578
Anal. Chem. 1985, 57,578-580
Microprocessor-Controlled ex Vivo Monitoring of Sodium and Potassium Concentrations in Undiluted Urine with Ion-Selective Electrodes Stephan Dutsch, Hans-Beat Jenny, Konrad J. Schlatter, Philippe M. J. PBrisset, Gunther Wolff,' Jean Thomas Clerc? Ern0 Pretsch, and Wilhelm Simon* Department of Organic Chemistry, Swiss Federal Institute of Technology, CH-8092Zurich, Switzerland
A mlcroprocessor-controlled system performlng automatic ex VIVO measurements of Na' and K+ In urine uslng lon-selectlve liquid membrane electrodes was realized. A slngle drop flow-through measuring chamber allows for stable readouts even In undliuted urine samples and In the presence of sedlments and gas bubbles. Ion concentrations are calculated by uslng an automatic callbratlon system and an Iterative procedure for the evaluatlon of activlty coefficlents. Over a concentration range of 50-250 mmol L-' (Na') and 5-120 mmoi L-' (K') the relatlve standard devlatlons between values measured with this system and by flame photometry were below 10%.
For routine measurements of sodium and potassium in clinical chemistry, flame photometry is successively replaced by ion-selective electrode techniques ( I ) . Although there exist a series of instruments using ion-selective electrodes for Na+ and K+ determinations in urine (2-9), measurements in undiluted urine exhibited until recently a high bias (IO,11). With adequate membrane technology, this interference by lipophilic ions may be circumvented (12). This opens up the possibility for continuous ex vivo measurements of sodium and potassium in undiluted urine of catheterized patients. Efforts in this direction have recently been described (13,24). There however still remain problems due to sediments, air bubbles, and extreme variation of ionic strength of the sample. Here we report on an automatic system resolving these sampling, calibration, and evaluation problems.
EXPERIMENTAL SECTION Scheme of Instrument. A scheme of the instrument developed is given in Figure 1. Urine samples were taken by means of a peristaltic pump from a siphon, which was part of the bladder catheter assembly. This siphon separated the patient from the monitor to prevent any contamination. The sample then passed through a urine flow meter consisting of a drop counting, L1, and a drop volume calibrating device, L2 and L3 in Figure 1. The latter is necessary since the drop volume of undiluted urine can vary in a wide range. It consisted of a glass tube (0.d. 7 mm, i.d. 5 mm, length 12 cm) installed vertically at the outlet of the drop counter. At its lower end, the tube was equipped with an electromagnetically driven pinch valve (V8 in Figure 1;Angar Pinch Valve 373-12-5 E, Angar Scientific, Cedar Knolls, NJ) which allowed damming up the sample in the tube. Near the lower and the upper ends of the tube two optical levels detectors (L2 and L3 in Figure 1)were installed. Each of them consisted of a light emitting diode and of a phototransistor (TIL31 and TIL81, Texas Instruments, Dallas, TX) which were mounted at an angle 120" to each other. In this arrangement the receiver detected the light reflected at the inner side of the glasswall of the empty tube.
-
Present address: Klinische Physiologie der Klinik fur Herz- und Thoraxchirur ie, Kantonsspital Basel, 4031 Basel, Switzerland. Present aidress: Pharmazeutisches Institut, Universitat Bern, Baltzerstrasse 5, 3012 Bern, Switzerland.
