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Polarographic determination of total serum protein
J. Electroanal. Chem., 81 (1977) 1 5 1 - - 1 5 9
151
© Elsevier Sequoia S.A., Lausanne -- Printed in The Netherlands
P O L A R O G R A P H I C D E T E R M I N A T I O N OF T O T A L SERUM P R O T E I N
P.W. A L E X A N D E R , R. HOH and L.E. S M Y T H E
Department of Analytical Chemistry, University o f New South Wales, P.O. Box 1, Kensington, N.S. W. 2033 (Australia) (Received 29th J u n e 1976, in revised f o r m 7th S e p t e m b e r 1976)
ABSTRACT The total serum protein is accurately d e t e r m i n e d with high sensitivity by a catalytic polarographic m e t h o d with trans-dichloro-bis(N,N'-dimethylethylenediamine) Rh(III) chloride as reagent. It is s h o w n that microlitre serum samples can be analyzed, giving correlation with the biuret colorimetric t e c h n i q u e which is the r e c o m m e n d e d standard total protein m e t h o d of analysis. Analysis of 17 h u m a n serum samples with varying albumin/globulin ratios gave a correlation coefficient (r) of 0.9910 relative to the biuret procedure.
INTRODUCTION
Total protein content of blood serum is c o m m o n l y determined colorimetrically by the biuret method [1], b u t in addition there are other colorimetric methods available including the Lowry m e t h o d [2] and modifications [3] and biuret modifications [4,5]. Electrochemical methods for total protein are n o t commo.nly used in clinical laboratories, b u t a number of methods are available including the Brdi6ka catalytic method [6] and more recent polarographic [7] and ion selective electrode methods [8,9]. The catalytic activity of a rhodium(III) complex of N,N'-dimethylethylenediamine has been shown [10] to be highly sensitive to serum proteins with response of individual proteins being dependent on buffer pH. In this paper we extend the studies of this effect to the determination of total protein in blood serum. A method is developed giving excellent correlation with the biuret method and highly sensitive determination of total serum protein with micro quantities of serum suitable for pediatric and other clinical studies requiring micro-sampling and high sensitivity. EXPERIMENTAL
Reagents For polarographic analysis, a stock solution (1.0 X 10 - 3 M) of the complex,
trans-dichloro-bis(N,N'-dimethylethylenediamine) Rh(III) chloride, hereafter referred to as Rh-sdmen, was made up in distilled water. All ammonia buffers were prepared with 1.0 M concentration of ammonium chloride with the pH values 8.2, 8.6, 9.2, 9.6 and 10.0 b y varying the concentration of 15 M ammo-
152 nia in the range 0.06 M to 5.62 M in 500 ml. As a surfactant, a Triton X-100 solution (2%) was used and diluted to 80 pg m1-1 just prior to addition to protein solutions. Protein and serum solutions were prepared using human albumin, fraction V, from Nutritional Biochemical Corporation and human serum, Q-Pak Chemistry Control Serum 1, lot No. 0369T001A1 (Hyland Co. Ltd.). A stock solution of albumin (500 pg m1-1) was prepared freshly and the serum control samples were reconstituted just prior to use. A standardized albumin solution (9.9%) was obtained from Miles Laboratories Inc. Code No. 81-017, Lot No. 19. For colorimetric analysis, the biuret reagent was prepared by the m e t h o d described by Wolf et al. [11]. I n s t r u m e n ta tion
A Princeton Applied Research Model 174 polarographic analyzer equipped with a drop-timer Model 174/70 and a Mace Laboratory Recorder FBQ100 was used for polarographic analysis. The control settings for the Model 174 are described in a previous paper [10] for operation in both the D.C. and differential pulse (D.P.P.) modes with controlled drop times of 2 s and 1 s, respectively. Polarographic waves were measured with a three-electrode system consisting of a Pt-metal auxiliary electrode, a saturated calomel reference electrode and a dropping mercury electrode as the indicator electrode with the following characteristics: drop time 2.91 s and flow rate 3.73 mg s-~ at a Hg-column height of 21.0 cm. Unless otherwise stated, all polarographic measurements were made at 20°C in a glass cell with a water jacket for temperature control. Colorimetric determinations were made with an Hitachi Model No. 139 U.V.visible spectrophotometer fitted with 10-mm quartz cuvettes. Procedure
