IIrlrr Ilr
A new electrochemical reference cell shows promisefor sta nda rdized measurements of ionized calcium in plasma
Masao Umemoto Wataru Tani Chemicals Inspection and Testing lnstitute
Katsuhiko Kuwa University of Tsukuba
Yusuke Ujihira The University of Tokyo 352 A
C
alcium exists in serum in three forms: free, unbound ions; protein-bound; and complex-bound. It is generally recognized that the fundamental role of calcium in many physiological processes is exerted mainly by the free, unbound calcium ions, the so-called ionized calcium (iCa"). Before the development of calcium ion-selective electrodes (ISEs), no practical method for measuring iCa2+in plasma existed; various methods for measuring the concentration of diffusible calcium (1)in plasma require equilibrium conditions and are time-consuming (2).The development of calcium ISEs (3) has made it possible to assay serum iCa2+ rapidly and directly (4). Calcium ISEs have become widely used and, as reported by Bowers, Brassard, and Sena (3,iCa2+constituted 88% of all clinical calcium measurement requests compared with 11%for total calcium and l%for urine calcium at Hartford Hospital ((3').However, because there is no worldwide reference method established for accurately determining iCa2+in plasma, large variations remain in iCa2+ values measured with various commercial analyzers. To establish consistency in measured iCa2+,several groups, including the European Working Group on ISEs, the American Association for Clinical Chemistry, and the Japan Society of Clinical Chemistry, formed the International Working Group on Ion-Selective Electrodes to act as an advisorv bodv to the International Federation of Clinical Chemistry (IFCC) Scientific Committee Expert Panel on pH/ Blood Gases and Electrolytes. A reference method for the determination of iCa2+in serum, plasma, or whole blood has been recommended for IFCC adoption. "
Analytical Chemistry, Vol. 66, No. 6, March 15, 1994
I
----
The reference method is based on a cell that serves to accurately determine the amount of iCa2+in plasma and to eliminate disagreement between commercial analyzers. The proposed reference cell consists of an external reference electrode with a saturated KC1 junction in combination with a calcium ion-selective membrane electrode of defined construction and performance. It also incorporates the capillary junction that originated from the investigations by Guggenheim (6) and Siggaard-Andersen and co-workers (7) on pH measurement. Although laboratories throughout the world have worked to fabricate a reference cell satisfying these criteria, problems such as lack of reproducibility, protein effect, and effects of membrane compositions and electrode configurations have been manifested (8-11).We have developed an optimum reference cell design that not only meets the IFCC criteria, but also solves these problems (12).Factors that cause errors in iCa2+measurements are clarified, and protein effects and discrepancies derived from membrane compositions are eliminated. The reference cell
The original design of the reference cell was proposed by Covington and Kelly (13) and remains fundamentally the same in this study (Figure 1).The vertical flow path over the calcium electrode is the most efficient configuration for cleaning the electrode surface. However, the vertical position of the inlet and outlet tubing relative to the calcium electrode is not easily applicable to whole-blood samples because hemolysis of blood cells can occur as the result of turbulence caused by the sharp bends in the tubing. Angled in0003 - 270019410366-352Al$04.50/0 0 1994 American Chemical Society
let and outlet tubes are used with wholeblood samples. Another disadvantage of the vertical tubing is that air bubbles tend to become trapped at the sharp corners; This can be avoided by introducing the sample into the electrode chamber slowly. The sample introduction tubing (inner tube) from the junction capillary to the inlet of the calcium electrode chamber is kept at 37 "C by circulating thermostated water between the outer and the inner tubes. This reduces the time it takes for the electrode to reach a steady state. When a cold sample or air is rapidly introduced into the electrode, a drop in temperature and hysteresis will occur on the electrode surface, and considerable systematic error may arise. Calcium electrode. It has been a matter