When the tube was filled with any aqueous solution, no optical total reflection occurred and therefore no signal was detected by the receiver. While the pinch valve V8 was held closed, the urine sample was transferred to the measuring chamber by means of a processor controlled peristaltic pump (P1 in Figure 1, capacity 11.7 mL/min). Excess sample flowed to the urine collector by opening the pinch valve V8. A pinch valve of the same type was applied after the measuring chamber (V7) which let urine samples pass to the urine collector and rinsing or calibratingsolutions pass to the waste. Ion-Selective Electrode Cell System. The silicone rubber membrane (15)of the K+-selective minielectrode (12) consisted of 85.3 wt % Silopren K1000,12.5 wt % cross-linking agent KA-1 (both obtained from Bayer AG, Leverkusen, GFR), and 2.2 wt % valinomycin (Fluka AG, Buchs, Switzerland). Electrode preparation and selectivity data are given in ref 12. The membrane of the Na+-selective minielectrode (16) was made of 66.0 wt % bis(1-butylphenyl)adipate (BBPA, Fluka AG, Bucks, Switzerland), 33.0 wt % PVC (S704,high molecular, Lonza AG, Visp, Switzerland), and 1.0 w t % sodium ionophore ETH 227 (l,l,l-tris[l'-(2'-oxa-4'-oxo-5'-aza-5'-methyl)dodec~yl]prop~e (17), Fluka AG, Buchs, Switzerland). Electrode preparation is given in ref 16. A slight improvement of the selectivity vs. K+ in respect to reported values (16) was achieved through the choice of the plasticizer BBPA (log K z K = -1.94). A saturated calomel reference electrode Philips R44/2 (Joint removed, N. V. Phillips' Gloeilampenfabrieken, Eindhoven, The Netherlands) was used. Its liquid junction was formed by a zirconium oxide diaphragm at the end of a glass capillary (0.d. 1.1mm, i.d. 1.0 mm, length 30 mm) filled with a 3 M NH&l solution as bridge electrolyte. This glass capillary was fixed on the reference electrode body with a PVC gasket. A platinum wire of 0.5 mm diameter served as a low impedance common electrode. The potential measurements were carried out in a differential mode (18, 19). The four electrodes were arranged in the measuring chamber in such a way that it was possible to build up a hanging sample drop contacting all the electrodes but without touching the cell walls (see Figures 2 and 3). This setup allows samples containing gas bubbles as well as sediments to pass the sensitive area around the electrode tips without causing any troubles. Furthermore only small sample volumes of 50-70 ,uL are needed in spite of the relatively large dimension of the sensing electrodes of about 3 mm diameter. This dimension is necessary because of the high impedance ( lo9 Q) of the silicone rubber electrode. In order to increase signal stability, the outside of the cell was covered with an aluminum sheet which was electrically grounded. The stray capacitance was reduced by connecting the shield to the output of the impedance converter. The common electrode was also grounded so that no static potential could build up in the sample drop. The impedance converters (AD515KH, Analog Devices, Norwood, MA) are mounted directly on the electrodes. To each channel (two measuring and one reference electrode) its own bessel filter of fourth order is added (3 dB, frequency 16 Hz). The dominant part of the low frequency noise is synchronous to the line (50 Hz). It could be eliminated by taking the mean value of an even number of readings triggered by the two half waves of the line. With these precautions the drift and the noise of the measuring system were minimized so that a standard deviation of f30 ,uV ( N = 15) was measured over a time period of 5 h. N
0003-2700/85/0357-0578$01.50/00 1985 Amerlcan Chemical Society
,ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985 URINE FROM PATIENT
PRINTER
0
MICROCOMPUTERSYSTEM
u n n u
URINE
1
579
4
ANALOG DIGITAL INTERFACE INTERFPCE
@
I
II ii 1
WASTE
-
1
URINE, RINSING AND CALIBRATION SOWTkWS AIR 3 DIGITAL SIGNALS URINE D ANALOG SIGNALS COLLECTOR
Figure 1. Scheme of the instrument: V1-V6, magnetic valves; V7, V8, pinch valves; L1, drop counter; L2, L3, optlcal level detectors; P1, peristaltic pump; P2, air pump; WR, rinsing solutlon; WL, WM, WH, calibration solutions.
5
5
5
Flgure 2. Cross section of the measuring chamber (front view): 1, impedance converters with minielectrodes; 2, common electrode (platinum wire); 3, reference electrode; 4, polypropylene body, the top and bottom part are screwed; 5, screwthreads for flxlng the electrodes; 6, sample drop; 7, joints for outlet drains; 8, bore for pressure equalization.
