REPORT
Measuring Calcium in Plasma A new electrochemical reference cell shows promise for standardized measurements of ionized calcium in plasma
Masao U m e m o t o Wataru Tani Chemicals Inspection and Testing Institute
Katsuhiko K u w a University of Tsukuba
Y u s u k e Ujihira The University of Tokyo 352 A
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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 (iCaz+). 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 (5), iCa2+ constituted 88% of all clinical calcium measurement requests compared with 11% for total calcium and 1% for urine calcium at Hartford Hospital (CT). 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 advisory body 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
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 - 2700/94/0366 -352A/$04.50/0 © 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-w-octylphenyl) phosphate and di-M-octyl phenylphosphonate from a Radiometer F2121 electrode for an ionized calcium analyzer (Radiometer A/S) (14), ETHlOOl neutral carrier membranes prepared in our laboratory (15,16), and a membrane from a BP0360 calcium electrode for a Sysmex 984ISE 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(2-ethylhexyl)sebaacate (DOS) as the plasticizer and two using o-nitrophenyloctyl ether (oNPOE) from different sources (FlukaandDojindo). Calibration solutions containing Ca2+, Mg2*, Na+, K+, CI", and HEPES (W-2-hydroxyethylpiperazine-JV'-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 3 5 3 A
REPORT
was added at 1-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 1-mL plastic needle syringe, and - 200 μι was introduced through a glass capillary and dipped into the bridge solution. In - 60 $, the emf reached a constant value and the value was read. For analysis of serum samples, the cell was calibrated by introducing the 1.25-mM calibration solution prior to the introduction of the sample. After the po tential attributable to the serum sample was measured, the electrode was rinsed with 0.1 mL of the 1.25-mM calibration solution five times in 60 s. The overall cy cle 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, pro tein 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, KCl crystallized out of solu tion. We found that the crystallized KCl did not fully redissolve without stirring, even when the thermostat was switched on again. The concentration of the satu rated KCl bridge solution decreased to 3.5 M in winter; a 1-M decrease in KCl con centration resulted in a 0.01-0.02-mM de crease in measured iCa2+ concentration. pH. The ion-exchange membrane (Ra diometer F2121) showed a considerable pH dependence (8), and the ΕΤΗ 1001 membrane prepared using the Dojindo oNPOE plasticizer showed a far stronger dependence. On the other hand, the AVL BP0360 membrane and the ΕΤΗ 1001 membrane prepared using DOS plasti cizer had no dependence on pH, whereas the ETH1001 membrane prepared using Fluka oNPOE showed a small depen dence, as indicated in Figure 2. Grima and Brand (17) reported in 1977 that a neutral carrier membrane con taining oNPOE plasticizer was strongly 354 A
affected by pH. However, the results of by changing the size of the O-ring in the their work are inconsistent with ours. We electrode shown in Figure 1. The experi suggest that these effects, as well as those ment was carried out with electrodes us exhibited by the Dojindo oNPOE, result ing the Radiometer F2121 membrane and from impurities contained in the mem the ETH1001 membrane that contained brane. DOS. A dependence of measured iCa2+ Electrode fabrication. We compared thevalues on the chamber volume was some times observed only for the Radiometer reproducibility of two methods for elec membrane electrode and only for serum trode fabrication. One method mounts a disk cut out of the ETH1001 master mem samples. Because the phenomenon ap brane to the electrode (18), and the other peared only when the Radiometer mem brane was used, it may be attributable to a involves directly casting the ETH1001 Donnan distribution at the cellophane membrane to the tip hole of the electrode membrane. However, Fogh-Andersen et (19). The reproducibility of electrodes al. (20) tested the effect of the Donnan obtained by the first method was inferior to that obtained with electrodes fabricated distribution on iCa2+ concentration using an ion-exchange membrane electrode cov by using the casting method. All elec ered with cellophane. They observed a trodes obtained by the casting method negligible effect. showed good reproducibility and the same performance; thus, we adopted the Our results show that the effect of the casting method for this study. The mem chamber volume is associated with pro brane area should be as small as possible tein adsorption on the cellophane mem to reduce protein effects; we selected a tip brane. The Radiometer electrode, which hole 1 mm in diameter. has a larger membrane surface area than Electrode chamber volume. We investithe other electrode, is apt to be contami gated the effect of reducing the volume of nated by protein, and the protein-rinsing efficiency is low for a smaller chamber the cylindrical sample chamber or elec volume. trode chamber volume (see Figure 1 in set) to avoid carryover and trapped air Magnesium level. We noticed a signifi bubbles. The chamber volume was altered cant Mg effect in this investigation. Fresh
Thermistor \ Plexiglas jacket
stopcock Peristaltic pump
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Calcium electrode l
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Glass capillary.. Water bath
Inner reference solution (37 -C)
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Figure 1 . Reference cell. Inset: detail of the ETH1001 membrane electrode. (Adapted with permission from Reference 12.)
Analytical Chemistry, Vol. 66, No. 6, March 15, 1994
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F i g u r e 2 . E f f e c t of p H o n e m f f o r various calcium electrodes. Key: 1 : ΕΤΗ 1001/DOS; 2: AVL BP0360; 3: ΕΤΗ 1001/Fluka; 4: Radiometer F2121 (batch CN-1); 5: Radiometer F2121 (batch BD-1); 6: ΕΤΗ 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 in creased to 0.05-0.1 mV. When the clean ing 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 poten tial jump phenomenon was first reported by Fogh-Andersen et al. (20). In our study the potential jump Δ£ρΓΟ, defined as the poten tial difference observed for the same aque ous calibration solution before and after serum exposure, was also observed for both the ion-exchange membrane electrode with cellophane and the prepared ETH1001 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 sam ples. After more than 100 measurements, several cellophane membranes showed a potential jump 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 con tinues 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 expo sures, causing large systematic errors.
Once a membrane shows a large jump it should not be used. Discrepancy derived from 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 ΕΤΗ 1001 electrode (AVL BP0360 membrane) was only 0.01 mM. However, because the preparation methods of these two commercially avail able membranes are unknown, the pre pared ETH1001 electrodes containing DOS or Fluka oNPOE were also exam ined (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 cali bration solutions (e.g., sodium, magne sium, and pH) were kept close to those of serum samples.
The performance of the reference cell meets the IFCC criteria, as shown in Ta ble 2. Good between-day reproducibility was obtained with both the Radiometer F2121 and the prepared ETH1001 elec trodes; the range of between-day varia tions 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 fil tered with a 0.2-μηι filter and tonometered with 5.75% C0 2 to give pH 7.41 and 26 mM HCO3. Sodium, potassium, chlo ride, and magnesium concentrations were adjusted to 145, 4.4,106, and 0.9 mM, re spectively, and total protein and albumin were 76 and 46 g/L, respectively. Calci um-binding substances (e.g., citrate, lac tate, phosphate, and sulfate) were at their normal concentrations. Plasma water
T a b l e 1 . C o m p a r i s o n of m e a s u r e d i C a 2 + v a l u e s a n d p r o t e i n - i n d u c e d potential jumps â£pro
Commercial electrode Radiometer F2121 AVL BP0360 membrane Prepared ETH1001 membrane electrode DOS plasticizer Fluka oNPOE plasticizer
mV (bias)
Measured iCa2* value9 (mM)
Fresh or used membrane (mV)
Deteriorated" membrane
1.24210.005 1.25010.011
