ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
of cobalamins in samples 7 and 8. These results are reverse of what is expected from the cobalamin specificities of the two bioassay organisms (uide supra). One explanation of these unexpected findings would be that the samples 7 and 8 contain ectocrines which inhibit the growth of clone 13-1 but not that of 3H. In case the ectocrines are growth promoting substances, then they would be enhancing the growth of clone 3H specifically. The most important finding of this investigation is that, compared to bioassays, the isotopic methods give 4-10 times higher values for the concentrations of cobalamins. The disparity in the results of these methods may be explained as follows. It has been reported that vitamin BIZ,its various analogues and transformation products (e.g. lactam BIZ,lactone BIZ,etc.) all compete on almost an equal basis for binding to the porcine intrinsic factor ( 2 1 ) . I t may then be expected that isotopic methods would determine the sum of the concentration of all these molecules. Of the cobalamins detectable by isotopic methods, the transformation products of vitamin B12and its analogues exhibit little or no biological activity. In view of this, it may be suggested that out of 18-50 pg/mL of cobalamins and cobalamin-like molecules determined by the isotopic methods only 25-4070 are vitamin B,,, pseudo-vitamin Biz, and their analogues. The remaining 60-75% of the material may be a mixture of transformation products formed by the action of light and seawater on cobalamins. However, the possibility cannot be excluded that this 6 0 ~ 7 5 %of the
199
material may actually be some types of biologically produced cobalamins which are not utilized by the bioassay organisms used in these investigations. What is the actual chemical nature of this material and what is its ecological role is a subject of future research.
LITERATURE CITED A. F. Carlucci, "Handbook of Phycological Methods", J. R . Stein, Ed., Cambridge University Press, 1973,pp 387-394. D. M. Mathews, R. Gunasegararn, and J. C. Linnel. J . Clin. Pathol.. 20,
683-685 (1967). H.-Y. Shum, A. M. Streeter. B. J. O'Neill, and M. C. Path, Med. J . Aust.,
1, 1144-1148 (1970). R. A. Beck, Anal. Chem., 50, 200-202 (1978). A. Zettner, Clin. Chem. ( Winston-Salem, N.C.), 19, 699-705 (1973). A. Zettner and P. E. Duly, C/in. Chem. ( Winston-Salem, N.C.), 20, 5-14
(1974). J. D. H. Strickbnd and T . R . Parsons, "A Practical Handbook of Seawater Analysis", J. C. Stevenson, Ed., Fisheries Research Board of Canada, Ottawa, 1968. K. S. Lau, C. Gottlieb, L. R . Wasserman, and V. Herbert, Blow', 26, 202
(1965). David Rodbard, Clin. Chem. ( Winston-Saiem, N.C.). 20, 1255-1270
(1974). R. R. L. Guiliard, J . Phycol., 4, 59-64 (1968). M. B. Bunge and R. F. Schilling, Proc. SOC.Exp. &'io/. Med., 96, 587 ( 1957).
RECEIVED for review May 19,1978. Accepted October 30,1978. Work supported by New York State Contract No. C114053 and by grants from Nassau and Suffolk Counties. NYOSL Contribution No. 97.
Enzymatic Determination of Urea in Serum Based on pH Measurement with the Flow Injection Method J. Ruzicka," E. H.
Hansen, and Animesh
K. Ghose'
Chemistry Department A, The Technical University of Denmark, Building 207, DK-2800 Lyngby, Denmark
H. A. Mottola Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma
A method based on the Flow Injection Analysis system incorporating a flow-through capillary pH-electrode is described and used for the enzymatic determination of urea in aqueous and serum samples. By maintaining a constant buffering capacity of the carrier stream solution, a linear relationship between the recorded pH signal and the urea content is obtained. Attaining the analytical readout within 30 s of sample introduction, the sampling rate was 60 samples per hour and the reproducibility of measurement was h0.0029 pH unit corresponding to &0.52 %. When soluble urease was used in the carrier stream, the consumption was 25 units per analysis. The possibility of further reducing the enzyme consumption by using the merging zone principle in the Flow Injection stop-flow system is discussed.
Many enzyme catalyzed reactions involve proton exchange with the background electrolyte. Therefore, a pH sensor is 'Present address: D e p a r t m e n t of Chemistry, U n i v e r s i t y of Allahabad, Allahabad-211002, I n d i a . 0003-2700/79/0351-0199$01 OO/O
74074
the most appropriate as well as the simplest monitoring detector as it does not require an additional coupling of the primary reaction to an indicator reaction, such as, e.g., NAD-NADH. T h e drawbacks of pH sensing are, however, serious because (a) the resulting pH change may inhibit the enzyme function; (b) the pH response is a logarithmic function of the analyte concentration; and (c) any buffer present in the background electrolyte affects the pH response. The first drawback is the least serious one since, as the pH change is usually small, one can design the chemistry so that only the initial part of the reaction is used for determination; but above all, by handling the process of mixing and measuring automatically, the small undesired influence of inhibition can be built into the calibration curve because each sample is handled in exactly the same way. The second drawback of the pH electrode sensing is more serious as the logarithmic response is inherently less reproducible-an objection often made to all ion-selective electrode measurements-and very difficult to use, e.g., for derivative techniques where the measurement has to be made a t a fixed signal level, while the fixed time method using a system with a logarithmic response is not readily applicable (1). C 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
As enzymatic assays are of principal interest in clinical and biochemical analyses, where sample materials frequently contain several buffer systems in different concentrations, one inevitably encounters considerable and curia ble background effects which can be summarized as a variable blank affecting both the position as well as the slope of the Calibration curve. Apart from these drawbacks, yet another group of well known difficulties is encountered if the enzymes are used on an immobilized form, whether contained in columns (2-51, tubular reactors (6) (where the enzymes are attached to the tube walls), or sensors covered by enzyme layers (such as enzyme amperometric ( 7 ) or potentiometric (8)electrodes, or enzyme covered thermistors (9, 1 0 ) ) . To avoid this second group of problems, it was decided to use a soluble enzyme in this work. This approach is justified not only because urease is rather inexpensive, but mainly because the recently designed multiinjection technique ( 1 1 ) allows the use of the merging zones principle (12) in Flow Injection enzymatic analysis (13, 14) where as little as 0.5 unit of enzyme in 25 pL of reagent solution is consumed per determination (15). It was Mosbach and his co-workers (16,171 who first made an enzyme electrode based on pH measurements with the conventional hydrogen ion glass electrode covered with a layer of gel (or trapped liquid) containing glucose oxidase, urease, or penicillinase. Already in this pioneering work, the influence of varying the background buffer concentration was observed and later confirmed in a more detailed study on the penicillinase electrode ( 1 7 ) ,which was used in practice for the continuous control of fermentation processes. Their sensor was reported to have a logarithmic response and was affected both by the buffering capacity within the enzyme layer as well as by the measured fermentation liquid. However, a linear response can be obtained in two ways. both being based upon the concept of the buffering capacity d = dCB/dpH that is, this can be achieved either: (a) by keeping pH constant and volumetrically determine the amount of base (or acid) dCB used in the course of a measurement; or (b) by keeping the buffering capacity d constant and measure the pH change which thus becomes a linear function of the amount of protons produced or consumed. The most recent example of the first approach is the work of Adams and Carr (5) who used an electrochemical pH stat (where protons were generated coulometrically) in connection with tubular and column reactors containing immobilized urease. Apart from the usual difficulties associated with the use of an immobilized enzyme, this careful study confirmed the interfering effect of variable buffering capacity of individual samples, which in the case of serum was exaggerated to such a degree that the determination of urea could not be practically performed. T h e second approach was first used bs Papariello and co-workers ( 4 ) who designed an immobilized enzyme continuous flow system in which the p H changes on a stream of constant buffering capacity were measured. Although their sample turnover was low (6 samples/h), sample consumption high (1.5 mL), and the blank so variable that they had to resort to clean-up by dialysis, their work indeed confirmed that one can obtain a useful linear range, and their theory correctly predicts the main factors influencing the readout. When measuring with the glass electrode in the Flow Injection system, the recorded peak height reflects the p H increase between the base line pHbase line and the pH,,, measured atop a peak: (2) dpH = p H m a x - PHbase line = dCB'l/lj and this response can be related to the substrate concentration by means of the Michaelis equation:
d_ P dt
v,s +S
(3)
K,
where P is the product concentration, t is the reaction time, K, is the Michaelis constant, and S is the substrate concentration, while V, is the maximum velocity of the reaction. Using an excess of enzyme (K, >> S) and relating d P to dCB ( d P = (l/q)dCB),one obtains-in the case that the product is a base-the expression: Ti
(4) where dt is the resident time T of a sample plug in the Flow Injection Analyzer (13) and q is a constant. Equation 4 predicts that there will be a linear relationship between peak height (dpH) and substrate concentration ( S ) , provided that the p H change is measured at the initial stages of the reaction with sufficient activity of enzyme present (Le., that the V,/K,,, ratio is constant). It also follows that an increase of the buffering capacity will decrease the slope of the calibration line. Thus high 13 is clearly undesirable, yet its constancj is a prerequisite for obtaining a linear response, since at very low or no buffering capacity, the electrode response would be logarithmic:
pH = const
+ log S
(5)
uhere S = k[OH ] (if the product is a strong base) leading to:
pH = log S
~
log K,.