of controversy whether electrode configurations and membranes are responsible for the discrepancies in measured values. Three types of membranes were examined in an attempt to settle this debate: an ion-exchange membrane containing calcium bis (di-n-octylphenyl)phosphate and di-n-octyl phenylphosphonate from a Radiometer F2121 electrode for an ionized calcium analyzer (Radiometer A/S) ( 1 4 , ETHlOO1 neutral carrier membranes prepared in our laboratory (15,16),and a membrane from a BP0360 calcium electrode for a Sysmex 984 ISE electrolyte analyzer (AVL Scientific). Electrodes were prepared by placing each membrane in the body of the Radiometer F2121 electrode. The ion-exchange membrane from the Radiometer
F2121 electrode was covered with a cellophane membrane, and the AVL BP0360 membrane was covered with the ETHlOOl neutral carrier membrane (15, 16).Three types of ETHlOOl neutral carrier membranes were prepared: one using bis (Zethylhexyl) sebaacate (DOS) as the plasticizer and two using o-nitrophenyloctyl ether (oNPOE) from different sources (Fluka and Dojindo). Calibration solutions containing Ca2', M$+, Na', K', C1-, and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) were prepared according to the IFCC reference method. However, because sodium has been shown to have an effect on the electrode (8),the sodium concentration was decreased compared with that of the reference method. HEPES
Analytical Chemistry, Vol. 66, No. 6, March 15, 1994 353 A
was added at l-mM concentration to buffer the calibration solutions, and the pH of the solutions was adjusted to 7.4. Measurement procedure. Each sample was drawn into a l-mL plastic needle syringe, and - 200 p.L was introduced through a glass capillary and dipped into the bridge solution. In - 60 s, the emf reached a constant value and the value was read. For analysis of serum samples, the cell was calibrated by introducing the 1.25mM calibration solution prior to the introduction of the sample. After the potential attributable to the serum sample was measured, the electrode was rinsed with 0.1 mL of the 1.25mM calibration solution five times in 60 s. The overall cycle was sometimes repeated two or three times until the residual potential jump was reduced to a level of 0.05 mV or less. This rinsing procedure plays an important role in the performance of the reference cell. Error factors Several factors, including changes in KC1 concentration, pH, electrode fabrication, electrode chamber volume, Mg level, protein level, and membrane discrepancy, can cause errors in the iCa2+value. Changes in KCl concentration. When the thermostat of the water bath was switched off, KC1 crystallized out of solution. We found that the crystallized KC1 did not fully redissolve without stirring, even when the thermostat was switched on again. The concentration of the saturated KC1 bridge solution decreased to 3.5 M in winter; a l-M decrease in KCl concentration resulted in a 0.01-0.02-mM decrease in measured iCa2+concentration. PH. The ion-exchange membrane (Radiometer F2121) showed a considerable pH dependence (8),and the ETHlOOl membrane prepared using the Dojindo oNPOE plasticizer showed a far stronger dependence. On the other hand, the AVL BP0360 membrane and the ETHlOOl membrane prepared using DOS plasticizer had no dependence on pH, whereas the ETHlOOl membrane prepared using Fluka oNPOE showed a small dependence, as indicated in Figure 2. Grima and Brand (17) reported in 1977 that a neutral carrier membrane containing oNPOE plasticizer was strongly
affected by pH. However, the results of their work are inconsistent with ours. We suggest that these effects, as well as those exhibited by the Dojindo oNPOE, result from impurities contained in the membrane. Electrodefabrication. We compared the reproducibility of two methods for electrode fabrication. One method mounts a disk cut out of the ETHlOOl master membrane to the electrode (18),and the other involves directly casting the ETHlOOl membrane to the tip hole of the electrode (19).The reproducibility of electrodes obtained by the first method was inferior to that obtained with electrodes fabricated by using the casting method. All electrodes obtained by the casting method showed good reproducibility and the same performance; thus, we adopted the casting method for this study. The membrane area should be as small as possible to reduce protein effects; we selected a tip hole 1mm in diameter. Electrode chamber volume. We investigated the effect of reducing the volume of the cylindrical sample chamber or electrode chamber volume (see Figure 1inset) to avoid carryover and trapped air bubbles. The chamber volume was altered
