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(9) Tsunoda, Y.; Seko, M.; Watanabe, M.; Ehara, R.; Misumi, T. Japanese Tokkyo Koho 4143, 1957. (IO) Pepper, K. W.; Paisley, H. M.; Young, M. A. J . Chem. Soc. 1953, 4097. (11) Helfferich, F. "Ion Exchange"; McGraw-Hill: New York, 1962; p 512. (12) Teorell, T. Prog. Biophys. Biophys. Chem. 1953, 3 ,305. (13) Diamond, R. M. J . fhys. Chem. 1963, 6 7 , 2513. (14) Lindenbaum, S.;Boyd, G. E. J . fhys. Chem. 1964, 68,911. (15) Boyd, G. E.; Schwarz, A,; Lindenbaum, S. J . fhys. Chem. 1966, 7 0 , 821. (16) Gregor, H. P.; Belle, J.; Marcus, R. A. J . Am. Chem. Soc. 1955, 7 7 , 27 13. (17) Ishibashi, N.; Kohara, H.; Uemura, N. BunsekiKagaku 1972, 27, 1072. (18) Kamo, N.; Hazemoto, N.; Kobatake, Y. Taianta 1977, 2 4 , 111. (19) Camman, K. "Working with Ion-Selective Electrodes"; Springer-Verlag: Berlin, Heidelberg, New York, 1979; Chapter 3. (20) Buck, R. P. The University of North Carolina, Chapel Hill, NC, private communication. (21) Srinivasan, K.; Rechnitz, G. A . Anal. Chem. 1968, 4 0 , 509. (22) Moody, G. J.; Thomas, J. D. R. "Selective Ion Sensitive Electrodes"; Merrow Publishing Co.: Watford, England, 1971. (23) Baum, G. J . Phys. Chem. 1972, 76, 1872.
(24) Blck. S.;Sandblom, J. Anal. Chem. 1973, 4 5 , 1680. (25) Morf, W. E.; Amman, D.; Pretsh, E.: Simon, W. Pure Appl. Chem. 1973. 36,421. (26) Reinsfelder, R. E.; Schultz, F. A. Anal. Chim. Acta 1973, 6 5 . 425. (27) Yoshida, N.; Ishibashi. N. Chem. Lett. 1974, 493. (28) Shean, G.; Sollner, K. J . Membr. Bioi. 1972, 9 , 297. (29) Jyo. A.; Torikai, M.; Ishibashi, N. Bull. Chem. Soc. Jpn. 1974, 47, 2862. (30) Jyo, A.; Mihara, H.; Ishibashi, N. Denki Kagaku 1976, 4 4 , 268. (31) Yamauch, A.; Minematsu, T.; Kimizuka, H. Maku 1977, 2, 69. (32) Yoshida, N.: Ishibashi, N. Bull. Chem. SOC. Jpn. 1977, 50, 3189. (33) Shean, G. M. J . Membr. Sci. 1977. 2 , 133.
RECEn'ED for review January 7,1980. Accepted July 17,1980. This work was presented in part at the 26th International Congress of Pure and Applied Chemistry, Tokyo, Sept 1977 (Paper No. 8A610). This work was partially supported by Grants-in-Aid for Special Project Research (Grant No. 011911, No. 111211, No. 210410) from the Ministry of Education.
Automated Continuous-Flow Determination of Serum Albumin by Differential Pulse Polarography P. W. Alexander* and M. H. Shah Department of Analytical Chemistry, University of New South Wales, P.O. Box 1, Kensington, 2033 New South Wales, Australia
A polarographic study of the interaction of serum albumin with potassium titanium(1V) oxalate is reported over a buffer range of pH 4-5. The shift in the half-wave potential of the titanium reagent after reaction with albumin is used to develop a novel automated method for determination of albumin in blood serum. Polarographic analysis in a continuous-flow system is operated in the differential pulse mode at a fixed potential of -0.68 V. Under controlled solution conditions at pH 4.89, albumin is shown to selectively react with the Ti(1V) reagent without serious interference from other serum components including the major globulin and glycoprotein fractions unless they are present in excess of the albumin concentration. The polarographic method for analysis of 15 human serum samples is compared to the bromocresol green spectrophotometric dye-binding method for albumin determination, giving a correlation coefficient of 0.9934.