For both polarographic and colorimetric determination of total protein, the stock solution of human albumin (500 pg m1-1) was standardized against the Miles 9.9% standard by the biuret method. For the polarographic calibration, this solution was initially diluted to 50 pg m1-1 in isotonic saline. Aliquots of 0.2, 0.4, 0.6, 0.8 and 1.0 ml were then added to 50 ml volumetric flasks, followed by 0.9 ml of 1.0 × 10 _5 M Rhsdmen polarographic reagent, 5 ml of 1.0 M ammonia buffer at a selected pH and 0.4 ml of Triton X-100 solution (80 gg m1-1 ) and made up to volume with distilled water. For analysis of serum, the samples were first diluted by a factor of 103 by addition of 10 pl to 10 ml isotonic saline. Aliquots of the diluted serum in the range 0.2--1.0 ml were then added to the buffered Rh-sdmen solution as for the albumin standard. A blank solution of the Rh-sdmen reagent was prepared similarly but without the addition of the protein. Polarograms were recorded for each solution after deaeration of a 25 ml aliquot for 10 rain with pure nitrogen gas after pre-saturation of the nitrogen with the appropriate buffer. Catalytic currents for protein solutions were then determined from the equation: ip = i s -- i B
153 where ip is the protein catalytic current, is the total catalytic current, and i B the catalytic current of the blank solution. A similar procedure was followed for the study of pH and temperature effects. For colorimetric determination of total protein of the serum controls and samples, the biuret m e t h o d described by Wolf et al. was followed [11 ]. RESULTS The previous studies [10] on individual protein solutions showed that the polarographic response varied according to the nature of the protein, pH of the buffer and the presence of surfactants such as other proteins or Triton X-100. Hence, in a multi-component solution such as serum, the polarographic response of the total protein c o n t e n t of the serum was completely unpredictable. We therefore investigated the effect of pH on a control serum of k n o w n total protein content, prepared calibrations and compared the serum response to standard albumin response. In addition, studies of temperature dependence have shown that improved sensitivity can be obtained at high solution temperatures.
Effect o f p H Figure 1 compares the polarographic response of human albumin to human control serum as a funtion of ammonia buffer pH, with Rh-sdmen reagent and Triton X-100 concentrations at 1.8 × 10 -7 M and 0.64 pg m1-1, respectively. Polarograms were measured in both the D.C. and D.P.P. operational modes. We found that albumin gave a similar response to the total serum protein in the pH range 8.2--8.6, with a marked decrease in catalytic protein current occurring at higher pH values to almost zero current beyond pH 9.5. The pH curves for albumin and serum, however, began to diverge at pH > 8.6 and the divergence was considerably larger in the D.P.P. mode. Furthermore, in t h e D.P.P. mode, the catalytic current for the total serum protein was lower than in the D.C. mode, a similar result having been obtained for a-globulin at pH 9.2 in the previous study [10]. These pH effects for total serum protein as compared to albumin giving similar response at pH 8.2 were quite different from the results for individual proteins [10] where almost equal response was observed at a much higher pH at 9.5.
Calibration curves To further confirm these pH effects, calibration curves for b o t h standard albumin and total serum protein were constructed at two different:pH values, 8.2 and 9.2, in the D.C. and D.P.P. operational modes. Figures 2 and 3 show the calibrations measured with Rh-sdmen reagent and Triton X-100 concentrations kept constant as for the pH studies. In the D.C. mode, the calibrations in Fig. 2 for albumin and total serum protein were almost identical up to 0.8 pg m1-1 protein concentration at pH 8.2. At pH 9.2, however, the calibrations differed significantlY over the entire concentration range recorded. In addition, as shoWn in Figs. 2 and 3, the calibrations in the D.P.P. mode differed at b o t h pH values, the total serum protein
154 14
12
10-
8
%
4
o
2
I 8"5
8-0
gO
9~
IOD
pH Fig. 1. Effect of pH on the protein catalytic wave. Rh-sdmen, Triton X-100 and protein concentrations were 1.8 × 10 - 7 M, 0.64 pg m1-1 and 0.6 pg m1-1, respectively. A, B, C, D are human albumin (D.P.P. mode), human serum protein (D.C. mode), human albumin (D.C. mode) and human serum protein (D.P.P. mode).
25 a
A
B C
2O
15
10
%
o
o.z
0.4
Gprotein/fig
06
o~
~.o
m/I
Fig. 2. Calibration of human albumin (A, B) and human serum protein (C, D) using D.P.P. (A, D) and D.C. (B, C) modes at pH 8.2. Rh-sdmen and Triton X-100 as in Fig. 1.