by changing the size of the O-ring in the electrode shown in Figure 1.The experiment was carried out with electrodes using the Radiometer F2121 membrane and the ETHlOOl membrane that contained DOS. A dependence of measured iCa2+ values on the chamber volume was sometimes observed only for the Radiometer membrane electrode and only for serum samples. Because the phenomenon appeared only when the Radiometer membrane was used, it may be attributable to a Donnan distribution at the cellophane membrane. However, Fogh-Andersen et al. (20) tested the effect of the Donnan distribution on iCa2+concentration using an ion-exchange membrane electrode covered with cellophane. They observed a negligible effect. Our results show that the effect of the chamber volume is associated with protein adsorption on the cellophane membrane. The Radiometer electrode, which has a larger membrane surface area than the other electrode, is apt to be contaminated by protein, and the protein-rinsing efficiency is low for a smaller chamber volume. Magnesium level. We noticed a significant Mg effect in this investigation. Fresh
Figure 1. Reference cell. Inset: detail of the ETH1001 membrane electrode. (Adapted with permission from Reference 12.)
354 A Analytical Chemistry, Vol. 66, No. 6, March 75, 1994
Figure 2. Effect of pH on emf for various calcium electrodes. Key: 1: ETH 1001/DOS; 2: AVL BP0360; 3: ETH 1001/Fluka; 4: Radiometer F2121 (batch CN- 1); 5: Radiometer F2121 (batch BD-I); 6: ETH 1001/Dojindo oNPOE
membranes showed a small Mg effect of 0-0.02 mV but, once the membrane was exposed to a serum sample, the effect increased to 0.05-0.1 mV. When the cleaning of the electrodes was inadequate, it increased to < 0.2 mV. The Mg effect can be avoided both by addition of Mg2+to the calibration solutions and by adequate cleaning procedures. Protein level. The protein-induced potential jump phenomenon was fist reported by Fogh-Andersen et al. (20).In our study the potentialjump hEpro,defined as the potential difference observed for the same aqueous calibration solution before and after serum exposure, was also observed for both the ion-exchange membrane electrode with cellophane and the prepared ETHlOOl membrane electrodes. The potential jumps were < 0.2 mV except for rare cases but gradually increased with an increase in the number of measurements of serum samples. After more than 100 measurements, several cellophane membranes showed a potentialjump of 0.5 mV. The measured iCa2+values and protein-induced potential jumps for four different electrode types are listed in Table 1. A large potential jump apparently can be eliminated by repeated introduction of a calibration solution. Note, however, that an electrode exhibiting a large jump continues to show a large systematic error even after repeated rinsing. The protein effect is attributable to the asymmetric potential induced by the adsorption of protein onto the membrane surface (21). Protein adsorption from one exposure to serum will be enhanced by repeated exposures, causing large systematic errors.
Once a membrane shows a large jump it should not be used. Discrepancy derived fiom membranes. Measurements were performed only when the protein jumps of the electrodes were small. The discrepancy between the measured iCa2+concentration obtained with the Radiometer F2121 electrode and that obtained with the ETHlOOl electrode (AVL BP0360 membrane) was only 0.01 mM. However, because the preparation methods of these two commercially available membranes are unknown, the prepared ETHlOOl electrodes containing DOS or Fluka oNPOE were also examined (Table 1).Discrepancies between the membranes were small (< 0.02 mM) as long as protein-induced potential jumps met the criteria shown for fresh or used membranes and the compositions of calibration solutions (e.g., sodium, magnesium, and pH) were kept close to those of serum samples.