Although determination of albumin is one of the most common of analytical requirements in clinical laboratories, there are at present no procedures reported for its polarographic determination in blood serum. The polarographic reagents available for determination of serum proteins are Brdicka's hexaaminecobalt(II1) chloride ( I ) and a rhodium(111)-substituted ethylenediamine complex ( 2 , 3). Both of these, however, react with a wide range of serum proteins and cannot be used for specific determination of albumin without a prior separation step. We report here a method using potassium titanium(1V) oxalate as a reagent for polarographic determination of serum albumin in an automated continuous-flow system with differential pulse (dp) operation. T h e most commonly used methods for determination of serum albumin are spectrophotometric dye binding techniques for which bromocresol green, BCG ( 4 ) , HABA (2-(4'hydroxyazobenzene)benzoic acid) ( 5 ) ,or methyl orange (6) 0003-2700/80/0352-1896$01.OO/O
are selective reagents. In this paper, the Ti(1V) reagent is used for d p polarographic determination of albumin in a series of serum samples, and the results are compared with t h e BCG method. Good agreement between t h e results for the two methods is obtained, and the polarographic method is shown to be of comparable sensitivity to the BCG method. We recently reported (7) the use of the same Ti(1V) reagent for polarographic determination of serum glycoproteins in a continuous-flow system in an acidic solution at p H 1.6. Under these conditions, albumin and globulins in serum precipitated out and did not interfere with the glycoprotein determination. In this paper, by further study of p H effects on the response of various proteins with Ti(IV), we show that, a t higher p H values in a phosphatecitrate buffer, the reverse determination is possible. Albumin reacts with the reagent but glycoproteins and globulins have little effect. Study of a range of interferences shows that albumin can be determined selectively a t pH 4.89, and the analysis has been automated in a continuous-flow system.
EXPERIMENTAL SECTION Reagents and Stock Solutions. All reagents used were A.R. Grade, and solutions were prepared in distilled water throughout. A stock 0.1 M solution of the reagent, potassium titanium(1V) oxalate, was prepared after dissolving the salt in warm water. Working reagent solutions were then prepared by appropriate dilution of the stock solution to give Ti02+concentrations in the range (1.0-8.0) X M in a disodium hydrogen phosphate/citric acid buffer. To study the effect of buffer pH in the range pH 4-5, we varied the concentration of disodium hydrogen phosphate between 4.0 X M while the citric acid concentration was and 3.0 X kept constant at 4.0 X M. Values of pH >5.0 were not studied because of precipitation of the reagent. Bovine albumin (fraction V), bovine glycoprotein (Cohn fraction VI), bovine 0-globulin (fraction IV), bovine P-globulin (fraction 111), and human y-globulin (fraction 11),all from Miles Laboratories, Inc., were used in this work. Stock solutions of each (lo00 mg L-I) were prepared in distilled water, and appropriate dilutions S 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
were made fresh just prior to use. Other organic compounds used were vitamin B, and lysine (ICN Pharmaceuticals) and bilirubin (Calbiochem). Interference effects were studied by using 1 mg mL-' stock solutions of the organic compounds. Aliquots of 0.5, 1.0, and 1.5 mL of these stock solutions were added to 50-mL volumetric flasks containing 5 mL (1mg mL-') of bovine albumin solution. The volume was then made up to 50 mL by addition of distilled water. Apparatus. Polarographic data were recorded with a Princeton Applied Research Model 174 polarographic analyzer equipped with a drop timer (Model 174/70) and a Mace Type FBQ-100 chart recorder. The polarographic cell was fitted with a threeelectrode system consisting of a platinum auxiliary electrode, a saturated calomel electrode (SCE), and a dropping mercury electrode (DME) as the indicator electrode. Electrode characteristics of the DME glass capillary were m = 1.2 mg SKI and t = 5.5 s a t a mercury column height of 56.8 cm measured in potassium titanium(1V) oxalate (5.0 X lo-' M) at -0.68 V at room temperature. For polarographic analysis, the Model 174 instrument controls were set as follows: current range 100 PA, modulation amplitude 100 mV, droptime 1 s, fixed potential 4 . 