-
log h
Therefore, depending on the ratio between the buffering capacity and the amount of base (or acid) produced by the enzymatic reaction the calibration curve will have a shape described by Equation 3 or 5 and will also have a region of a mixed type of response. In order to obtain the widest possible range of linear response (Equation 4) and maximum slope of the calibration curve, the buffer selected as carrier stream should have a pK value close to the pH,,, value generated by the enzymatic conversion of a sample of medium concentration (i.e., in clinical analysis that of a normal sample), and its maximum buffering capacity d,,, (= 0.076 c) should be equimolar with the amount of base (or acid) generated from the least concentrated sample. The p H of the carrier stream containing this buffer should then be adjusted (by addition of strong acid or base) to be 0.2 p H unit from this carrier buffer pK value in the opposite direction than the expected pH change. In this way will be 94.8% at the base line, 100% for a tyyical or normal sample, and will decrease again to 94.8% when pHmax = PHbase line + o.4 (88.9% 3rnm at pHma, = pH base line + 0.5)(18). If there is another buffer present in various amounts in the individual samples, an additional requirement arises, that is, that the buffering capacity of this interfering buffer must be much smaller than that of the carrier stream so that its variations do not affect the overall buffering capacity of the carrier stream. This can be achieved by two means: (a) by diluting the sample solution by the carrier solution prior to injection; (b) by choosing a carrier buffer with a pK value and a pH-base-line value which both are much different from the pK of the buffer present in the sample solution. Thus if the pK of the interfering buffer differs by 2 p H units from the pK of the carrier stream buffer, the interfering buffer will have 25 times lower buffering capacity even if both buffers have the same molarities (for 1.5 p H units difference, the buffering capacity will be ten times lower). In clinical analysis, the main interfering buffer is bicarbonate (pK, = 6.3, pK2 = 10.3) and therefore the pK of
ANALYTICAL CHEMISTRY, VOL 51, NO 2, FEBRUARY 1979
i-
H
Figure 1. Flow Injection Analyzer for the enzymatic determination of urea by pH measurement. (S) Injection rotary valve (30 pL) furnished with a bypass, (a) reaction coil (0.75 m long, 0.5-mm i.d.) placed in a thermostated water jacket; (pH) capillary glass flow-through electrode; (REF) reference electrode. The overflow from t h e reference electrode vessel is pumped to waste (W). All connecting lines were 0.5-mm i.d.
2
7
201
1
-8
( D , = 3.2, T = 30 s)
t h e carrier stream buffer should be around 8. For t h e determination of urea in the range of 2 t o 20 mmol/L. the buffering capacity should be around 0.5 mmol/L to allow for t h e noncomplete enzymatic conversion of substrate. By choosing Tris as carrier buffer ( K = 8.06 a t 25 "C: 7.72 a t 37 "C) and pHbane = 7.70. t h e above requirements will be fulfilled a s during t h e enzymatic conversion of urea: H,NCOr\;H, + 2 H 2 0 + H+ -=z 2YH4++ HC0,3-,one proton is consumed. and this results in a shift of the p H through the region of maximum buffering capacity. By diluting the serum sample in the ratio 1:s with 1 X 10-'M Tris adjusted to p H 7.7 ( 3 = 0.5 mmol/L), t h e typical serum sample will yield a concentration of 4 mmol of bicarbonate/L which a t this pH will correspond to a buffering capacity of approximately 0.3 mmol/L, and this value will further decrease with increasing pH. Therefore, the buffering capacity will remain practically constant within the measuring range as the pK of the principal buffer (Tris) is situated almost exactlv halfway between the two pK values of t h e interfering buffer. Additionally, the dispersion in the Flow Injection system which is a result of mixing of carrier solution and sample zone. will further affect the ratio between the interfering buffer and the carrier stream buffer in favor of the latter one. Thus in the system depicted in Figure 1 the total dispersion (D,) measured by t h e color method (18) was found t o be 3.2, i.e.. while $buffer remains constant will decrease to approximately one third of the nominal value of t h e diluted serum samples. T h e presence of bicarbonate, however: will also influence the starting pH. which would be different from the pHbaseline of the carrier stream containing only Tris. Therefore, the peak height d p H must be corrected for the blank, pHblank,to obtain t h e correct answer for the urea content:
dpH
=
pH,,,
-
pHblank= Saconst
(7)
EXPERIMENTAL The peristaltic pump was a four channel IShlATEC Minipuls. type 840. The pH electrode was a capillary glass Radiometer G299 A, of the type commonly used for blood gas measurement. The electrode was checked in the Flow Injection system by injecting 2 Radiometer Precision Buffer Solutions (Sl510, pH = 5.383, and Sl500, pH = 6.841). It is important to note that to avoid electrical noise the thermostating solution surrounding the pH glass capillary was made conductive by addition of KNO, (to - 0 . 5 7 ~ ) and electrically connected to the reference socket of the pH meter. The calomel reference electrode, type K401, the pH meter. type 64, as well as the Servograph recorder. type 310. furnished with an REA unit 100, were all made by Radiometer. A Heto thermostat 053623 was used as a supply of circulating thermostated water. The injection valve was of the rotary t p e with a volumetric bore of 30 pL, furnished with a bypass of lo-cm, 0.5-mm i.d. polyethylene tubing (13). The Flow Injection manifold is described in detail in Results and Discussion. Reagents. The carrier buffer solution containing 1 X M Tris in 0.14 M NaCl had a pH of '7.70 and was prepared daily from a stock buffer by 50-fold dilution with 0.14 M NaC1. The stock buffer was made by mixing 50 mL of 0.1 M Tris (containing 12.114 g L-' of Tris(hydroxymethy1)aminomethane)with 36.6 mL of 0.1
Figure 2. Dependence of t h e peak height H(i.e.,dpH) on the reaction coil length L (in crn). Note that the peak height increases with increasing resident time ( r ) , passes through a maximum at L = 125 cm, and then decreases because of the prevailing effect of the sample zone dispersion (Dl)