155 12
. f k
°
/
8
AB
A ~
I
02
I
0.4
G protein /jug
I
1
06
0-e
I
1.0
m1-1
Fig. 3. Calibration of human albumin (B, C) and human serum protein (A, D) using D.P.P. (B, D) and D.C. (A, C) modes at pH 9.2. Rh-sdmen and Triton X-100 concentrations as in Fig. 1.
calibration being lower while the albumin calibration was higher than obtained with the D.C. mode. These results show that total protein can be determined in serum with albumin as a standard by measurement of the polarographic catalytic Rh(III) waves at pH 8.2 in the D.C. operational mode.
Temperature effects Because of possible effects of temperature variation on the accuracy of polarographic protein analysis, we studied temperature effects on the Rh-sdmen catalytic wave. Figure 4 shows the temperature dependence of total catalytic wave heights on solutions of albumin at 1.0 pg m1-1 and blank Rh-sdmen solutions with the reagent concentrations given previously. Significant changes in wave height were observed in both the D.C. and D.P.P. operational modes. In the D.C. mode, the catalytic current for the blank solution increased only slightly up to 30°C but decreased at higher temperatures. For the protein solutions, there was a marked increase in wave height up to 35°C. In the D.P.P. mode (100 mV pulse amplitude) however, b o t h the blank and protein wave heights increased quite sharply with temperature and remained at constant values above 30°C. It is interesting to note that a split wave was observed in the D.P.P. mode above 30°C with peaks at --1.45 and --1.53 V. The resolution in the D.C. mode is not sufficient for this effect to be recorded, but the effect
156 100 A .
80
60
4O
2O
0
I
10
20
Temperature
30
/°O
4O
Fig. 4. T e m p e r a t u r e d e p e n d e n c e o f the h u m a n a l b u m i n catalytic wave at p H 8.2. A and B are a l b u m i n / R h - s d m e n s o l u t i o n s in the D.P.P. and D.C. m o d e s , and C and D are R h - s d m e n blanks in t h e D.P.P. m o d e and D.C. m o d e . R h - s d m e n , T r i t o n X-100 and p r o t e i n c o n c e n t r a tions as in Fig. 1.
TABLE 1 Total p r o t e i n d e t e r m i n a t i o n s in h u m a n s e r u m samples Sample no.
Biuret m e t h o d /gdl -1
Polarographic m e t h o d a /g dl--1
P.H.H. L a b o r a t o r y results b /g dl--1
03858 03814 03749 03767 03866 03657 03685 25855 25921 25900 25894 25866 25906 25919 25922 75923 75909c
9.41 7.63 7.43 7.55 7.43 6.67 8.10 8.54 10.01 7.55 6.16 6.28 9.25 5.44 8.26 7.76 6,6
9.23 7.68 7.45 7.71 7.16 6.42 8.22 8.53 10.13 7.53 5.95 6.02 9.20 5.64 8.00 7.88 6.5
9.4 7.5 7.6 7.5 7.4 6.7 8.5
± 0.11 ± 0.04 +- 0.05 ± 0.12 ± 0.12 ÷ 0.08 ± 0.04 + 0.26 ± 0.09 ± 0.13 ± 0.06 -+ 0.12 ± 0.28 ± 0.10 ± 0.12 ± 0.26 ± 0.20
(2.1 (2.8 (3.6 (3.4 (3.2 (3.0 (2.8
: : : : : : :
7.3) 4.7) 4.0) 4.1) 4.2) 3.7) 5.7)
a R e s u l t s f r o m triplicate analysis. b ResuRs f r o m Prince H e n r y Hospital, S y d n e y , N.S.W. A l b u m i n t o globulin ratios are s h o w n in brackets. c R e c o n s t i t u t e d C o n t r o l S e r u m L o t No. 0 3 6 9 T 0 0 1 A 1 (Hyland).
157
is similar to temperature dependence observed by Ruttkay-Nedecky and Anderl o v a [ 12] for Brdicka-type protein double waves which show greater voltage separation. The temperature coefficient in the range 15--30°C was approximately 4 pA °C-1, and the results indicate that improved sensitivity can be achieved by measurement at elevated temperatures. However, the temperature coefficient is relatively high and it is therefore necessary to control the temperature during the analysis. We carried out all other measurements in this study at 20.0 +- 0.1°C because of easier control of temperature near room values. Analysis of human serum samples To compare the polarographic and biuret methods, serum samples obtained from Prince Henry Hospital Clinical Laboratory were analyzed for total protein content. Seventeen samples were analyzed, some with normal and other with abnormal albumin : globulin ratios with independent laboratory values available, as shown in Table 1. For polarographic analysis, reagent concentrations were kept constant at the values given for the calibrations with 0.1 M ammonia buffer
12
10
~-
6
3
4
.=_ oJ
2
Y=0"30+
0.97x
r 2= 0.9821 n =17
1 0
2
i 4
t 6
Cprot ein ( polar ographic)/g
i
i
8
10
12
d1-1
Fig. 5. Comparison of patient sample data for polarographic and hiuret method. Data from Table 1.