6 8 V, potential scan rate 5 mV s-l (when required), and chart speed 500 m h-'. The differential pulse operational mode was used throughout this work unless otherwise specified. The flow system used for continuous-flow analysis has been previously described (7, 8). All features were kept identical for this study including the pump speed, pump tubing sizes, and flow cell, except that the reagent solution conditions were altered. Procedure. The reagent solution consisting of potassium titanium(1V) oxalate at a concentration of 6.0 X lo-* M in 1.2 X lo-' M disodium hydrogen phosphate and 4.0 X lo4 M citric acid was initially pumped through the flow system and mixed with the wash solution (distilled water). Nitrogen gas was used for bubble segmentation and also for continuous purging of the reagent and wash solutions. The solution was pumped through the polarographic flow cell after debubbling a t a constant flow rate of 9.2 mL min-', and a voltage scan was recorded in the differential pulse operational mode from +0.2 to -1.2 V. The effect of individual proteins on the reagent was then monitored by aspirating a protein sample solution instead of the wash solution into the flow system for 3-5 min and rescanning the voltage range from +0.2 to -1.2 V. For continuous-flow analysis at a fixed potential, an appropriate potential value ( 4 . 6 8 V) was chosen after inspection of the above polarograms. A steady base line was then established by continuous pumping of the reagent and wash solution through the flow cell at a flow rate of 9.2 mL mi&. Cui-rent response to sample solutions of standard bovine albumin in the concentration range 50-500 mg L-' was then monitored by using manual sampling with a 30-s sampling time and a 30-s wash time between each sample, equivalent to 60 samples per hour with a 1:l sample to wash ratio. Calibration curves were then constructed by plotting the peak height for each albumin solution as a function of albumin concentration where the peak height represents the difference between the peak current and the base line current. For polarographic determination of albumin in human blood serum, 10-pL aliquots of each sample were diluted in 2 mL of distilled water in a 3-mL centrifuge tube. After being thoroughly mixed by swirling in a centrifuge, the diluted samples were aspirated directly into the flow system for 30 s, again followed by a 30-s wash of distilled water, and the differential pulse current response was monitored at -0.68 V. For comparison, the same serum samples were also analyzed by spectrophotometry using the BCG method of Doumas and Biggs ( 4 ) with standard bovine serum albumin solution prepared in aqueous sodium azide solution (500 mg L-I) in the concentration range 20-60 g L-'. An aliquot (10 pL) of an albumin standard or serum sample was added t o the working dye solution (0.06 mg of BCG/mL). After the sample was mixed and allowed to stand for 10 min, the absorbance of each solution was measured at 628 nm with an Hitachi Perkin-Elmer UV-Vis spectrophotometer.
RESULTS T h e differential pulse (dp) polarogram of the reagent, potassium titanium(1V) oxalate, in oxalate buffer a t p H 1.6 has
1897
E
'02
0
-02
-04
-06
-08
-10
E ( v o l t s ) vs S C E
Figure 1. Continuous-flow differential pulsis polarographic scans of the
Ti02+ reagent (6.0 X
M) buffered a1 pH 4.89 with the following albumin concentrations (mg L-'): (A) 0, (BI 20, (C)50, (D) 100, (E) 200
previously been reported ( 7 ) . Two peaks were observed a t -0.10 and -0.51 V. In this work, the effect of changing the buffer was studied over a wider p H range than previously reported (7). Figure 1 shows the d p polarographic scan for the reagent solution (curve A) in phosphate-citrate buffer a t p H 4.89 pumped continuously through the flow cell. The peak potentials were little changed from the values a t the lower pH, but there was a distinct change in peak heights, the peak at -0.51 V being decreased in height a t p H 4.89. T h e effect of albumin and other proteins on the Ti(1V) reagent at p H 4.89 was then studied by continuously aspirating protein sample solutions into the flow system. T h e d p polarographic scans for albumin a t various concentrations, as shown in Figure 1, indicate the appearance of a new peak at -0.78 V and shift of the second Ti(IV) peak to more negative potentials. This effect differed from the effect of glycoprotein on the Ti(IV) reagent a t low p H where the protein shifted the second Ti(1V) peak b u t there was rio separate peak for the protein. Effect of Other Proteins. T h e reaction of the Ti(IV) reagent was studied with a series of other proteins including bovine glycoprotein (Cohn fraction VI) and human y-globulins. As shown previously ( 7 ) , addition of glycoprotein to the reagent in unbuffered solution caused a shift in peak potential to -0.81 V, but the effect was sharply dependent on p H . At p H >4, the sensitivity of the Ti(IV) reagent t o glycoprotein was markedly reduced. In a phosphate-citrate buffer a t p H 4.89, we found t h a t the glycoprotein a t the relatively high concentration of 200 mg L-' caused a shift in the Ti(1V) reagent peak, giving a double peal; a t -0.60 and -0.73 V, similar to the albumin peaks. T h e sensitivity of the glycoprotein, however, was significantly less than the albumin effect on the reagent. y-Globulin, in contrast, when added t o the reagent stream in phosphate-citrate buffer was found t o precipitate to give a turbid solution At concentrations less than 1000 mg L-', y-globulin had no effect on the Ti(1V) reagent peak a t -0.51 V. Choice of Conditions for Fixed-Potential Continuous-Flow Operation. From the voltage scans shown in Figure 1, it is clear that the appearance of the new peak a t 4 . 7 8 V is near the minimum in the polarogram for the reagent alone. In fixed potential operation for continuous-flow analysis, however, the increase in Icurrent with increasing albumin concentration was found to he most sensitive a t 4 . 6 8
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980 lOOr
500
1
Albumin Conc., mg C'
Figure 2. Albumin calibration curves determined from continuous-flow measurements with a fixed Ti0" reagent concentration (6.0 X lo-' M) at buffer pH 4.89 and at the following fixed potentials (V): (A) -0.75, (B) -0.55, (C) -0.60, (D) -0.70, (E) -0.68
Flgure 4. Continuous readout of albumin sample peaks at a sampling rate of 60 per hour recorded at a fixed potential of -0.68 V with a reagent concentration of 6.0 X lo-' M in buffer at pH 4.89: (A) calibration for the range 50-500 mg L-'; (8) carry-over between sequential samples of 50, 400, and 50 mg L-'; (C) replicates for 400 mg L'' samples; (D) steady-state reading at 400 mg L-'
501 A
LO.
50
-
LO
-
30.
4
B
-
6
20.
i, 3 0 -
I -@SO
-0.60
-070
-080
Etvolts) vs SC.E
Figure 3. Dependence of peak height of an albumin sample solution (300 mg L-') on the fixed monitoring potential measured with a reagent concentration of 6.0 X lo-' M at pH 4.89 Albumin C o n c .
V rather than a t the peak voltage of -0.78 V. Albumin calibrations were plotted a t several fixed voltage values, as shown in Figure 2. T h e slope of the calibration was found to markedly increase for operation a t voltages from -0.51 t o -0.68 V. In another approach, as shown in Figure 3, the albumin sample concentration was kept constant, and the sample peak height (Ai)was measured a t various fixed voltage values. Ai represents the difference in current between the blank reagent and the reaction product and clearly reaches a maximum a t -0.68 V. T h e continuous peak readout a t 4 . 6 8 V is shown in Figure 4 a t a sampling rate of 60 per hour. By use of a flow rate through the polarographic cell of 9.2 mL min-', the sample peaks reached 92% of the steady-state reading. The resulting precision for replicate determination of albumin sample solutions was 1.2% RSD with a carry-over of 2, and hence no interference was observed from this protein. Some interference was observed from lysine, vitamin B,, and bilirubin, as shown in Table I. However, the interferences occurred a t ratios well in excess of the weightto-weight ratios found in normal serum, given in Table I as approximate interferent-to-albumin ratios ( 9 , l O ) . y-Globulin was studied in more detail later, as shown in Tables I1 and
111. In addition to these possible interferences, the other proteins present as a significant percentage of the total protein content of serum are the a-, /3-, and y-globulins. The effect of these proteins on the reagent response to albumin is shown in Tables I1 and 111. Table 11 indicates little interference to the albumin peak height when albumin is in excess of the total globulin concentration, as occurs in normal serum. In synthetic mixtures of proteins where the globulin concentrations approach and exceed the albumin, significant interference to the albumin determination is found, as shown in Table 11. However, we found that the individual globulins had different effects on the albumin response. As shown in Table 111, a- and (3-globulins both increased the albumin peak height while y-globulin decreased the peak. These differences can be attributed to the purity and nature of globulin fractions, as discussed further below.
