Automatic Digital System for Quantitative Kinetic AnalysisApplication to Catalytic Determination of Thyroid Hormones Gunter Knapp Department of General Chemistry, Micro- and Radiochemistry, Technical University, Graz, Austria
Hans Leopold Department of Physical Chemistry, University of Graz, Austria
An automatic digital system for quantitative kinetic analysis is described. The instrument system is applicable to any reaction system for which the detector response can be made to change linearly with time. The system is applied to the determination of traces of thyroid hormones after separation from blood serum by column chromatography. After a short calibration procedure, the system indicates the hormone concentration of blood serum on a numerical display and prints it out automatically, the through-put rate of the analyzer being 36 samples per hour. Read-out of the concentration of the thyroid hormones in pg/100 mi is obtained within 100 sec after mixing sample and reagents. The system allows determination of as little as 0.20 ng of T3 and 0.10 ng of T4.
The system to be described in the present paper may be used for the automatic performance of various kinds of catalytic analyses. The only requirement of the digital measuring system is that it be supplied with a signal which changes linearly with time. The system delivers the mean value of the slope of the mentioned signal plotted us. time in digital representation by using the “fixed time” method ( I ) and a rate-controlled low-pass filter providing for a high degree of noise immunity, thereby allowing high precision of a single measurement. The system also includes a special purpose calculator in order to convert reaction rates on-line into calibrated concentration values. An incorporated control-unit sequences the sampling, measuring, and calculation procedures. In principle, the problem of determination of thyroid hormones (T3 and T4) may be solved by determining their iodine concentration if present in isolated form. If, however, as for instance in clinical applications, the thyroid hormones are present together with other iodine compounds, a column-chromatographic separation of the individual hormone prior to the iodine determination is necessary. This separation has recently been demonstrated in a convenient manner suitable #for clinical requirements ( 2 ) . In any case, the sensitivity of the determination of the iodine concentration must be rather high in order to enable sufficient accuracy in determining the hormone content of human blood serum. As far as serum-triiodothyronine (normal range 0.9 to 1.7 ng per ml serum) is concerned, a method faster and more sensitive than the common catalytic determination of iodine (3, 4 ) is desirable. A new modified catalytic reaction ( 5 ) increases the sensitivity-speed product appreH V . Malmstadt C. J. Delaney. and E. A. Cordos, Anai. Chem., 44 1121. 79A 11972). G. Knapp. H Spitzy. and H Leopold, Anai. Chem.. 46, 724 (1974). E. B Sandell and I M . Kolthoff, J. Amer. Chem. SOC.,5 6 , 1426 (1934). E. B. Sandell and I . M . Kolthoff, Mikrochim. Acta, 1, 9 (1937) G . Knapp and H . Spitzy, Taianta, 16, 1353 (1969)
ciably, thus making possible automatic determination of the concentration of thyroid hormones by means of a reaction rate system. The redox reaction
+
-
+
2Ce“ As3+ 2Ce” As” (1) is catalyzed by traces of iodine (3, 4 ) . Thus, the reaction rate measured with a spectrophotometer can be used as a measure of the iodine concentration [I]. It is generally assumed that reaction 1 is first order in Cer(1V) under certain reaction conditions (6-9). The above reaction normally takes place in a medium of sulfuric acid. By using a medium af nitric acid instead, the catalytic activity of iodine is increased 20-fold. Therefore. measuring times in the range of a few seconds are possible. This modified reaction also has been found to be of first order in Ce(IV) by varying the ratio [As(III)/Ce(IV)]from 2.5 to 10. Those observations are discussed quantitatively elsewhere ( 5 ) . As the reaction also takes place in the absence of iodine, we may assume the reaction rate to be additively composed of two terms which are governed by the constants kl and k2:
Keeping in mind that the absorbance A is proportional to the Ce4f concentration (Beer’s law), we obtain after solving Equation 2:
The term to the left represents the slope of the straight line which will be obtained by plotting the natural logarithm of the absorbance us. time. According to Equation 3, the iodine concentration can be thus evaluated:
(4) The new constants k ‘ l and k ’ z , replacing k l and k z , can be determined by a simple calibration procedure.
EXPERIMENTAL Apparatus. The system developed for automatic determination of the Concentration of thyroid hormones (based on Equation 4) performs the following steps. To start the sequence of operations, the reaction mixture is aspirated into the thermostated cuvette of the photometer by the sampler. After a certain interval to allow for the establishment of thermal equilibrium, the data processing unit measures the average slope of the In A u s . time signal, converts it to a digital number, and evaluates the iodine or hormone concentration, using the constants k’l and k ’ z which are stored in a permanent form. Thereafter, the result appears on a digital read-out and the print(6) A. L. Chaney, Ind. Eng. Chem., Anal. Ed.. 12, 179 (1940) (7) A. Lein and N. Schwartz, Ana/. Chem.. 23, 1507 (1951) (8) N. E. Kontaxis and D. E. Pickering. J. Ciin. Endocrinoi Metab., 18, 774 (1958). (9) P. A . Rodriguez and H . L. Pardue, Anai. Chem., 41, 1369 (1969).
ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1974
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J n
lo recordrr
1
reodoul
Figure 1. Block diagram of the data processing unit
Figure 2. Block diagram of the dynamic filter
voiloge
4
counter 2
gale 2
Figure 3. Slope detector with timing diagram 720
ANALYTICAL CHEMISTRY, V O L . 46, NO. 6, MAY 1974
num sw/lch “K,” num switch
”
nine’s complement
I
p a r o / / e / input
output register
Figure 4. Block diagram of t h e calculator
er is activated. At the same time, the sampler removes the reaction mixture from the cuvette and rinses the cuvette with distilled water. All these activities are controlled by the sequence determinator of the processing unit. Sampler. Essentially, the sampler consists of a peristaltic pump driven by a reversible synchronous motor. The filling of the cuvette takes place during a counterclockwise rotation of the pump. The sampling time is chosen so t h a t the mixture will only fill the cuvette, but does not reach the pump. A clockwise rotation of the p u m p empties the mixture out of the measuring cell and at the same time passes water for rinsing through it. The action of t h e sampler is controlled by the processing unit. Photometer. In the present case, the inexpensive Perkin-Elmer Model 44 set to 365 nm was used as a linear absorbance spectrophotometer. A high level output is provided in order of facilitate the data transfer to the processing unit. Data Processing Unit. Figure 1 shows a block diagram of this unit. T h e photometer signal first passes a rate-controlled dynamic low-pass filter. The filter is inserted in order t o reject spurious signals picked up by the highly sensitive input of the photometer amplifier. The operation of the filter is based on rate discrimination. If the photometer output voltage shows a higher differential quotient with respect to time than can be expected by the reaction itself, the filter circuit immediately interrupts the signal path between photometer and data-processing unit and holds the last undisturbed value in a sample-and-hold circuit. The circuit switches back to the sample mode 0.5 second after the differential quotient has returned to a normal value. This delay was introduced as it is not possible to distinguish between the tail-end of a spurious signal of exponential form and an actual reaction rate. Figure 2 shows a block diagram of the filter circuit. The 33 msec low-pass filter rejects high frequency noise; the sample-hold is made u p of an operational transconductance amplifier and a field effect transistor, the remaining part of conventional operational amplifiers. In the case of the reaction described, the filter rejected any interference perfectly, even in a high noise environment. After passing the described filter, the logarithm of the absorbance is formed by a log amplifier. A recorder may be connected to the output of the log amplifier in order to confirm t h a t the reaction behaves monomolecularly. The principle of the slope detector and t h e corresponding timing diagram are shown in Figure 3. It is essential to note that the analog-to-digital conversion is performed in this unit. t o is the moment when the actual measurement is started. Reset of counter 1 and counter 2 is therefore inhibited. At that time. the output voltage of the log amplifier must be more positive than the output of the digital-to-analog converter (D/A), which converts the information of the ( a t this point still zeroed) binary counter 1 into an analog voltage, causing the comparator output to go high. As a consequence, the clock pulses derived from a crystal-controlled oscillator (frequency 1 kHz, pulse width 100 nsec) may pass gate 1 and will count u p counter 1 until the out-
put voltage of the D/A surpasses log A , causing the comparator output to go low. Now the count is inhibited until the increasing log voltage itself surpasses once more the output of the D/A. Therefore, the content of counter 1 follows step by step the magnitude of the increasing log voltage. The amplitude of 1 step (10 mV) is given by the maximum analog voltage divided by the modulus of counter 1 (21°). At tl, the sequence determinator enables gate 2 for exactly 40, 80, or 160 seconds, thus counting up the BCD counter 2 for a content proportional to the mean value of the slope of the input voltage. At t z , gate 2 is disabled, the numerical value (d (In A ) ) / d t being stored in counter 2 as a 12-bit BCD word. The calculator which was specially designed for this instrumentation performs the arithmetical procedure according to Equation 4. The constants k’l and k‘z are stored in two 3-digit (12-bit BCD numerical switches. As the computer repeats its program every 5 milliseconds, any change of the content of the numerical switches causes an immediate alternation of the read-out, thus facilitating the calibration procedure. As a detailed description of the computer would go beyond the scope of this article, we . shall limit ourselves to discussing its main features. Essentially, the calculator (Figure 4) consists of two 16-bit parallel in/serial out shift registers (the operand O P and the accumulative register AK); a single-bit full adder with series correcting network, thus performing the serial BCD-addition/subtraction in one cycle while allowing parallel load with input data and constant: a 12-bit output register; and an operational control-unit containing the clock pulse generator; the bit- and digit-timing circuits; the multiplication control with the numerical switch “ k ’ s ” ; and the program control. The wired program consists of only 4 steps: 1) Transfer (d (In A))/dt AK; Transfer nine’s complement of k’l OP. 2) Add; check, if end around carry. 