R.1 HC1 making the volume to 100 mL with distilled water ( 1 8 ) . The urea standards were. for the preliminary experiments, prepared from a reagent grade urea (Merck) by dissolving the appropriate amount of urea in 0.14 M KaC1 to obtain a 100 mM stock solution. For serum analyses, however. this stock solution was further diluted by a 0.14 hl NaC1-0.02 >I NaHC03 solution to obtain standards in the range of 4.0 to 20.0 mM urea. Both types of standards were. prior to injection. diluted 1:s with the carrier solution. The urease (LVorthington Biochemical Corp.) contained 110 units,mg and was dissolved in the carrier solution in an amount of 30 mg,!100 mL. The serum standards were Monitrol I (lot LTD 144) and Monitrol I1 (lot PTD 464), reconstituted according to manufacturers' instruction (Dade and Merz, Bern). It was, however, found that while Monitrol I contained the specified amount of bicarbonate (12 mM after reconstitution) Monitrol I1 did not contain any bicarbonate (it was not stated as a constituent either). For this reason, Monitrol I was dissolved in distilled water, while Monitrol I1 was dissolved in 0.02 M NaHC03 solution. RESULTS AND DISCUSSION All measurements were execut.ed in t h e very simple experimental setup schematically shown in Figure 1. The carrier stream was pumped by the small peristaltic pump a t a rate of 0.76 mL/min continuously through the injection valve which was furnished with a bypass (10 cm-long, 0.5 mm-i.d.) and which had a volumetric bore of 30 pL. While the sample was being filled into t h e valve (see Figure 11, t h e carrier solution streamed through the bypass and, after the valve was turned, the sample was injected into the system, carried through the thermostated coil a (0.5-mm i.d. polyethylene tubing) and then the resulting pH was measured during t h e passage of t h e sample zone through t h e capillary electrode. T h e calomel reference electrode was situated close by in a small vessel in which a constant level was maintained by back pumping (2 mL/min). T h e optimum coil length was found by injecting 10 m M urea standards in 0.14 M NaCl into the carrier stream of Tris containing 30 mg urease/100 mL. T h e plot of peak height vs. coil length (Figure 2) yielded a typical curve with maximum a t L = 125 cm. As there is not much difference between L = 125 and L = 100 cm, the latter coil length was used in all subsequent experiments as it would yield a resident time of 30 s and a sampling frequency of 60 samples/h. In order t o find t h e optimum urease concentration while saving the M Tris to which urea was enzyme, a carrier stream of 1 X added to the level of 10 m M was continuously pumped through the system. By injecting Tris solutions containing .?, 10, 15, 20, 25, 30. 40, and 50 mg of urease/100 mL, it was found t h a t t h e peak height increased approximately loga-
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
C
B
Table I. Regression Analysis of Urea-Calibration Curves in the Concentration Range 4-15 mM a t Different Levels of Bicarbonate Added to the Sample Solutione [HCO,] level added to the sample s o h . pHbase line regression (mmol/L) const. (cure, = 0 ) dpHb& coefficient
800-
0 5 10 20
4 Figure 3. Recorder output showing from right to left: a series of serum blanks (B) recorded without urease in the carrier stream; a series of serum analyses (S);and a series of aqueous standards of urea (C). All samples injected in triplicate. The normal (N) and elevated (E) ranges of the urea levels in blood are indicated on the arrow between series S. and 6. The blank value (B') is that of an aqueous sample containing no urea, but only the interfering buffer (20 mM bicarbonate). All samples were diluted 1:5 with the carrier buffer prior to injection. The total dispersion in t h e system (D,) was 3.2
rithmically with the "half-concentration'' of enzyme corresponding to 10.8 m g / l O mL. As little is gained in peak height when increasing t h e enzyme concentration over three "half-concentrations", 30 mg of enzyme per 100 mL Tris were used as carrier solution (Le., 33 units/mL) in all subsequent enzymatic assays. Additionally, higher concentrations of enzyme will lead to increased consumption of protons resulting in a shorter range within which the d p H will be a linear function of the urea concentration (Equation 7 ) . T o develop a method which could be used for serum analysis, all three drawbacks of p H sensing outlined at the beginning of this paper must be overcome. T h e first two disadvantages are readily detected and evaluated from the calibration curve obtained in the absence of the interfering buffer, that is, by injecting urea standards prepared in Tris-0.14 M NaCl solution into the carrier stream consisting of Tris-0.1 M NaC1-urease. It was found that the recorded pH,,, value, that is, the analytical readout is available 30 s after sample injection and the base line is reached within another 30 s, thus permitting a sampling rate of 60 determinations per hour. When the d p H (peak height) was plotted vs. the concentration of urea within the range of 4 to 15 mM, each sample solution being injected in triplicate (see Figure 3 C), the regression coefficient was 0.9998, the standard error of estimate amounting to 0.0029 p H unit. The reason for the 20 m M urea d p H value being lower than what would be obtained by extrapolating the straight line was found not to be due to the lack of enzymatic activity (or inhibition), but due to the fact that the amount of base produced affected the constancy of the buffering capacity of Tris. This conclusion was reached after plotting the same d p H values vs. the logarithm of the urea concentrations where it was found that the slope was 95% of the Nernstian value for the linear portion of the calibration curve situated between 15 and 30 mM urea. As would be expected, below 15 m M the deviation from linearity was the larger the lower the concentration of urea was, and at 4 mM the slope of the pH-log urea curve was close t o zero. T h u s it was confirmed that with the present experimental conditions (a) there is no inhibition of the enzyme function and (b) the p H response is a linear function of the urea concentration, can be obtained within 30 s, and is reproducible to 0.0029 p H unit. For serum analysis, the main obstacle is the interfering effect of bicarbonate-which is the major component governing the serum buffering capacity-which still had to be overcome. Therefore, four series of urea standards, encompassing the range 4 to 20 mM urea, were prepared in 0.14
0.0404 0.0402 0.0396 0.0376
7.730
0.000 0.036 0.089 0.183
0.9997 0.9994 0.9997 0.9996
All samples (4.0, 6.0, 8.0, 10.0, 12.0, and 15.0 mM) were prediluted 1 : 5 with carrier buffer solution (1 X M Tris in 0.14 M NaCl) and were injected in triplicate. dpH = pH,,, dpH = pHm, - pHbhnk = const:C,,,,. PHbas line - (pHblank - PHbase line) = const.'Curea, i.e., P H m a = const.'Curea -k PHbase line -t dpHbh&.