For analog to digital conversion a 16-bit ADC (MN6280, Micro Network Corp., Worcester, MA) working by the method of successive approximation with a conversion time of less than 100 pus is used. The full scale is *500 mV which corresponds to a resolution of 15.3 pV. The electrodes were cleaned and recalibrated automatically with a 0.1 M H3P04rinsing solution (WR, Figure 1) and three calibrating solutions I to I11 containing NaCl and KC1 within the physiological range of urine. The concentrations of NaCl and KCl were as follows: (given in units of mmol/L): I (WL) 60 and 40; I1 (WM) 100 and 66.7; I11 (WH) 180 and 120, respectively. All the solutions were transferred to the measuring cell by a slight overpressure produced by an aquarium air pump while they were selected by electromagnetic valves (LF AA 1200618H;The Lee Co., Westbrook, CT) controlled by the microprocessor. Microcomputer System. The microcomputer system is based on an iSBC 80/20-4 (Intel Corp., Santa Clara, CA) single board. The 8080A microprocessor and the following components of this board are essential: 4K static RAM for storing program parameters and the measured results between two plot outputs; sockets for EPROM 2716; the software written in assembly level resides on 2 EPROMS (2732A-4),using about 7.5 kbytes; two parallel interface adapters 8255 for connecting the analog (multiplexer and ADC) and the digital (valves and pumps) interface; one serial interface adapter 8251 for driving the printer; one interrupt controller 8259 for generating the jump vectors of the five possible
Flgure 3. Cross section of the measuring chamber (side view): 1, reference electrode; 2, screwthreads for fixing the reference electrode; 3, inlet for rinsing and calibration solutions and air; 4, inlet for urine; 5, polypropylene body; 6, sample drop; 7, common electrode.
interrupts and for resolving their priority: (1)interrupt switch, (2) line trigger (100 Hz real time clock), (3) drop counter, (4) raising tube level detectors, (5) serial interface interrupt. Furthermore an AMC 95/6011 (Advanced Micro Computers, Santa Clara, CA) arithmetic processor board is added to perform 16- and 32-bit fixed point and 32-bit floating point arithmetic. Although not necessary in routine operation there is a primitive way of changing program parameters by four BCD switches which are read when the interrupt switch on the front panel of the system has been pressed. Output in listed and plotted form is to a printer, Swiss-Print Matrix, Wenger Datentechnik, Basel, Switzerland. Flame Photometry. Flame photometric determinations were performed at the Medico-Chemical Central Laboratory of the University Hospital Zurich by using a IL-343 flame photometer (Instrumentation Laboratory Inc., Lexington, MA). The urine samples were mixed with a 1 M LiCl, 1 M Li2S04solution and with double distilled water in the ratio of 1:1:200. The photometer was operated with a propane/air flame and calibrated with a solution of NaCl and KC1 (120 mmol/L each).