158
at pH 8.2. Human albumin was used as the standard for preparation of a calibration with the working range 0.1--0.8 pg m1-1. Serum samples were diluted to this working range, added to the Rh-sdmen reagent solution and measured polarographically in the D.C. operational mode at 20°C. For comparison, total protein in each sample was also determined eolorimetrically with the biuret reagent and the results are given in Table 1. The final correlation plot is shown in Fig. 5 and indicates excellent agreement between the polarographic and colorimetric methods with a correlation coefficient of 0.9910. DISCUSSION
The polarographic Rh-sdmen reagent has been shown to be applicable as a highly sensitive reagent for accurate determination of total protein in serum. At present, there are no other methods available that correlate accurately with the biuret m e t h o d and at the same time offer high sensitivity for analysis of micro-serum samples. The biuret method is now recognised [13] as the standard total protein method of analysis, but is insensitive and requires relatively high protein concentrations. The senmtlve colonmetrm and fluorometrm methods available are the Lowry [2] and Udenfriend et al. [14] methods but these do not correlate accurately with the biuret m e t h o d because of differences in molar absorptivities and emission intensities of individual protein molecules. There are however a number of points requiring discussion about the proposed polarographic method. The reasons for the good agreement between albumin and total serum protein in the D.C. mode at pH 8.2 and the divergences in the D.P.P. mode are difficult to interpret. We attribute these differences to the measurement of pulse currents at the end of the life of the Hg-drop. For adsorption processes it has been shown [15] that the instantaneous limiting adsorption current is inversely proportional to t 1/a. As the drop grows, the adsorption current decreases and hence pulse current measurements at the end of the drop life show differences between adsorption currents for albumin and total serum protein. These differences cannot be detected by D.C. polarography which records measurements over the whole drop life. Temperature studies confirmed that the protein adsorption currents were affected in different ways by various operational modes. Here we showed clearly that, although the total catalytic wave height increased with temperature in both operational modes, the value of ip for the protein effect was smaller in the D.P.P. mode than in the D.C. mode because the blank Rh-sdmen wave increased significantly at the higher temperatures. The blank D.C. wave on the other hand decreased at the highest temperature measured. With temperature variation allowing sensitivity to be improved, if necessary, the polarographic determination of total protein with the Rh-sdmen catalytic reagents offers a simple accurate means for analysis of micro-samples of serum. The possibility of automating the procedure is at present being investigated, since it has recently been found [161 that catalytic reactions can be monitored automatically in a continuous-flow polarographie system. .
.
.
.
.
.
.
lIP
ACKNOWLEDGEMENT
We gratefully acknowledge the supply of serum samples with analytical data from the Clinical Laboratories, Prince Henry Hospital, University of N.S.W.