DISCUSSION From the results of the correlation study with the BCG method, we have shown that the polarographic method described here offers a n accurate method for determination of serum albumin in human serum. The method is sensitive, requiring only 10 pL of serum sample, and overcomes some deficiencies of colorimetric methods; viz., lipemic and turbid samples do not cause interference problems, bilirubin, haemoglobin, amino acids, vitamins, and glycoproteins do not interfere a t the levels normally present in serum, and the method can be automated giving rapid analysis in a simpler flow-manifold than the automated BCG method ( 4 ) . In
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
EiVOltS)
vs S C E
Figure 8. Continuous-flow dc polarographic scans of the Ti02+ reagent (6.0 X lo-' M) in buffer at pH 4.89 with the following albumin concentrations (mg L-'): (A) 0, (B) 25, (C) 100
routine use of the method, however, there is the need for accurate p H control since, as shown in Figure 6, a small change in p H of as little as 0.09 p H units can alter the response to a fixed concentration of albumin by as much as 50%. T h e pH of the buffered reagent solution therefore must be strictly controlled, and separate calibration is required for each batch of reagent solution used. This requirement restricts the routine use of the method as a general substitute for the bromocresol green method. We attribute the interaction between albumin and the Ti(IV) reagent to be formation of a metal complex which shifts the polarographic Ti(1V) wave to more negative potentials. Figure 8 shows the dc polarograms recorded for the reagent before and after reaction with albumin. The very low dc wave a t -0.56 V shifted t o -0.78 V and increased in height as the albumin concentration was increased. T h e wave finally observed at 4 . 7 8 V (Figure 8C) is of low slope with a pronounced maximum, indicating an irreversible adsorption mechanism a t the electrode surface.
However, the polarographic method was not completely specific for albumin, and interferences from other serum proteins, particularly from y-globulin, were found. y-Globulin a t or above the concentration level of 100 mg L-' reduced the albumin peak height, indicating some interaction between albumin and y-globulin a t the dropping mercury electrode, while in contrast, the individual effects of a- and (3-globulins were found to increase the albumin response (Table 111). We attribute this increase partly to the impure fractions of a- and @-globulinsused in this study. The commercially available fractions (fractions IV and 111, Miles Laboratories) were known to be contaminated with albumin. The preparation of aglobulin may be made up of as much as 50% albumin ( I I ) , whereas the y-globulin was certified 98% pure by electrophoresis ( 1 1 ) . Doumas and Biggs ( 4 ) also reported problems with the impurity of commercially available a- and 0-globulin fractions when attempting to check the specificity of the BCG method for albumin determination. T h e results obtained here in Tables I1 and I11 are therefore of only limited value in assessing the specificity of the polarographic method. The specificity, however, can also be established by comparing results with established methods for analysis of real samples. For serum analysis we have obtained polarographic results in agreement with the BCG method for albumin values in the range 32.0-54.0 g L-l.
ACKNOWLEDGMENT We gratefully acknowledge the supply of serum samples from K. Kenrick, N.S.W. State Blood Bank. LITERATURE CITED (1) Brdicka, R.; Brezina. M.; Kalous, V. Talanta 1965, 12, 1149. (2) Alexander, P. W.; Hoh, R.; Srnythe, L. E. J . Electroanal. Chem. 1977, 80, 143. (3) Alexander, P. W.; Hoh, R.; Smythe, L. E. J . Electroanal. Chem. 1977, 87,152. (4) Doumas, B. T.; Biggs, H. G. Stand. Methods Clin. Chem. 1972, 7 , 175. (5) Rutstein, D. D.; Ingenito, E. F.; Reynolds, W. E. J . Clin. Invest. 1954, 33, 211. (6) Wren, H. T.; Fetchmeyer, T. V. Am. J . Clin. Pathol. 1958, 26, 960. (7) Alexander, P. W.; Shah, M. H. Anal. Chem. 1979, 57, 2139. (8) Alexander, P. W.; Shah. M. ti. Talanta 1979, 26, 97. (9) Tietz, N. W., Ed. "Fundamentals of Clinical Chemistry"; W. B. Saunders Co.: Philadelphia, 1970. (10) Searcy, R. L. "Diagnostic Biochemistry"; McGraw-Hili: New York, 1969. (1 1) Miles Biochemicals Catalogue, Research Products Division: Ekhart, IN, 1977; p 92.
RECEIVED for review April 17, 1980. Accepted June 25, 1980. The authors thank the Australian Government for assistance to M.H.S. from a Colombo Plan Fellowship.