RESULTS AND DISCUSSION Operation of the System. After program start up no further operator interaction is required. A measuring cycle is started by closing the valve V8 (Figure 1)at the lower end of the raising tube. When the urine reaches the upper level detector (L2), a concentration determination is initialized and the actual drop volume is calculated. For a concentration determination the urine is transferred by the peristaltic pump P1 from the lower end of the tube into the measuring chamber. A new cycle is then started by opening the valve V8 and closing it again when the tube is empty. Urine flow is measured by a drop counter (see Experimental Section). This measurement allows an estimate of the time, when a sample in the measuring cell has left the kidney. For this purpose each time 1 mL of urine has passed the drop counter, the data in the shift register representing the volume between the kidney and the measuring chamber are shifted by one position and the actual value of time is entered at the beginning of the register. The synchronization of the measurement with the line frequency was performed with a line trigger interrupt. A/D conversion is started immediately after entry to this interrupt routine. Then the clock was updated and on exit the value from the ADC is stored if required. Every hour the system performed a rinsing and a three point calibration of the electrodes. The stability of each potential and the quality of the linear regression applied to the calibration points of each
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ANALYTICAL CHEMISTRY, VOL. 57,
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MARCH 1985
T a b l e I. I n f l u e n c e of t h e I t e r a t i v e E v a l u a t i o n of C o n c e n t r a t i o n s on t h e L i n e a r C o r r e l a t i o n b e t w e e n t h e R e s u l t s of P o t e n t i o m e t r i c and F l a m e P h o t o m e t r i c M e a s u r e m e n t s of S o d i u m in U r i n e
coefficientsn
values n o t corrected for ionic strengthb
A B
-6.01 f 2.79 mM 1.05 f 0.02
4.1 mM
S
values corrected
100 -
for ionic strengthc -0.35 f 2.25 mM 1.00 f 0.001 3.3 mM
50 -
+
Y = A (AsA) B (fs&X: Y , ion selective electrode measurements; X , flame photometric measurements, range 26-265 mM, mean 165 mM; s, residual standard deviation; number of samples, 21. Calibration based o n concentration values. Calbration based o n a c t i v i t y values.
electrode were checked before the values were accepted, Evaluation of the Concentrations. Since the ionic strength of urine samples can vary over a range of more than 1 order of magnitude, no reliable direct determination of the concentration is possible. The measured values of activities were therefore converted into concentrations by an iterative procedure. Starting with assumed single ion activity coefficients of yi = 0.7, a fiit estimate of Na+ and K+ concentrations was calculated. Assuming that only Nat, K+, and C1- are present, the ionic strength I was then evaluated. With this ionic strength value the next estimate of the activity coefficients was computed by the DebyeHuckel formalism (20-22)
log y+ = -
0.5108p/2 1
+ 0.328AiI1j2
+ Bil
(1)
y+ is the mean ion activity coefficient. Since monovalent ions dominate in urine, we assume that yi = ya. Ai and Bi are 4.35 and 0.026 for NaCl and 3.90 and 0.004 for KC1, respectively. This allowed a next estimation of the ionic strength. After three cycles of this iteration procedure the calculated yi values were stable within 1 %. This procedure improves the linear correlation between the results of potentiometric and flame photometric measurements of sodium significantly (see Table I). Application to Open Heart Surgery. The system described has been repeatedly used in intensive care units and during open heart surgery. A fully automatic run obtained during an operation for valve replacement at extracorporeal circulation is represented in Figure 4 together with values (dota) determined by flame photometry. Artifacts may be due to the rather large dead space volume between the localization of glomerular filtration of urine and sensor assembly. This dead space volume consists of the intrarenal volume of the tubular system and the pyelon, of the ureters, emptying the peristaltic contraction, of a very small residual volume of the drained urine bladder and of the constant volume of the urine catheter itself including the draining and measuring system outside the patient. Total dead space volume of both kidneys estimated after intravenous injection of a rapidly filtered indicator sums up to 30-40 mL in the healthy adult. The contribution of the catheter amounts to about 90%. Flame photometric reference values were taken from the samples in the raising tube after performing the potentiometric measurements. Although the samples for both measurements were not strictly identical, the agreement with a relative standard deviation of about 7% is compatible with the values given in Table I which, after conversion to the relative scale, are about 2%. This compares well with the corresponding value of 1.8% obtained earlier with a different cell assembly (23). In about 20 runs up to 24 h of a development and test phase of the monitor, no deterioration of the sensor performance
0I
,
0
60
,
,
120
,
I
180
1
zio
'
[mi, TIME
Figure 4. Automatic on-line determination of Na+ and K+ concentrations in urine during open heart surgery. The symbols 0 and 0 mark flame photometric determinations.
was observed, if the rinsing procedure described above was strictly applied. In all systems applying liquid membrane electrodes, the inherent problem of possible interferences of lipophilic ionic species must however be kept in mind (IO, I I ) .