Figure 4. Calibration curves obtained by plotting dpH values from Figure 3, series C. Curve a includes the blank value B' so that dpH = pH,, - pHbase ,,w, while curve b is corrected by the 6' (so that dpH = pH,, - pHblank). The latter curve is then used to read the urea contents in Monitrol I (MI) and Monitrol I1 (MII); for detail see text
M NaCl solution, the first series containing no bicarbonate, the second series 5 x M NaHC03, the third series 10 x M NaHC03. M NaHCO, and the fourth series 20 X Each sample from these series was diluted 1:5with the carrier M Tris in 0.14 M NaCl) and assayed buffer solution (1 X by Flow Injection Analysis in the manifold described above (Figure l), using the carrier stream containing 30 mg urease/100 mL. When plotted, the resulting values of d p H yielded a series of straight lines within the range of 4 to 15 mM urea which were practically parallel and shifted toward higher pH with increasing contents of bicarbonate (Table I). With no bicarbonate present, the straight line intersects the ordinate of zero content of urea a t a p H which corresponds to p H base line of the carrier stream, that is, d p H is zero (see also Figure 4). With increasing bicarbonate content, the blank increases also and its value, pHblank,found by extrapolation, is in good agreement with the value found by injecting the blank solution of Tris-NaC1-NaHC03 with no urea present. T h e serum analysis developed on the basis of the previous experiments comprised three steps as shown in Figure 3. First, a series of blank experiments was run (B) by injecting the samples into the carrier buffer solution without urease and by pumping them through the manifold described above (Figure 1). The second series of serum samples (S)was then injected into the carrier buffer solution containing 30 mg urease/100 mL. Finally a series of aqueous standards containing 0.0, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0, and 20 m M urea,
ANALYTICAL CHEMISTRY, VOL. 5 1 ,
respectively, and 20 m M bicarbonate was injected to obtain the calibration curve (C). All serum samples as well as aqueous M Tris + 0.14 M NaCl standards were diluted 1:5 with 1 X carrier buffer solution prior to injection, and were all injected in triplicate. T h e calibration curve, plotted as peak height vs. millimoles of urea: is shown in Figure 4, line a, while the same curve, corrected by blank, B’, (dpHblank= 0.183 pH), i.e.:
dpH = pH,,
-
pHblank= C,,,,.const
is shown in Figure 4, line b. T h e const. has a value of 0.0372 p H / m M urea and the intersect of line b for zero urea content is 7.73 p H , i.e. only 0.03 pH unit higher than the pHbase of the carrier stream. Thus, for accurate assay, the correction for blank values of individual serum samples must be made by using Equation 7 (or calibration line Figure 4b) as otherwise the urea values in the normal range would be elevated. This can be illustrated by referring to the two samples, represented by the first six injections on the right hand side of groups B and S in Figure 3, of which the first three represent the standard serum material Monitrol I and the second three are Monitrol 11. The urea content of Monitrol I (lot LTD 144) was 4.64 mM (confidence limits 3.57-5.71) and of Monitrol I1 (lot P T D 46A) was 12.4 m M (confidence limits 10.5-14.3) as declared and found by the manufacturer on the basis of enzymatic assay. By the present method, comprising blank correction, 4.20 mM urea was found for Monitrol I, while 12.8 mM urea was found for Monitrol 11. Without blanking, Monitrol I1 was found to contain 12.9 mM, while Monitrol I was elevated to 7.94 m M urea. For the rapid screening or simple monitoring of the changes of the urea content in blood, the blanking could be avoided if the error caused by the variation of the bicarbonate content in the individual serum samples would be acceptable. This can be illustrated by refering to samples 1-4 which were injected following the above mentioned Monitrol serum standards. As their “average blank value” is 0.075 p H unit higher than pHbimk(value B’ in the calibration group C), the use of Equation 9 or calibration curve b of Figure 4 would yield results higher by 2 mM urea, compared to the actual content. Considering the fact that the analytical readout is available within 30 s after sample injection and the instrument can be started up within a few minutes, this approach could well be considered for an emergency screening, by mean of a simple instrument. The Flow Injection system with p H detection can be easily assembled from commercially available parts of high reliability. This is not the least true for the sensor, the capillary glass electrode, which has been used for years as a standard part of the Radiometer clinical gas blood analyzers. The use of soluble enzymes, a fresh portion of which is mixed with each sample during t h e analysis, is much more reliable than use of enzyme columns or enzyme covered electrodes in which the enzyme activity gradually decreases and which are difficult to maintain and prepare. (Use of insolubilized enzymes also makes the blanking more difficult.) The reagent consumption is moderate, considering that the carrier stream containing 33 units of urease/mL is pumped a t a rate of 0.76 mL/min, while the sampling rate is 60 determinations per hour. T h e enzyme consumption could, however, be further reduced by a factor of five or even ten by using the recently developed multi-injection technique termed merging-zone Flow Injection Analysis ( I I , 1 2 ) . In this
NO. 2, FEBRUARY 1979
203
variation, the carrier stream is buffer solution or water, into which both sample and reagent (urease) are injected simultaneously by means of a double injector, and the analytical reaction takes place as sample and reagent merge downstream on their way to the detector. Such a procedure is most economical with respect to consumption of reagent. This approach will be used either in (a) the continuous flow mode described above, or (b) in the Stop-flow Injection system (13). In method (a), a separate sample zone, along with a second sample zone and an enzyme zone which merge and react in a coil, are injected simultaneously. While sample zone no. 1 allows the determination of the blank value (pHblank), the subsequent passage of the two merging zones through the flow cell renders the analytical readout (pH,,). In method (b), sample and reagent zones are injected, allowed to merge immediately before entering the detector, in which they are stopped automatically by means of an electronic device which locates the top of the peak. The enzymatic reaction is monitored continuously, the change in p H as a function of time yielding the analytical result. T h e working cycle is completed when the pump is restarted and the sample thus flushed out from the system. The experience, gained during the work on the spectrophotometric determination of glucose in serum using glucose dehydrogenase coupled with NADH (15), suggests that a similar approach could be successfully applied when measuring urea by urease, provided that the sample zone could be stopped reproducibly within the p H sensing glass capillary electrode. T h e drawbacks of the p H sensing mentioned a t the beginning of this paper were successfully overcome, yet it is realized that a more exact choice of the concentration of the carrier buffer components can be made following the mathematical approach oulined in the theory of “one point titration” suggested by Johansson and co-workers (19, 20). Such an approach would further increase the linear response range and might even lead to higher sensitivity of detection which might be needed for assays of and with enzymes of lower activity than the urease used in this work.
LITERATURE CITED H. H. Bauer, G. D. Christian and J. E. O’Reilly, ”Instrumental Analysis”, Allyn and Bacon, Boston, 1978, Chapter 18. S. J. Updike and G. P. Hicks, Science, 158. 270 (1967). G. G. Guiibault, “Handbook of Enzymatic Methods of Analysis”, Marcel Dekker, New York, 1976. J. F. Rusling, G. H. Luttrell, L. F. Cuilen, and G. J. Papariello, Anal. Chem., 48, 1211 (1976). R. E. Adams and P. W. Carr, Anal. Chem., 5 0 , 944 (1978). L. P. Leon, M. Sansur, L. R . Snyder and C. Horwath, Clin. Chem. (Winston-Salem, N . C . ) , 23, 1556 (1977). L. C. Clark and C. Lyons, Ann. N .Y . Acad. Sci., 102, 29 (1962). G. G. Guilbault and J. Montaivo, J . Am. Chem. Soc., 91, 2164 (1969). B. Mattiasson, B. Danielson, and K. Mosbach, Anal. Lett., 9, 217 (1976). L. D. Bowers, S. S . Schifreen, and P. W. Carr, Clin. Chem. (Winston-Salem, N . C . ) , 22, 1427 (1976). J. Mindegaard, Anal. Chim. Acta, in press. H. Bergamin, E. A. Zagatto, F. J. Krug, and B. F. Reis, Anal. Chim. Acta, 101, 17 (1978). J. Ruzicka and E. H. Hansen, Anal. Chim. Acta, 99, 37 (1978). D. Betteridge, Anal. Chem., 5 0 , 832A (1978). J. Ruzicka and E. H. Hansen, Anal. Chim. Acta, in press. H. Nilsson, A. Ch. Akerlund, and K. Mosbach, Biochim. Siophys. Acta, 320, 529 (1973). H. Nilsson, K. Mosbach, S. 0. Enfors, and N. Molin, Biotechnol. Bioeng., 20, 527 (1978). D. D. Perrin and B. Dempsey, “Buffers for pH and Metal Ion Control”, Chapman and Hall, London, 1974. G. Johansson and W. Backen, Anal. Chim. Acta. 69, 415 (1974). 0. Astrom, Anal. Chim. Acta, 88, 17 (1977).