159 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
T.E. W e i c h s e l b a u m , A m e r . J. Clin. P a t h o l . ( T e c h . S u p p l . ) , 16 ( 1 9 4 6 ) 4 0 . O.}-I. Lowry~ N.J. R o s e h r o u g h , A . L . F a r r a n d R. R a n d a l l , J. Biol. Chem., 193 ( 1 9 5 1 ) 2 6 5 . E.F. Hartree, Anal. Biochem., 48 (1972) 422. K. D o e t s c h , Clin. C h e m . , I S ( 1 9 7 2 ) 2 9 6 . K. D o e t s c h a n d R . H . G a d s d e n , Clin. C h e m . , 19 ( 1 9 7 3 ) 1 1 7 0 . R. B r d i c k a , M. B r e z i n a a n d V. K a l o u s , T a l a n t a , 12 ( 1 9 6 5 ) 1 1 4 9 . P.W. A l e x a n d e r , J. E l e c t r o a n a l . C h e m . , 36 ( 1 9 7 2 ) A p p . 21. P.W. A l e x a n d e r a n d G . A . R e c h n i t z , A n a l . C h e m . , 4 6 ( 1 9 7 4 ) 2 5 0 . P.W. A l e x a n d e r a n d G . A . R c c h n i t z , A n a l . C h e m . , 4 6 ( 1 9 7 4 ) 8 6 0 . P.W. A l e x a n d e r , R. H o h a n d L.E. S m y t h e , J. E l e c t r o a n a l . C h e m . , 8 0 ( 1 9 7 7 ) 1 4 3 . P.L. Wolf, D. Williams, T. T s u d a k a a n d L. A c o s t a , M e t h o d s a n d T e c h n i q u e s in Clinical C h e m i s t r y , Wiley-Interscience, New York, 1972, pp. 353--357. G. R u t t k a y - N e d e c k y a n d A. A n d e r l o v a , N a t u r e , 2 1 3 ( 1 9 6 7 ) 5 6 4 . T. Peters, Clin. C h e m . , 1 4 ( 1 9 6 8 ) 1 1 4 7 . S. U d e n f r i e n d , S. S t e i n , P. B S h l e n , W. D a i r m a n , W. L e i m g r u b e r a n d M. Weigele, S c i e n c e , 1 7 8 ( 1 9 7 2 ) 871. S.G. M a i r a n o v s k i i , C a t a l y t i c a n d K i n e t i c Waves in P o l a r o g r a p h y , P l e n u m Press, N e w Y o r k , 1 9 6 8 , p. 8 7 . P.W. A l e x a n d e r a n d S.H. Q u r c s h i , J. E l e c t r o a n a l . C h e m . , 71 ( 1 9 7 6 ) 2 3 5 .
151
© Elsevier Sequoia S.A., Lausanne -- Printed in The Netherlands
P O L A R O G R A P H I C D E T E R M I N A T I O N OF T O T A L SERUM P R O T E I N
P.W. A L E X A N D E R , R. HOH and L.E. S M Y T H E
Department of Analytical Chemistry, University o f New South Wales, P.O. Box 1, Kensington, N.S. W. 2033 (Australia) (Received 29th J u n e 1976, in revised f o r m 7th S e p t e m b e r 1976)
ABSTRACT The total serum protein is accurately d e t e r m i n e d with high sensitivity by a catalytic polarographic m e t h o d with trans-dichloro-bis(N,N'-dimethylethylenediamine) Rh(III) chloride as reagent. It is s h o w n that microlitre serum samples can be analyzed, giving correlation with the biuret colorimetric t e c h n i q u e which is the r e c o m m e n d e d standard total protein m e t h o d of analysis. Analysis of 17 h u m a n serum samples with varying albumin/globulin ratios gave a correlation coefficient (r) of 0.9910 relative to the biuret procedure.
INTRODUCTION
Total protein content of blood serum is c o m m o n l y determined colorimetrically by the biuret method [1], b u t in addition there are other colorimetric methods available including the Lowry m e t h o d [2] and modifications [3] and biuret modifications [4,5]. Electrochemical methods for total protein are n o t commo.nly used in clinical laboratories, b u t a number of methods are available including the Brdi6ka catalytic method [6] and more recent polarographic [7] and ion selective electrode methods [8,9]. The catalytic activity of a rhodium(III) complex of N,N'-dimethylethylenediamine has been shown [10] to be highly sensitive to serum proteins with response of individual proteins being dependent on buffer pH. In this paper we extend the studies of this effect to the determination of total protein in blood serum. A method is developed giving excellent correlation with the biuret method and highly sensitive determination of total serum protein with micro quantities of serum suitable for pediatric and other clinical studies requiring micro-sampling and high sensitivity. EXPERIMENTAL
Reagents For polarographic analysis, a stock solution (1.0 X 10 - 3 M) of the complex,
trans-dichloro-bis(N,N'-dimethylethylenediamine) Rh(III) chloride, hereafter referred to as Rh-sdmen, was made up in distilled water. All ammonia buffers were prepared with 1.0 M concentration of ammonium chloride with the pH values 8.2, 8.6, 9.2, 9.6 and 10.0 b y varying the concentration of 15 M ammo-
152 nia in the range 0.06 M to 5.62 M in 500 ml. As a surfactant, a Triton X-100 solution (2%) was used and diluted to 80 pg m1-1 just prior to addition to protein solutions. Protein and serum solutions were prepared using human albumin, fraction V, from Nutritional Biochemical Corporation and human serum, Q-Pak Chemistry Control Serum 1, lot No. 0369T001A1 (Hyland Co. Ltd.). A stock solution of albumin (500 pg m1-1) was prepared freshly and the serum control samples were reconstituted just prior to use. A standardized albumin solution (9.9%) was obtained from Miles Laboratories Inc. Code No. 81-017, Lot No. 19. For colorimetric analysis, the biuret reagent was prepared by the m e t h o d described by Wolf et al. [11]. I n s t r u m e n ta tion