3) Transfer AK OP. 4 ) Multiply. The first 3 steps take 16 clock pulses each, the multiplication 480. There is no multiplicand register, as the contents of the several digits of the numerical switch k‘z are compared sequentially to the digit counter of the multiplication control. Serial data outp u t takes place during the last shift operation of the multiplication. A separate 12-bit serial in/parallel out shift register statisizes the output word and stores it during the repeated sequence of the program. It seems worth noticing that the calculator is on a single printed board and uses only 47 packages of TTL integrated circuits, mostly of medium scale integration. The sequence determinator driven by a real-time clock pulse is responsible for the correct timing of all operations. I t consists of a number of decade counters, decoders, and other gating to perform the following sequence of commands: 0 Stand by; wait for ext. start 1 Sampling; duration adjustable by means of a n external switch to 8, 10, or 1 2 seconds 2 Wait for thermal equilibrium for 8 seconds 3 Measure for 40,80, or 160 seconds
-
- -
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 6, M A Y 1 9 7 4
721
100k
Bo--
I
60-LO-
10
--
IO-8-6
-.
4-
2-
t
,
36
.ia
32
32
2i
33
20 31
16
oc
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IO-^+
Figure 5. Reaction rate as a function of 1 / T
Table I. M e a s u r e m e n t of B l a n k Tests (Bo)a n d Ta Solutions (6 ng TI)(23,) at Various Temperatures. Measuring Period, 40 sec Temperature of reaction, “C
36 32 28 24 20 16 12
Bo (Digit)
46 27 17 10
6 3-4 2
Bo (Digit)
AVr
74 54 39 28 20 14
0.61
RESULTS AND DISCUSSION
1.30 1.80 2.33 3 .OO 4.00
The thyroid hormones (T3, T4), as well as iodine, have a catalytic effect on the redox reaction 1. This effect is probably due to the fact that in the catalytic mixture some of the iodine atoms from the hormone molecule are split off through oxidation (11, 12). Some authors have investigated the varying catalytic effect of the different iodine amino acids (13-15). These papers, however, do not indicate what percentage of the organically bound iodine atoms is set free. In order to do that, we have in the present paper made comparative measurements of iodide and TS and T4 solutions, respectively, with the same iodine content. This showed that 54% in T3 and 62% in T4 of the organically bound iodine atoms are catalytically active. These values were obtained for the reaction mixture with nitric acid, which we described. Other factors of importance for the sensitivity of the catalytic determination of T3 and T4 are the temperature of the reaction and the. reaction time. Attempts were made to optimize these two parameters. If we express the sensitivity of a catalytic method of analysis as
10
1 .oo
4 Print 5 Wash for 40 seconds The sequence determinator is set to its stand-by position by switching on the power, and will return to stand-by after each cycle and wait for the next start either by pushing the button or by the ext. start input. The read-out is formed by 3 Nixie tubes t o display the iodine or thyroid hormone content in concentration units and a sign indicator tube which shows + if the measurement was correct, - if for some reason the constant k ‘ l was > (d In A))/dt, and X if counter 1 (Figure 3 ) flows over before the measuring time has elapsed. The latter would mean that the iodine concentration was higher than the measuring range. The digital printer activated by the seauence determinator is fed with the same data as the read-out, inc-luding the signs. Reagents. Ceric Reagent. Three grams of Ce ( S 0 4 ) 2 . 4 H20 are dissolved in 100 ml ofdistilled water and 10 ml of-cbncentrated sulfuric acid. This solution is then diluted to 500 ml with distilled water. Arsenious Reagent. Four grams of As203 are dissolved in 50 ml of 1N NaOH. After adding 2 ml of concd HzS04 and 2 grams of NaC1. the solution is diluted to 500 ml with distilled water. T3 Solution. One milligram of 3-5-3’-triiodothyronine is dissolved in 1000 ml of NaOH (0.1N). One milliliter of this solution is diluted to 100 ml with XaOH (0.1N). The latter solution contains 10 ng Ts/ml. T4-Solution. This is prepared in the same way as the T3 solution, but starting wit,h 1.0 mg of very pure m-thyroxine. Procedure. Determination of T3 and T4. The T3 or Tq standard solution, or eluted material from a chromatographic thyroid hormone separation (2, I O ) , is mixed with 0.2 ml of arsenious reagent, 0.5 ml of “ 0 3 (l5M), 1.5 ml of distilled water, and 0.08 ml of ceric reagent. This reaction mixture is then measured by means of the apparatus described. Calibration of t h e Digital Calculator. A calibration procedure is necessary to determine k ’ l and k ‘ z . For this purpose, the switches for k ’ l are set to 00.0 and for k ‘ 2 to 1.00. After a blank reaction, (10)
722
the numerical indicator will show a number equal to the desired constant k ’ l . Setting switch k’l to this value, the read-out will show 00.0. This setting may be checked by running several blanks. The measurement of a standard sample will give the value for k ‘ 2 . Switch k ’ z is to be adjusted so that the read-out indicates the concentration of the standard. The latter step should also be repeated.