RECEIVED for review September 5 , 1978. Accepted November 1, 1978. The authors express their gratitude to the Scientific Affairs Division of NATO which through Grant No. 1492 made this work possible. The gift of two capillary electrodes from Radiometer A / S , Copenhagen, is gratefully acknowledged.
of cobalamins in samples 7 and 8. These results are reverse of what is expected from the cobalamin specificities of the two bioassay organisms (uide supra). One explanation of these unexpected findings would be that the samples 7 and 8 contain ectocrines which inhibit the growth of clone 13-1 but not that of 3H. In case the ectocrines are growth promoting substances, then they would be enhancing the growth of clone 3H specifically. The most important finding of this investigation is that, compared to bioassays, the isotopic methods give 4-10 times higher values for the concentrations of cobalamins. The disparity in the results of these methods may be explained as follows. It has been reported that vitamin BIZ,its various analogues and transformation products (e.g. lactam BIZ,lactone BIZ,etc.) all compete on almost an equal basis for binding to the porcine intrinsic factor ( 2 1 ) . I t may then be expected that isotopic methods would determine the sum of the concentration of all these molecules. Of the cobalamins detectable by isotopic methods, the transformation products of vitamin B12and its analogues exhibit little or no biological activity. In view of this, it may be suggested that out of 18-50 pg/mL of cobalamins and cobalamin-like molecules determined by the isotopic methods only 25-4070 are vitamin B,,, pseudo-vitamin Biz, and their analogues. The remaining 60-75% of the material may be a mixture of transformation products formed by the action of light and seawater on cobalamins. However, the possibility cannot be excluded that this 6 0 ~ 7 5 %of the
199
material may actually be some types of biologically produced cobalamins which are not utilized by the bioassay organisms used in these investigations. What is the actual chemical nature of this material and what is its ecological role is a subject of future research.
LITERATURE CITED A. F. Carlucci, "Handbook of Phycological Methods", J. R . Stein, Ed., Cambridge University Press, 1973,pp 387-394. D. M. Mathews, R. Gunasegararn, and J. C. Linnel. J . Clin. Pathol.. 20,
683-685 (1967). H.-Y. Shum, A. M. Streeter. B. J. O'Neill, and M. C. Path, Med. J . Aust.,
1, 1144-1148 (1970). R. A. Beck, Anal. Chem., 50, 200-202 (1978). A. Zettner, Clin. Chem. ( Winston-Salem, N.C.), 19, 699-705 (1973). A. Zettner and P. E. Duly, C/in. Chem. ( Winston-Salem, N.C.), 20, 5-14
(1974). J. D. H. Strickbnd and T . R . Parsons, "A Practical Handbook of Seawater Analysis", J. C. Stevenson, Ed., Fisheries Research Board of Canada, Ottawa, 1968. K. S. Lau, C. Gottlieb, L. R . Wasserman, and V. Herbert, Blow', 26, 202
(1965). David Rodbard, Clin. Chem. ( Winston-Saiem, N.C.). 20, 1255-1270
(1974). R. R. L. Guiliard, J . Phycol., 4, 59-64 (1968). M. B. Bunge and R. F. Schilling, Proc. SOC.Exp. &'io/. Med., 96, 587 ( 1957).
RECEIVED for review May 19,1978. Accepted October 30,1978. Work supported by New York State Contract No. C114053 and by grants from Nassau and Suffolk Counties. NYOSL Contribution No. 97.
Enzymatic Determination of Urea in Serum Based on pH Measurement with the Flow Injection Method J. Ruzicka," E. H.
Hansen, and Animesh
K. Ghose'
Chemistry Department A, The Technical University of Denmark, Building 207, DK-2800 Lyngby, Denmark
H. A. Mottola Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma
A method based on the Flow Injection Analysis system incorporating a flow-through capillary pH-electrode is described and used for the enzymatic determination of urea in aqueous and serum samples. By maintaining a constant buffering capacity of the carrier stream solution, a linear relationship between the recorded pH signal and the urea content is obtained. Attaining the analytical readout within 30 s of sample introduction, the sampling rate was 60 samples per hour and the reproducibility of measurement was h0.0029 pH unit corresponding to &0.52 %. When soluble urease was used in the carrier stream, the consumption was 25 units per analysis. The possibility of further reducing the enzyme consumption by using the merging zone principle in the Flow Injection stop-flow system is discussed.
Many enzyme catalyzed reactions involve proton exchange with the background electrolyte. Therefore, a pH sensor is 'Present address: D e p a r t m e n t of Chemistry, U n i v e r s i t y of Allahabad, Allahabad-211002, I n d i a . 0003-2700/79/0351-0199$01 OO/O
74074
the most appropriate as well as the simplest monitoring detector as it does not require an additional coupling of the primary reaction to an indicator reaction, such as, e.g., NAD-NADH. T h e drawbacks of pH sensing are, however, serious because (a) the resulting pH change may inhibit the enzyme function; (b) the pH response is a logarithmic function of the analyte concentration; and (c) any buffer present in the background electrolyte affects the pH response. The first drawback is the least serious one since, as the pH change is usually small, one can design the chemistry so that only the initial part of the reaction is used for determination; but above all, by handling the process of mixing and measuring automatically, the small undesired influence of inhibition can be built into the calibration curve because each sample is handled in exactly the same way. The second drawback of the pH electrode sensing is more serious as the logarithmic response is inherently less reproducible-an objection often made to all ion-selective electrode measurements-and very difficult to use, e.g., for derivative techniques where the measurement has to be made a t a fixed signal level, while the fixed time method using a system with a logarithmic response is not readily applicable (1). C 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
As enzymatic assays are of principal interest in clinical and biochemical analyses, where sample materials frequently contain several buffer systems in different concentrations, one inevitably encounters considerable and curia ble background effects which can be summarized as a variable blank affecting both the position as well as the slope of the Calibration curve. Apart from these drawbacks, yet another group of well known difficulties is encountered if the enzymes are used on an immobilized form, whether contained in columns (2-51, tubular reactors (6) (where the enzymes are attached to the tube walls), or sensors covered by enzyme layers (such as enzyme amperometric ( 7 ) or potentiometric (8)electrodes, or enzyme covered thermistors (9, 1 0 ) ) . To avoid this second group of problems, it was decided to use a soluble enzyme in this work. This approach is justified not only because urease is rather inexpensive, but mainly because the recently designed multiinjection technique ( 1 1 ) allows the use of the merging zones principle (12) in Flow Injection enzymatic analysis (13, 14) where as little as 0.5 unit of enzyme in 25 pL of reagent solution is consumed per determination (15). It was Mosbach and his co-workers (16,171 who first made an enzyme electrode based on pH measurements with the conventional hydrogen ion glass electrode covered with a layer of gel (or trapped liquid) containing glucose oxidase, urease, or penicillinase. Already in this pioneering work, the influence of varying the background buffer concentration was observed and later confirmed in a more detailed study on the penicillinase electrode ( 1 7 ) ,which was used in practice for the continuous control of fermentation processes. Their sensor was reported to have a logarithmic response and was affected both by the buffering capacity within the enzyme layer as well as by the measured fermentation liquid. However, a linear response can be obtained in two ways. both being based upon the concept of the buffering capacity d = dCB/dpH that is, this can be achieved either: (a) by keeping pH constant and volumetrically determine the amount of base (or acid) dCB used in the course of a measurement; or (b) by keeping the buffering capacity d constant and measure the pH change which thus becomes a linear function of the amount of protons produced or consumed. The most recent example of the first approach is the work of Adams and Carr (5) who used an electrochemical pH stat (where protons were generated coulometrically) in connection with tubular and column reactors containing immobilized urease. Apart from the usual difficulties associated with the use of an immobilized enzyme, this careful study confirmed the interfering effect of variable buffering capacity of individual samples, which in the case of serum was exaggerated to such a degree that the determination of urea could not be practically performed. T h e second approach was first used bs Papariello and co-workers ( 4 ) who designed an immobilized enzyme continuous flow system in which the p H changes on a stream of constant buffering capacity were measured. Although their sample turnover was low (6 samples/h), sample consumption high (1.5 mL), and the blank so variable that they had to resort to clean-up by dialysis, their work indeed confirmed that one can obtain a useful linear range, and their theory correctly predicts the main factors influencing the readout. When measuring with the glass electrode in the Flow Injection system, the recorded peak height reflects the p H increase between the base line pHbase line and the pH,,, measured atop a peak: (2) dpH = p H m a x - PHbase line = dCB'l/lj and this response can be related to the substrate concentration by means of the Michaelis equation:
d_ P dt
v,s +S
(3)
K,
where P is the product concentration, t is the reaction time, K, is the Michaelis constant, and S is the substrate concentration, while V, is the maximum velocity of the reaction. Using an excess of enzyme (K, >> S) and relating d P to dCB ( d P = (l/q)dCB),one obtains-in the case that the product is a base-the expression: Ti
(4) where dt is the resident time T of a sample plug in the Flow Injection Analyzer (13) and q is a constant. Equation 4 predicts that there will be a linear relationship between peak height (dpH) and substrate concentration ( S ) , provided that the p H change is measured at the initial stages of the reaction with sufficient activity of enzyme present (Le., that the V,/K,,, ratio is constant). It also follows that an increase of the buffering capacity will decrease the slope of the calibration line. Thus high 13 is clearly undesirable, yet its constancj is a prerequisite for obtaining a linear response, since at very low or no buffering capacity, the electrode response would be logarithmic:
pH = const
+ log S
(5)
uhere S = k[OH ] (if the product is a strong base) leading to:
pH = log S
~
log K,.