A Princeton Applied Research Model 174 polarographic analyzer equipped with a drop-timer Model 174/70 and a Mace Laboratory Recorder FBQ100 was used for polarographic analysis. The control settings for the Model 174 are described in a previous paper [10] for operation in both the D.C. and differential pulse (D.P.P.) modes with controlled drop times of 2 s and 1 s, respectively. Polarographic waves were measured with a three-electrode system consisting of a Pt-metal auxiliary electrode, a saturated calomel reference electrode and a dropping mercury electrode as the indicator electrode with the following characteristics: drop time 2.91 s and flow rate 3.73 mg s-~ at a Hg-column height of 21.0 cm. Unless otherwise stated, all polarographic measurements were made at 20°C in a glass cell with a water jacket for temperature control. Colorimetric determinations were made with an Hitachi Model No. 139 U.V.visible spectrophotometer fitted with 10-mm quartz cuvettes. Procedure
For both polarographic and colorimetric determination of total protein, the stock solution of human albumin (500 pg m1-1) was standardized against the Miles 9.9% standard by the biuret method. For the polarographic calibration, this solution was initially diluted to 50 pg m1-1 in isotonic saline. Aliquots of 0.2, 0.4, 0.6, 0.8 and 1.0 ml were then added to 50 ml volumetric flasks, followed by 0.9 ml of 1.0 × 10 _5 M Rhsdmen polarographic reagent, 5 ml of 1.0 M ammonia buffer at a selected pH and 0.4 ml of Triton X-100 solution (80 gg m1-1 ) and made up to volume with distilled water. For analysis of serum, the samples were first diluted by a factor of 103 by addition of 10 pl to 10 ml isotonic saline. Aliquots of the diluted serum in the range 0.2--1.0 ml were then added to the buffered Rh-sdmen solution as for the albumin standard. A blank solution of the Rh-sdmen reagent was prepared similarly but without the addition of the protein. Polarograms were recorded for each solution after deaeration of a 25 ml aliquot for 10 rain with pure nitrogen gas after pre-saturation of the nitrogen with the appropriate buffer. Catalytic currents for protein solutions were then determined from the equation: ip = i s -- i B
153 where ip is the protein catalytic current, is the total catalytic current, and i B the catalytic current of the blank solution. A similar procedure was followed for the study of pH and temperature effects. For colorimetric determination of total protein of the serum controls and samples, the biuret m e t h o d described by Wolf et al. was followed [11 ]. RESULTS The previous studies [10] on individual protein solutions showed that the polarographic response varied according to the nature of the protein, pH of the buffer and the presence of surfactants such as other proteins or Triton X-100. Hence, in a multi-component solution such as serum, the polarographic response of the total protein c o n t e n t of the serum was completely unpredictable. We therefore investigated the effect of pH on a control serum of k n o w n total protein content, prepared calibrations and compared the serum response to standard albumin response. In addition, studies of temperature dependence have shown that improved sensitivity can be obtained at high solution temperatures.
Effect o f p H Figure 1 compares the polarographic response of human albumin to human control serum as a funtion of ammonia buffer pH, with Rh-sdmen reagent and Triton X-100 concentrations at 1.8 × 10 -7 M and 0.64 pg m1-1, respectively. Polarograms were measured in both the D.C. and D.P.P. operational modes. We found that albumin gave a similar response to the total serum protein in the pH range 8.2--8.6, with a marked decrease in catalytic protein current occurring at higher pH values to almost zero current beyond pH 9.5. The pH curves for albumin and serum, however, began to diverge at pH > 8.6 and the divergence was considerably larger in the D.P.P. mode. Furthermore, in t h e D.P.P. mode, the catalytic current for the total serum protein was lower than in the D.C. mode, a similar result having been obtained for a-globulin at pH 9.2 in the previous study [10]. These pH effects for total serum protein as compared to albumin giving similar response at pH 8.2 were quite different from the results for individual proteins [10] where almost equal response was observed at a much higher pH at 9.5.
