised in the quantitative determination of protein and there is the well known protein error of indicators utilised in “Albustix”. Thus changes in the absorption spectrum of pure BSP in buffer solution caused by the presence of protein, described by Se&on et al.4 and confirmed by us, were to be expected. The extent of the changes is dependent on the amount of protein present and Clin.
Chim.
Acta,
30
(1970)751-759
758
FLECK,
MORRISON
when the total serum protein concentration changes from 5 g/roe ml through 7 g/Ioo ml to IO g/xoo ml changes in the spectrum of the dye were detectable using the Unicam SP 800. These changes can however be virtually though not completely eliminated by the introduction of sodium fi-toluene sulphonate as suggested by Seligson et aL4 (see Fig. I). It was found to be advantageous to use twice the concentration of pTS advocated by Seligson, although the improvement is only slight. This action of pTS is possibly due to protein denaturation, since 5 M urea gives similar results (Fig. Z) as does heat (e.g. raising the temperature to 60”). This last, however, led to difficulties due to the development of turbidity and was regarded as an unsuitable procedure. It may also be anticipated that the presence of protein would alter the pH dissociation curve of the dye. This does not seem to have been taken into account in the dete~ination of BSP in plasma or serum by others, and clearly (see Fig. 2) influences the results. It is therefore suggested that a final pH of x1.2 is more appropriate for the determination of plasma or serum BSP than is the final pH of 10.5 as employed by Seligson et ah4 and Tovey 3. It was also concluded that because of these effects of protein the standard curve should be determined in the presence of serum or plasma. The proposed measures, however, do not completely overcome the effect of protein as is shown by standard curves prepared using sera containing different amounts of protein (Fig. 5). This would suggest that ideally the patient’s own serum should be used for the preparation of the standard curve, but as this is usually not practicable, the protein content should be estimated and taken into consideration when interpreting the results of the BSP test. The error introduced by generally ignoring this effect is small, particularly for levels close to 5% retention of dye (Pig. 5). Yet another possible difficulty could arise from the presence of conjugates of BSP in plasma. We have been unable to detect these in plasma using thin-layer chromatography even in samples from human subjects considered to have some degree of liver dysfunction due to alcoholism. This is in agreement with the conclusion of Higgins et aZ.l3who found that the greater part of the dye in plasma 40 or 60 min after injection in human subjects was unconjugated BSP. It was initially not anticipated that changes due to temperature and time after mixing reagents would be significant and we have no explanation for the observed effects. These are sufficiently large that it is advisable to mix reagents at room temperature, to avoid extremes of temperature and to read the absorption at a specified time after mixing which should not be less than 5 min, nor greater than 30 min and we suggest between IO and 20 min after mixing as ideal. After detecting the changes in colour intensity of dye-plasma-buffer mixtures with temperature and time, changes on storage were anticipated. Storage of the pure dye either in concentrated form or dilute solution in the refrigerator seems to be satisfactory for up to a month or more. This is not the case when protein is present. In the presence of protein storingin thedeep freeze at --IO’ even for a few days leads to marked changes. It follows that for satisfactory results BSP in plasma or serum should be determined on the same day as the blood sample was withdrawn. When care is taken over these various points it is possible to determine BSP with acceptable accuracy and precision in plasma; that is, to obtain over-all precision Clilz. Chiwz.Acta, 30 (1970) 7.j-759
BROMSULPHTHALEIN
IN SERUM
759
better than 5% and usually not greater than 2%, Claims of precision of better than 1% (ref. g) must be viewed with suspicion in view of our findings. In fact, our present results suggest that unless considerable care is taken the error at the level usually accepted as the upper limit of normal for the 45-min test (i.e. 5% retention) may be much greater than 5% and occasionally of the order of 50%. When employed with suitable care (see ref. 14) both with injection of dye and its determination in the laboratory, the “45-min” BSP test would seem to have a place as a sensitive and simple test of liver function. ACKNOWLEDGEMENTS
We wish to thank Mr. C. Bryce and Mrs. Margaret Lockhart for technical assistance. REFERENCES I S. SHERLOCK, A. G. BEAN, B. H. BILLING AND
J. C. S. PATERSON, J. Lab. Clin. Med., 35 (1950)
923. H. J. D. H. R. H. D. 8 G. g 0. IO A.
VARLEY, Practical Clinical Biochemistry, 4th ed., Heinemann, London, 1967, p. 383. E. TOVEY, C&z. Chim. Acta, 15 (1967) 149. SELIGSON, J. MARION AND E. DODSON, Clin. Chem., 3 (1957) 638. OTT AND D. PIRRWITZ, Clin. Chim. Acta, 22 (1968) 439. J. HENRY, Clinical Chemistry, Harper and Row, New York, 1964, p. 551. H. WILLARD, L. L. MERRITT AND 1. A. DEAN, Instrumental Methods of _ Analysis, _.. 4th ed., Van Nostrand, Princeton, N. J., 1966. F. LOTHIAN, Absorption Spectrophotometry, Adam Hilger Ltd., London, 1969. H. GAEBLER, Am. J. Clin. Pathol., 15 (1945) 452. H. BOWKER AND G. J. LIEBERMAN, Engineering Statistics, Prentice-Hall, Englewood Cliffs,
II 12 13 14
ZIEVE AND E. HILL, Gastroenterology, 28 (1955) 766. N. NAYYAR AND D. GLICK, J. Histochem. Cytochem., 2 (1954) 282. E. HIGGINS, W. T. FOULK AND J. L. BALLMAN. J. Clin. Invest., 38 (1960) 194. W. ASTIN, Bit. Med. J., ii (1965) 408.
2 3 4 5 6 7
N.J., 1959.
L. S. F. T.
Clin. Chim. Acta, 30 (1970) 751-759