-
log h
Therefore, depending on the ratio between the buffering capacity and the amount of base (or acid) produced by the enzymatic reaction the calibration curve will have a shape described by Equation 3 or 5 and will also have a region of a mixed type of response. In order to obtain the widest possible range of linear response (Equation 4) and maximum slope of the calibration curve, the buffer selected as carrier stream should have a pK value close to the pH,,, value generated by the enzymatic conversion of a sample of medium concentration (i.e., in clinical analysis that of a normal sample), and its maximum buffering capacity d,,, (= 0.076 c) should be equimolar with the amount of base (or acid) generated from the least concentrated sample. The p H of the carrier stream containing this buffer should then be adjusted (by addition of strong acid or base) to be 0.2 p H unit from this carrier buffer pK value in the opposite direction than the expected pH change. In this way will be 94.8% at the base line, 100% for a tyyical or normal sample, and will decrease again to 94.8% when pHmax = PHbase line + o.4 (88.9% 3rnm at pHma, = pH base line + 0.5)(18). If there is another buffer present in various amounts in the individual samples, an additional requirement arises, that is, that the buffering capacity of this interfering buffer must be much smaller than that of the carrier stream so that its variations do not affect the overall buffering capacity of the carrier stream. This can be achieved by two means: (a) by diluting the sample solution by the carrier solution prior to injection; (b) by choosing a carrier buffer with a pK value and a pH-base-line value which both are much different from the pK of the buffer present in the sample solution. Thus if the pK of the interfering buffer differs by 2 p H units from the pK of the carrier stream buffer, the interfering buffer will have 25 times lower buffering capacity even if both buffers have the same molarities (for 1.5 p H units difference, the buffering capacity will be ten times lower). In clinical analysis, the main interfering buffer is bicarbonate (pK, = 6.3, pK2 = 10.3) and therefore the pK of
ANALYTICAL CHEMISTRY, VOL 51, NO 2, FEBRUARY 1979
i-
H
Figure 1. Flow Injection Analyzer for the enzymatic determination of urea by pH measurement. (S) Injection rotary valve (30 pL) furnished with a bypass, (a) reaction coil (0.75 m long, 0.5-mm i.d.) placed in a thermostated water jacket; (pH) capillary glass flow-through electrode; (REF) reference electrode. The overflow from t h e reference electrode vessel is pumped to waste (W). All connecting lines were 0.5-mm i.d.
2
7
201
1
-8
( D , = 3.2, T = 30 s)
t h e carrier stream buffer should be around 8. For t h e determination of urea in the range of 2 t o 20 mmol/L. the buffering capacity should be around 0.5 mmol/L to allow for t h e noncomplete enzymatic conversion of substrate. By choosing Tris as carrier buffer ( K = 8.06 a t 25 "C: 7.72 a t 37 "C) and pHbane = 7.70. t h e above requirements will be fulfilled a s during t h e enzymatic conversion of urea: H,NCOr\;H, + 2 H 2 0 + H+ -=z 2YH4++ HC0,3-,one proton is consumed. and this results in a shift of the p H through the region of maximum buffering capacity. By diluting the serum sample in the ratio 1:s with 1 X 10-'M Tris adjusted to p H 7.7 ( 3 = 0.5 mmol/L), t h e typical serum sample will yield a concentration of 4 mmol of bicarbonate/L which a t this pH will correspond to a buffering capacity of approximately 0.3 mmol/L, and this value will further decrease with increasing pH. Therefore, the buffering capacity will remain practically constant within the measuring range as the pK of the principal buffer (Tris) is situated almost exactlv halfway between the two pK values of t h e interfering buffer. Additionally, the dispersion in the Flow Injection system which is a result of mixing of carrier solution and sample zone. will further affect the ratio between the interfering buffer and the carrier stream buffer in favor of the latter one. Thus in the system depicted in Figure 1 the total dispersion (D,) measured by t h e color method (18) was found t o be 3.2, i.e.. while $buffer remains constant will decrease to approximately one third of the nominal value of t h e diluted serum samples. T h e presence of bicarbonate, however: will also influence the starting pH. which would be different from the pHbaseline of the carrier stream containing only Tris. Therefore, the peak height d p H must be corrected for the blank, pHblank,to obtain t h e correct answer for the urea content:
dpH
=
pH,,,
-
pHblank= Saconst
(7)
EXPERIMENTAL The peristaltic pump was a four channel IShlATEC Minipuls. type 840. The pH electrode was a capillary glass Radiometer G299 A, of the type commonly used for blood gas measurement. The electrode was checked in the Flow Injection system by injecting 2 Radiometer Precision Buffer Solutions (Sl510, pH = 5.383, and Sl500, pH = 6.841). It is important to note that to avoid electrical noise the thermostating solution surrounding the pH glass capillary was made conductive by addition of KNO, (to - 0 . 5 7 ~ ) and electrically connected to the reference socket of the pH meter. The calomel reference electrode, type K401, the pH meter. type 64, as well as the Servograph recorder. type 310. furnished with an REA unit 100, were all made by Radiometer. A Heto thermostat 053623 was used as a supply of circulating thermostated water. The injection valve was of the rotary t p e with a volumetric bore of 30 pL, furnished with a bypass of lo-cm, 0.5-mm i.d. polyethylene tubing (13). The Flow Injection manifold is described in detail in Results and Discussion. Reagents. The carrier buffer solution containing 1 X M Tris in 0.14 M NaCl had a pH of '7.70 and was prepared daily from a stock buffer by 50-fold dilution with 0.14 M NaC1. The stock buffer was made by mixing 50 mL of 0.1 M Tris (containing 12.114 g L-' of Tris(hydroxymethy1)aminomethane)with 36.6 mL of 0.1
Figure 2. Dependence of t h e peak height H(i.e.,dpH) on the reaction coil length L (in crn). Note that the peak height increases with increasing resident time ( r ) , passes through a maximum at L = 125 cm, and then decreases because of the prevailing effect of the sample zone dispersion (Dl)