Calibration curves To further confirm these pH effects, calibration curves for b o t h standard albumin and total serum protein were constructed at two different:pH values, 8.2 and 9.2, in the D.C. and D.P.P. operational modes. Figures 2 and 3 show the calibrations measured with Rh-sdmen reagent and Triton X-100 concentrations kept constant as for the pH studies. In the D.C. mode, the calibrations in Fig. 2 for albumin and total serum protein were almost identical up to 0.8 pg m1-1 protein concentration at pH 8.2. At pH 9.2, however, the calibrations differed significantlY over the entire concentration range recorded. In addition, as shoWn in Figs. 2 and 3, the calibrations in the D.P.P. mode differed at b o t h pH values, the total serum protein
154 14
12
10-
8
%
4
o
2
I 8"5
8-0
gO
9~
IOD
pH Fig. 1. Effect of pH on the protein catalytic wave. Rh-sdmen, Triton X-100 and protein concentrations were 1.8 × 10 - 7 M, 0.64 pg m1-1 and 0.6 pg m1-1, respectively. A, B, C, D are human albumin (D.P.P. mode), human serum protein (D.C. mode), human albumin (D.C. mode) and human serum protein (D.P.P. mode).
25 a
A
B C
2O
15
10
%
o
o.z
0.4
Gprotein/fig
06
o~
~.o
m/I
Fig. 2. Calibration of human albumin (A, B) and human serum protein (C, D) using D.P.P. (A, D) and D.C. (B, C) modes at pH 8.2. Rh-sdmen and Triton X-100 as in Fig. 1.
155 12
. f k
°
/
8
AB
A ~
I
02
I
0.4
G protein /jug
I
1
06
0-e
I
1.0
m1-1
Fig. 3. Calibration of human albumin (B, C) and human serum protein (A, D) using D.P.P. (B, D) and D.C. (A, C) modes at pH 9.2. Rh-sdmen and Triton X-100 concentrations as in Fig. 1.
calibration being lower while the albumin calibration was higher than obtained with the D.C. mode. These results show that total protein can be determined in serum with albumin as a standard by measurement of the polarographic catalytic Rh(III) waves at pH 8.2 in the D.C. operational mode.
Temperature effects Because of possible effects of temperature variation on the accuracy of polarographic protein analysis, we studied temperature effects on the Rh-sdmen catalytic wave. Figure 4 shows the temperature dependence of total catalytic wave heights on solutions of albumin at 1.0 pg m1-1 and blank Rh-sdmen solutions with the reagent concentrations given previously. Significant changes in wave height were observed in both the D.C. and D.P.P. operational modes. In the D.C. mode, the catalytic current for the blank solution increased only slightly up to 30°C but decreased at higher temperatures. For the protein solutions, there was a marked increase in wave height up to 35°C. In the D.P.P. mode (100 mV pulse amplitude) however, b o t h the blank and protein wave heights increased quite sharply with temperature and remained at constant values above 30°C. It is interesting to note that a split wave was observed in the D.P.P. mode above 30°C with peaks at --1.45 and --1.53 V. The resolution in the D.C. mode is not sufficient for this effect to be recorded, but the effect
156 100 A .
80
60
4O
2O
0
I
10
20
Temperature
30
/°O
4O
Fig. 4. T e m p e r a t u r e d e p e n d e n c e o f the h u m a n a l b u m i n catalytic wave at p H 8.2. A and B are a l b u m i n / R h - s d m e n s o l u t i o n s in the D.P.P. and D.C. m o d e s , and C and D are R h - s d m e n blanks in t h e D.P.P. m o d e and D.C. m o d e . R h - s d m e n , T r i t o n X-100 and p r o t e i n c o n c e n t r a tions as in Fig. 1.
TABLE 1 Total p r o t e i n d e t e r m i n a t i o n s in h u m a n s e r u m samples Sample no.
Biuret m e t h o d /gdl -1
Polarographic m e t h o d a /g dl--1
P.H.H. L a b o r a t o r y results b /g dl--1
03858 03814 03749 03767 03866 03657 03685 25855 25921 25900 25894 25866 25906 25919 25922 75923 75909c
9.41 7.63 7.43 7.55 7.43 6.67 8.10 8.54 10.01 7.55 6.16 6.28 9.25 5.44 8.26 7.76 6,6
9.23 7.68 7.45 7.71 7.16 6.42 8.22 8.53 10.13 7.53 5.95 6.02 9.20 5.64 8.00 7.88 6.5
9.4 7.5 7.6 7.5 7.4 6.7 8.5
± 0.11 ± 0.04 +- 0.05 ± 0.12 ± 0.12 ÷ 0.08 ± 0.04 + 0.26 ± 0.09 ± 0.13 ± 0.06 -+ 0.12 ± 0.28 ± 0.10 ± 0.12 ± 0.26 ± 0.20
(2.1 (2.8 (3.6 (3.4 (3.2 (3.0 (2.8
: : : : : : :
7.3) 4.7) 4.0) 4.1) 4.2) 3.7) 5.7)
a R e s u l t s f r o m triplicate analysis. b ResuRs f r o m Prince H e n r y Hospital, S y d n e y , N.S.W. A l b u m i n t o globulin ratios are s h o w n in brackets. c R e c o n s t i t u t e d C o n t r o l S e r u m L o t No. 0 3 6 9 T 0 0 1 A 1 (Hyland).