ACKNOWLEDGMENT We thank R. Asper for performing flame photometric measurements in the Medico-chemical Central Laboratory of the University Hospital Zurich. Registry No. N a , 7440-23-5; K, 7440-09-7. LITERATURE CITED (1) Schindler, J. G.; Schindler, M. M. "Bioeiektrochemische MembranElektroden"; de Gruyter: Berlin, New York, 1983. (2) Epsteln, E. E. Clin. Chem. (Winston-Salem, N.C.) 1981, 27, 1111. (3) Chan, D. W.; White, J. Clln. Chem. (Wlnston-Salem, N . C . ) 1981, 27, 1111. (4) Blumenfeld, T. A.; Griffith, B. Clin. Chem. (Winston-Salem, N . C . ) 1980, 28, 1883. (5) Keiler, M.; Niederer, R.; Richter, W. Biomed. Tech. W80, 25, 42. (6) Peileg, A.; Levy, G. 8. Clin. Chem. (Wlnston-Salem, N.C.)1975, 21, 1572 (7) Koch; D. D.; Parrish, D.; Ladenson, J. H. Clin. Chem. (Winston-Sa/em, N.C.)1981, 27, 1110. (8) Ladenson, J. H. Clin. Chem. (Winston-Salem, N.C.) 1979, 25, 757. (9) Lambert, W. J.; Byrnes, R. J. Clin. Chem. (Winston-Salem, N.C.) 1881. 27. .- - ., . , 1110. . . .-. Koch, D. D.; Ladenson, J. H. Anal. Chem. 1983, 55, 1807. Koch, D. D.; Parrish, D.; Ladenson, J. H. Clln. Chem. (Winston-Sa/em, N.C.)1983, 29, 1090. Jenney, H.4.; Rless, C.; Ammann, D.; Magyar, 8.; Asper, R.; Simon, W. Mlkrochim. Acta 1980, I I , 309. 13) Nikkiso, Co., Ltd. Jpn. Kokai Tokkyo Koho, Japan Patent No. 58 155353; 18.9.1983. 14) Toshiba Corp., Jpn. Kokai Tokkyo Koho, Japan Patent No. 81 155850; 2.12.1981. 15) Lindner, E.; Wuhrmann, P.; Simon, W.; Pungor, E. Proceedings of the
2nd Symposium on Ion-Selective Electrodes, Matrafured, Hungary, Oct 18-21, 1978; "Ion-Selective Electrodes"; Pungor, E., Buzas, I., Eds.; Akadamiai Kiado: Budapest, 1977; p 159. 16) Jenny, H.-B,; Ammann, D.; Dorig, R.; Magyar, B.; Asper, R.; Simon, W. Mlkrochlm. Acta 1980, II, 125. (17) Qilggl, M.; Oehme, M.; Pretsch, E.; Simon, W. Helv. Chlm. Acta 1976,
59,2417. (18) Brand, M. J. D.; Rechnitz, G. A. Anal. Chem. 1970, 42,616. (19) Osswaid, H. F.; Asper, R.; Dimal, W.; Simon, W. Clin. Chem. (Winston-Salem, N.C.)1979, 25,39. (20) Robinson, R. A,; Stokes, R. M. "Electrolyte Solutions"; Butterworth: London, 1968. (21) Bates, R. G.; Aifenaar, M. I n "Ion-Selective Electrodes"; Durst, R. A. Ed.; National Bureau of Standards: Washlngton, DC, 1969; Special Publication 314. (22) Meier, P. C. Anal. Chim. Acta 1982, 736,363. (23) Anker, P.; Jenny, H.-8.; Wuthier, U.; Asper, R.; Ammann, 0.; Simon, W. Clin. Chem. (Winston-Salem, N.C.)1983, 29, 1508.
RECEIVED for review October 1, 1984. Accepted November 26, 1984. This work was partly supported by the Eidgenossische Stiftung zur Forderung der Schweizerischen Volkswirtschaft durch wissenschaftliche Forschung and by the Schweizerischer Nationalfonds zur Forderung der wissenschaftlichen Forschung.