R.1 HC1 making the volume to 100 mL with distilled water ( 1 8 ) . The urea standards were. for the preliminary experiments, prepared from a reagent grade urea (Merck) by dissolving the appropriate amount of urea in 0.14 M KaC1 to obtain a 100 mM stock solution. For serum analyses, however. this stock solution was further diluted by a 0.14 hl NaC1-0.02 >I NaHC03 solution to obtain standards in the range of 4.0 to 20.0 mM urea. Both types of standards were. prior to injection. diluted 1:s with the carrier solution. The urease (LVorthington Biochemical Corp.) contained 110 units,mg and was dissolved in the carrier solution in an amount of 30 mg,!100 mL. The serum standards were Monitrol I (lot LTD 144) and Monitrol I1 (lot PTD 464), reconstituted according to manufacturers' instruction (Dade and Merz, Bern). It was, however, found that while Monitrol I contained the specified amount of bicarbonate (12 mM after reconstitution) Monitrol I1 did not contain any bicarbonate (it was not stated as a constituent either). For this reason, Monitrol I was dissolved in distilled water, while Monitrol I1 was dissolved in 0.02 M NaHC03 solution. RESULTS AND DISCUSSION All measurements were execut.ed in t h e very simple experimental setup schematically shown in Figure 1. The carrier stream was pumped by the small peristaltic pump a t a rate of 0.76 mL/min continuously through the injection valve which was furnished with a bypass (10 cm-long, 0.5 mm-i.d.) and which had a volumetric bore of 30 pL. While the sample was being filled into t h e valve (see Figure 11, t h e carrier solution streamed through the bypass and, after the valve was turned, the sample was injected into the system, carried through the thermostated coil a (0.5-mm i.d. polyethylene tubing) and then the resulting pH was measured during t h e passage of t h e sample zone through t h e capillary electrode. T h e calomel reference electrode was situated close by in a small vessel in which a constant level was maintained by back pumping (2 mL/min). T h e optimum coil length was found by injecting 10 m M urea standards in 0.14 M NaCl into the carrier stream of Tris containing 30 mg urease/100 mL. T h e plot of peak height vs. coil length (Figure 2) yielded a typical curve with maximum a t L = 125 cm. As there is not much difference between L = 125 and L = 100 cm, the latter coil length was used in all subsequent experiments as it would yield a resident time of 30 s and a sampling frequency of 60 samples/h. In order t o find t h e optimum urease concentration while saving the M Tris to which urea was enzyme, a carrier stream of 1 X added to the level of 10 m M was continuously pumped through the system. By injecting Tris solutions containing .?, 10, 15, 20, 25, 30. 40, and 50 mg of urease/100 mL, it was found t h a t t h e peak height increased approximately loga-
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 2, FEBRUARY 1979
C
B
Table I. Regression Analysis of Urea-Calibration Curves in the Concentration Range 4-15 mM a t Different Levels of Bicarbonate Added to the Sample Solutione [HCO,] level added to the sample s o h . pHbase line regression (mmol/L) const. (cure, = 0 ) dpHb& coefficient
800-
0 5 10 20
4 Figure 3. Recorder output showing from right to left: a series of serum blanks (B) recorded without urease in the carrier stream; a series of serum analyses (S);and a series of aqueous standards of urea (C). All samples injected in triplicate. The normal (N) and elevated (E) ranges of the urea levels in blood are indicated on the arrow between series S. and 6. The blank value (B') is that of an aqueous sample containing no urea, but only the interfering buffer (20 mM bicarbonate). All samples were diluted 1:5 with the carrier buffer prior to injection. The total dispersion in t h e system (D,) was 3.2
rithmically with the "half-concentration'' of enzyme corresponding to 10.8 m g / l O mL. As little is gained in peak height when increasing t h e enzyme concentration over three "half-concentrations", 30 mg of enzyme per 100 mL Tris were used as carrier solution (Le., 33 units/mL) in all subsequent enzymatic assays. Additionally, higher concentrations of enzyme will lead to increased consumption of protons resulting in a shorter range within which the d p H will be a linear function of the urea concentration (Equation 7 ) . T o develop a method which could be used for serum analysis, all three drawbacks of p H sensing outlined at the beginning of this paper must be overcome. T h e first two disadvantages are readily detected and evaluated from the calibration curve obtained in the absence of the interfering buffer, that is, by injecting urea standards prepared in Tris-0.14 M NaCl solution into the carrier stream consisting of Tris-0.1 M NaC1-urease. It was found that the recorded pH,,, value, that is, the analytical readout is available 30 s after sample injection and the base line is reached within another 30 s, thus permitting a sampling rate of 60 determinations per hour. When the d p H (peak height) was plotted vs. the concentration of urea within the range of 4 to 15 mM, each sample solution being injected in triplicate (see Figure 3 C), the regression coefficient was 0.9998, the standard error of estimate amounting to 0.0029 p H unit. The reason for the 20 m M urea d p H value being lower than what would be obtained by extrapolating the straight line was found not to be due to the lack of enzymatic activity (or inhibition), but due to the fact that the amount of base produced affected the constancy of the buffering capacity of Tris. This conclusion was reached after plotting the same d p H values vs. the logarithm of the urea concentrations where it was found that the slope was 95% of the Nernstian value for the linear portion of the calibration curve situated between 15 and 30 mM urea. As would be expected, below 15 m M the deviation from linearity was the larger the lower the concentration of urea was, and at 4 mM the slope of the pH-log urea curve was close t o zero. T h u s it was confirmed that with the present experimental conditions (a) there is no inhibition of the enzyme function and (b) the p H response is a linear function of the urea concentration, can be obtained within 30 s, and is reproducible to 0.0029 p H unit. For serum analysis, the main obstacle is the interfering effect of bicarbonate-which is the major component governing the serum buffering capacity-which still had to be overcome. Therefore, four series of urea standards, encompassing the range 4 to 20 mM urea, were prepared in 0.14
0.0404 0.0402 0.0396 0.0376
7.730
0.000 0.036 0.089 0.183
0.9997 0.9994 0.9997 0.9996
All samples (4.0, 6.0, 8.0, 10.0, 12.0, and 15.0 mM) were prediluted 1 : 5 with carrier buffer solution (1 X M Tris in 0.14 M NaCl) and were injected in triplicate. dpH = pH,,, dpH = pHm, - pHbhnk = const:C,,,,. PHbas line - (pHblank - PHbase line) = const.'Curea, i.e., P H m a = const.'Curea -k PHbase line -t dpHbh&.