The performance of the reference cell meets the IFCC criteria, as shown in Table 2. Good between-day reproducibility was obtained with both the Radiometer E 1 2 1 and the prepared ETHlOOl electrodes; the range of between-day variations for serum samples was only 0.01 mM for 10 days (12). Evaluation of commercial analyzers
Serum reference material was used for evaluation. A human serum pool was filtered with a 0.2-pm filter and tonometered with 5.75%CO, to give pH 7.41 and 26 mM HCO;. Sodium, potassium, chloride, and magnesium concentrations were adjusted to 145,4.4,106, and 0.9 mM, respectively, and total protein and albumin were 76 and 46 g/L, respectively. Calcium-binding substances (e.g., citrate, lactate, phosphate, and sulfate) were at their normal concentrations. Plasma water
Nernstian slope(o/o Response time(s)
95' IOO'/O
-, ift (mV/h) Noise levela , , Calibration solution Serum Repeatabilit Calibratio 3lut Serum
lange of fluctuation of vithin da
:urvi
Analytical Chemistry, Vol. 66, No. 6, March 15, 1994 355 A
We thank A. K. Covington and P. M. Kelly of the University of Newcastle-upon-Tyne for invaluable discussions and comments. We also thank A.H.J. Maas of the Technical University of Eindhoven and N. Fogh-Andersen of Herlev Hospital for providing useful comments and measuring the serum reference material. This investigation was sponsored by the Chemicals Inspection and Testing Institute and was s u p ported by Japanese manufacturers and distributors, including Horiba Ltd., Jookoo Co., Shimadzu Corp., Ciba-Coming Diagnostics K. K., Radiometer Trading K. K., Mallinckrodt Japan Co., Toa Medical Electronics Co., Techno Medica Co., Taiyo Yuden Corp., TOA Electronics Co., Hitachi Ltd., Daiichi Pure Chemicals Co., and Baxter Ltd. Figure 3. Evaluation of instrumentto-instrument variation. (Adapted with permission from Reference 12.)
mass concentration was 0.930 kdL. . and base excess was close to zero. Instrument-to-instrument variations for commercial iCa2+analyzers were evaluated (Figure 3). The reference value was assigned with the reference cell using the prepared ETHlOOl membrane electrode containing DOS and calibration solutions in which sodium, potassium, and magnesium concentrations were close to those of the reference material. Deviations were typically k 0.05 mM, with the exception of two analyzers for which results were much less precise than those of other analyzers. The mean value of the other 12 analyzers is close to 1.25 mM. Although the reference cell described here may not be optimal, the results should guide development of a better cell and the study of iCa2+measurements with ISEs.
Europe-The WPAC Eurocurriculum on Analytical Chemistry Robert Kellner (Anal. Chem. 1994, 66,98 A-101 A) Contributors to the textbook based o the WPAC Eurocurriculum include J. G. Grasselli (US.), K. Fuwa (Japan), W. Va der Linden (The Netherlands), P. G Zambonin (Italy), B. Griepink (Belg L. Niinisto (Finland), D. PereaBendito (Spain), K. Toth (Hungary), E.A.H. Hall (U.K.), V. Krivan (Germany), N. M. Nibbering (The Netherlands), W.M.A. Niessen (The Netherlands), H. Friebolin (Germany), M. Grasserbauer (Austria), F. Scordari (Italy), W. Wegscheider (Austria), and G. D. Christian (US.). as well 2:
References (1) Rona, P.; Takahashi, D. Biochem. 2. 1911,31,336-44. (2) Dillman, L. M.; Visscher, M. B.J. Biol. Chem. 1933.103.791-99. (3) Ross, J. W. Science 1967,156, 1378-79. (4) Schwartz, H. D. Clin. Chim. Acta 1975, 64,227-39. (5) Bowers, G. N., Jr.; Brassard, C.; Sena, S. F. Clin. Chem. 1986,32,1437-47. (6) Guggenheim, E. A. A. J. Am. Chem. SOC. 1930,52,1315-36. (7) Siggaard-Andersen, 0.;Engel, K.; Jorgensen, K.;Astrup, P. Scand. J. Clin. Lab. Invest. 1960,12,172-76. (8) Umemoto, M.; Tani, W.; Kuwa, K. In Methodology and Clinical Applications of Blood Gases, pH, Electrolytes, and Sensor Technology;Aizawa, M.; Kuwa, K., Eds.; Gene Art Planning: Tokyo, 1992; pp. 27787. (9) Maas, A. H. In Methodology and Clinical Applications of Blood Gases, pH, Electrolytes, and Sensor Technology;Aizawa, M.; Kuwa, K., Eds.; Gene Art Planning: Tokyo, 1992; p ~357-74. . (10) Covington, A. K. et al. In Electrolytes, Blood Gases, and Other Critical Analytes: The Patient, the Measurement, and the Government;D’Orazio, P. et al., Eds.; Omnipress: Madison, WI, 1992; pp. 10-20. (11) D’Orazio, P.; Bowers, Jr., G. N. Clin. Chem. 1992,38,1332-39. (12) Umemoto, M.; Tani, W.; Kuwa, K. In Electrolytes, Blood Gases, and Other Critical Analytes: The Patient, the Measurement, and the Government;D’Orazio, P. et al., Eds.; Omnipress: Madison, WI, 1992; pp. 47-59. (13) Covington, A. K.; Kelly, P. M. In Methodology and Clinical Applications of Ion-Selective Electrodes;Maas, A.H.J. et al., Eds.; Elinkwijk: Ultrecht, The Netherlands, 1989; pp. 119-28. (14) Ruzicka, J.; Hansen, E. H.; Tjell, J. C. Anal. Chim. Acta 1973,67,155-78. (15) Anker, P. et al.Ana1. Chem. 1981,53, 1970-74. (16) “Ionophores for Ion-Selective Electrodes and Optodes,” Fluka Chemie AG, Bucho, Switzerland, 1991. (17) Grima, J. M.; Brand, M.J.D. Clin. Chem. 1977,23,2048-54. (18) Craggs, A.; Moody, G. J.; Thomas, J.D.R. J. Chem. Educ. 1974,51,541-44. (19) Osswald, H. F.; Dohner, R E.; Meier, T.;
356 A Analytical Chemistry, Vol. 66, No. 6, March 15, 1994
Meier, P. C.; Simon, W. Chimia 1 9 7 7 , 3 1 (a), 50-53. (20) Fogh-Andersen,N.; Christiansen, T. F.; Komarmy, L.; Siggaard-Andersen, 0. Clin. Chem. 1978,24,1545-52. (21) Diirselen, L.F.J.; Wegmann, D.; May, K.; Oesch, U.; Simon, W. Anal. Chem. 1988, 60,1455-58.
Masao Umemoto (left) received his Ph.D. in analytical chemistryfiom The University of Tokyo (Japan). A n expert member of the Committee on Blood Gases and Electrolytes of the Japan Society of Clinical Chemistry, he is head of the Research and Development Division of Biochemistry at the Chemicals Inspection and Testing Institute, 4-1-1 Higashi-mukojima, Sumida-ku, Tokyo 131, Japan. Wataru Tani (right) is a senior research associate in the Chemical Standards Division of the Chemicals Inspection and Testing Institute. His research interests include thermal ionization MS for isotope dilution analysis.
n
I Katsuhiko Kuwa (left) is an associate professor at the College of Medical Technology and Nursing, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305,Japan. He also serves as an expert member of the Committee on Blood Gases and Electrolytes of the Japan Society of Clinical Chemistry. Yusuke Vihira (right), who received his Ph.D. in industrial chemistry jkom The University of Tokyo (19641, is a professor at the Research Centerfor Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153, Japan. His research interests include chemical sensors, Mossbauer spectrometry, and positron annihilation.