157
is similar to temperature dependence observed by Ruttkay-Nedecky and Anderl o v a [ 12] for Brdicka-type protein double waves which show greater voltage separation. The temperature coefficient in the range 15--30°C was approximately 4 pA °C-1, and the results indicate that improved sensitivity can be achieved by measurement at elevated temperatures. However, the temperature coefficient is relatively high and it is therefore necessary to control the temperature during the analysis. We carried out all other measurements in this study at 20.0 +- 0.1°C because of easier control of temperature near room values. Analysis of human serum samples To compare the polarographic and biuret methods, serum samples obtained from Prince Henry Hospital Clinical Laboratory were analyzed for total protein content. Seventeen samples were analyzed, some with normal and other with abnormal albumin : globulin ratios with independent laboratory values available, as shown in Table 1. For polarographic analysis, reagent concentrations were kept constant at the values given for the calibrations with 0.1 M ammonia buffer
12
10
~-
6
3
4
.=_ oJ
2
Y=0"30+
0.97x
r 2= 0.9821 n =17
1 0
2
i 4
t 6
Cprot ein ( polar ographic)/g
i
i
8
10
12
d1-1
Fig. 5. Comparison of patient sample data for polarographic and hiuret method. Data from Table 1.
158
at pH 8.2. Human albumin was used as the standard for preparation of a calibration with the working range 0.1--0.8 pg m1-1. Serum samples were diluted to this working range, added to the Rh-sdmen reagent solution and measured polarographically in the D.C. operational mode at 20°C. For comparison, total protein in each sample was also determined eolorimetrically with the biuret reagent and the results are given in Table 1. The final correlation plot is shown in Fig. 5 and indicates excellent agreement between the polarographic and colorimetric methods with a correlation coefficient of 0.9910. DISCUSSION
The polarographic Rh-sdmen reagent has been shown to be applicable as a highly sensitive reagent for accurate determination of total protein in serum. At present, there are no other methods available that correlate accurately with the biuret m e t h o d and at the same time offer high sensitivity for analysis of micro-serum samples. The biuret method is now recognised [13] as the standard total protein method of analysis, but is insensitive and requires relatively high protein concentrations. The senmtlve colonmetrm and fluorometrm methods available are the Lowry [2] and Udenfriend et al. [14] methods but these do not correlate accurately with the biuret m e t h o d because of differences in molar absorptivities and emission intensities of individual protein molecules. There are however a number of points requiring discussion about the proposed polarographic method. The reasons for the good agreement between albumin and total serum protein in the D.C. mode at pH 8.2 and the divergences in the D.P.P. mode are difficult to interpret. We attribute these differences to the measurement of pulse currents at the end of the life of the Hg-drop. For adsorption processes it has been shown [15] that the instantaneous limiting adsorption current is inversely proportional to t 1/a. As the drop grows, the adsorption current decreases and hence pulse current measurements at the end of the drop life show differences between adsorption currents for albumin and total serum protein. These differences cannot be detected by D.C. polarography which records measurements over the whole drop life. Temperature studies confirmed that the protein adsorption currents were affected in different ways by various operational modes. Here we showed clearly that, although the total catalytic wave height increased with temperature in both operational modes, the value of ip for the protein effect was smaller in the D.P.P. mode than in the D.C. mode because the blank Rh-sdmen wave increased significantly at the higher temperatures. The blank D.C. wave on the other hand decreased at the highest temperature measured. With temperature variation allowing sensitivity to be improved, if necessary, the polarographic determination of total protein with the Rh-sdmen catalytic reagents offers a simple accurate means for analysis of micro-samples of serum. The possibility of automating the procedure is at present being investigated, since it has recently been found [161 that catalytic reactions can be monitored automatically in a continuous-flow polarographie system. .
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lIP
ACKNOWLEDGEMENT
We gratefully acknowledge the supply of serum samples with analytical data from the Clinical Laboratories, Prince Henry Hospital, University of N.S.W.
159 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
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