Figure 4. Calibration curves obtained by plotting dpH values from Figure 3, series C. Curve a includes the blank value B' so that dpH = pH,, - pHbase ,,w, while curve b is corrected by the 6' (so that dpH = pH,, - pHblank). The latter curve is then used to read the urea contents in Monitrol I (MI) and Monitrol I1 (MII); for detail see text
M NaCl solution, the first series containing no bicarbonate, the second series 5 x M NaHC03, the third series 10 x M NaHC03. M NaHCO, and the fourth series 20 X Each sample from these series was diluted 1:5with the carrier M Tris in 0.14 M NaCl) and assayed buffer solution (1 X by Flow Injection Analysis in the manifold described above (Figure l), using the carrier stream containing 30 mg urease/100 mL. When plotted, the resulting values of d p H yielded a series of straight lines within the range of 4 to 15 mM urea which were practically parallel and shifted toward higher pH with increasing contents of bicarbonate (Table I). With no bicarbonate present, the straight line intersects the ordinate of zero content of urea a t a p H which corresponds to p H base line of the carrier stream, that is, d p H is zero (see also Figure 4). With increasing bicarbonate content, the blank increases also and its value, pHblank,found by extrapolation, is in good agreement with the value found by injecting the blank solution of Tris-NaC1-NaHC03 with no urea present. T h e serum analysis developed on the basis of the previous experiments comprised three steps as shown in Figure 3. First, a series of blank experiments was run (B) by injecting the samples into the carrier buffer solution without urease and by pumping them through the manifold described above (Figure 1). The second series of serum samples (S)was then injected into the carrier buffer solution containing 30 mg urease/100 mL. Finally a series of aqueous standards containing 0.0, 4.0, 6.0, 8.0, 10.0, 12.0, 15.0, and 20 m M urea,
ANALYTICAL CHEMISTRY, VOL. 5 1 ,
respectively, and 20 m M bicarbonate was injected to obtain the calibration curve (C). All serum samples as well as aqueous M Tris + 0.14 M NaCl standards were diluted 1:5 with 1 X carrier buffer solution prior to injection, and were all injected in triplicate. T h e calibration curve, plotted as peak height vs. millimoles of urea: is shown in Figure 4, line a, while the same curve, corrected by blank, B’, (dpHblank= 0.183 pH), i.e.:
dpH = pH,,
-
pHblank= C,,,,.const
is shown in Figure 4, line b. T h e const. has a value of 0.0372 p H / m M urea and the intersect of line b for zero urea content is 7.73 p H , i.e. only 0.03 pH unit higher than the pHbase of the carrier stream. Thus, for accurate assay, the correction for blank values of individual serum samples must be made by using Equation 7 (or calibration line Figure 4b) as otherwise the urea values in the normal range would be elevated. This can be illustrated by referring to the two samples, represented by the first six injections on the right hand side of groups B and S in Figure 3, of which the first three represent the standard serum material Monitrol I and the second three are Monitrol 11. The urea content of Monitrol I (lot LTD 144) was 4.64 mM (confidence limits 3.57-5.71) and of Monitrol I1 (lot P T D 46A) was 12.4 m M (confidence limits 10.5-14.3) as declared and found by the manufacturer on the basis of enzymatic assay. By the present method, comprising blank correction, 4.20 mM urea was found for Monitrol I, while 12.8 mM urea was found for Monitrol 11. Without blanking, Monitrol I1 was found to contain 12.9 mM, while Monitrol I was elevated to 7.94 m M urea. For the rapid screening or simple monitoring of the changes of the urea content in blood, the blanking could be avoided if the error caused by the variation of the bicarbonate content in the individual serum samples would be acceptable. This can be illustrated by refering to samples 1-4 which were injected following the above mentioned Monitrol serum standards. As their “average blank value” is 0.075 p H unit higher than pHbimk(value B’ in the calibration group C), the use of Equation 9 or calibration curve b of Figure 4 would yield results higher by 2 mM urea, compared to the actual content. Considering the fact that the analytical readout is available within 30 s after sample injection and the instrument can be started up within a few minutes, this approach could well be considered for an emergency screening, by mean of a simple instrument. The Flow Injection system with p H detection can be easily assembled from commercially available parts of high reliability. This is not the least true for the sensor, the capillary glass electrode, which has been used for years as a standard part of the Radiometer clinical gas blood analyzers. The use of soluble enzymes, a fresh portion of which is mixed with each sample during t h e analysis, is much more reliable than use of enzyme columns or enzyme covered electrodes in which the enzyme activity gradually decreases and which are difficult to maintain and prepare. (Use of insolubilized enzymes also makes the blanking more difficult.) The reagent consumption is moderate, considering that the carrier stream containing 33 units of urease/mL is pumped a t a rate of 0.76 mL/min, while the sampling rate is 60 determinations per hour. T h e enzyme consumption could, however, be further reduced by a factor of five or even ten by using the recently developed multi-injection technique termed merging-zone Flow Injection Analysis ( I I , 1 2 ) . In this
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variation, the carrier stream is buffer solution or water, into which both sample and reagent (urease) are injected simultaneously by means of a double injector, and the analytical reaction takes place as sample and reagent merge downstream on their way to the detector. Such a procedure is most economical with respect to consumption of reagent. This approach will be used either in (a) the continuous flow mode described above, or (b) in the Stop-flow Injection system (13). In method (a), a separate sample zone, along with a second sample zone and an enzyme zone which merge and react in a coil, are injected simultaneously. While sample zone no. 1 allows the determination of the blank value (pHblank), the subsequent passage of the two merging zones through the flow cell renders the analytical readout (pH,,). In method (b), sample and reagent zones are injected, allowed to merge immediately before entering the detector, in which they are stopped automatically by means of an electronic device which locates the top of the peak. The enzymatic reaction is monitored continuously, the change in p H as a function of time yielding the analytical result. T h e working cycle is completed when the pump is restarted and the sample thus flushed out from the system. The experience, gained during the work on the spectrophotometric determination of glucose in serum using glucose dehydrogenase coupled with NADH (15), suggests that a similar approach could be successfully applied when measuring urea by urease, provided that the sample zone could be stopped reproducibly within the p H sensing glass capillary electrode. T h e drawbacks of the p H sensing mentioned a t the beginning of this paper were successfully overcome, yet it is realized that a more exact choice of the concentration of the carrier buffer components can be made following the mathematical approach oulined in the theory of “one point titration” suggested by Johansson and co-workers (19, 20). Such an approach would further increase the linear response range and might even lead to higher sensitivity of detection which might be needed for assays of and with enzymes of lower activity than the urease used in this work.
LITERATURE CITED H. H. Bauer, G. D. Christian and J. E. O’Reilly, ”Instrumental Analysis”, Allyn and Bacon, Boston, 1978, Chapter 18. S. J. Updike and G. P. Hicks, Science, 158. 270 (1967). G. G. Guiibault, “Handbook of Enzymatic Methods of Analysis”, Marcel Dekker, New York, 1976. J. F. Rusling, G. H. Luttrell, L. F. Cuilen, and G. J. Papariello, Anal. Chem., 48, 1211 (1976). R. E. Adams and P. W. Carr, Anal. Chem., 5 0 , 944 (1978). L. P. Leon, M. Sansur, L. R . Snyder and C. Horwath, Clin. Chem. (Winston-Salem, N . C . ) , 23, 1556 (1977). L. C. Clark and C. Lyons, Ann. N .Y . Acad. Sci., 102, 29 (1962). G. G. Guilbault and J. Montaivo, J . Am. Chem. Soc., 91, 2164 (1969). B. Mattiasson, B. Danielson, and K. Mosbach, Anal. Lett., 9, 217 (1976). L. D. Bowers, S. S . Schifreen, and P. W. Carr, Clin. Chem. (Winston-Salem, N . C . ) , 22, 1427 (1976). J. Mindegaard, Anal. Chim. Acta, in press. H. Bergamin, E. A. Zagatto, F. J. Krug, and B. F. Reis, Anal. Chim. Acta, 101, 17 (1978). J. Ruzicka and E. H. Hansen, Anal. Chim. Acta, 99, 37 (1978). D. Betteridge, Anal. Chem., 5 0 , 832A (1978). J. Ruzicka and E. H. Hansen, Anal. Chim. Acta, in press. H. Nilsson, A. Ch. Akerlund, and K. Mosbach, Biochim. Siophys. Acta, 320, 529 (1973). H. Nilsson, K. Mosbach, S. 0. Enfors, and N. Molin, Biotechnol. Bioeng., 20, 527 (1978). D. D. Perrin and B. Dempsey, “Buffers for pH and Metal Ion Control”, Chapman and Hall, London, 1974. G. Johansson and W. Backen, Anal. Chim. Acta. 69, 415 (1974). 0. Astrom, Anal. Chim. Acta, 88, 17 (1977).
RECEIVED for review September 5 , 1978. Accepted November 1, 1978. The authors express their gratitude to the Scientific Affairs Division of NATO which through Grant No. 1492 made this work possible. The gift of two capillary electrodes from Radiometer A / S , Copenhagen, is gratefully acknowledged.