G.Kessler and V . J. Pileggi, Ciin. Chem., 16, 382 (1970) ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1974
AC = B, - Bo where Bc is the test value of a reaction with catalyst, corresponding to the term (d (In A ) ) / d t in Equation 4 and Bo is the test value of a reaction without catalyst, corresponding to k f l in Equation 4, then we can show that the sensitivity increases with the reaction temperature (Table I) as well as with the reaction time. However, attention has to be paid to the fact that the reaction time and temperature must not be increased indefinitely, as otherwise the reaction will largely take place even without catalyst, (11) C. H. Bowden and N. F. Maclagan, Biochem. J . , 56, VI1 (1954). (12) C. H . Bowden, N. F. Maclagan, and J. H . Wilkinson, Biochem. J . , 59, 93 (1955). (13) C . H. Bowden, N. F. Maclagan. and J. H . Wilkinson, Biochem. J . , 67, 5 (1957). (14) G . Morreale de Escobar and E. Gutierrez Rios, Clin. Chim. Acta, 3, 548 (1958). (15) K . Muller, H. Skrube, and H . Spitzy, Mikrochim. Acta, 1962, 1081.
Table 11. M e a s u r e m e n t of B l a n k Tests (Bo) and T3Solutions (6 n g of T3) (B,) a t Various Temperatures5 Temperature of Adjusted measuring reaction, O C period, See
36 32 28 24 20 16 12
23.5 40 .O 63.5
108 .o 180 .O 360 .O 540 .O
Table 111. Comparison of Ta and T4 D e t e r m i n a t i o n s at Various Reaction T e m p e r a t u r e s and Measuring Periods
Bo (Digit)
B, (Digit)
27 27 27 27 27 27 27
43 54 62 76 90 126 135
Temp. of reaction Measuring period
20
160 sec
1.10 0.55 0.22 0.66 0.33 0.13
T3
T4
The measuring periods were chomn so that Bo is kept constant. a
and thus a major portion of the photometric measuring range will be lost. It is, therefore, advantageous to vary t h e parameters of time and temperature so that Bo will be kept a low constant. The temperature dependence of the reaction rate may be expressed by the Arrhenius equation:
(6) where k is the reaction rate constant and slope of the In A R is the general gas constant, E is the activation energy, and T is the absolute reaction temperature. Through logarithmic operation, we obtain the equation
us. time plot, respectively, k o is a constant,
In k = In k ,
E l - -
RT
(7)
Figure 5 illustrates the relation of catalytic reactions a t various temperatures, thus demonstrating the influence of varying the chloride and hormone concentrations. The slope of the curve is proportional to the activation energy E. This shows that with increased concentration of thyroid hormones or iodine, the activation energy of the redox reaction is reduced. An increased chloride ion concentration, on the other hand, has no effect on the acti,vation energy, even though it will result in a higher reaction rate. Figure 5 clearly demonstrates that the relative difference in reaction rates P u r between reactions with and without catalyst (e.g. thyroid hormones) increases with fa1ling temperature.
== SD,N
oc
=
f 0 . 1 6 0 ngn =k 0.096 ng f 0.088 ng =k 0 . 0 5 1 ng 0 . 0 4 3 ng f 0 . 0 3 3 ng
+
32
o c
40 sec
1.10 i-0.198ng 0 . 5 5 + 0 . 2 0 5 ng
0 . 6 6 i 0 . 1 1 2 ng 0 . 3 3 f 0.108ng
10
Table I shows the test values Bo and Bc (amount of catalyst: 6 ng T3) after a measuring period of 40 sec, as well as the relative differences of the reaction rate for variations of the temperature of reaction. As we have mentioned above, for reasons of accurate measurement, it will be advantageous to always use the photometric measuring range as much as possible. To that effect, the measuring period for any reaction temperature is chosen so t h a t Bo will be kept constant. Table I1 shows the adjusted measuring periods for Bo = 27 as well as corresponding B, values for 6 ng of Ts. Table I11 shows the results of Ta and Tq determinations a t 32 "C and 40 sec and at 20 "C and 160 sec within the range of the detection limits. In practice, one will choose according to purpose between a brief measuring period or a lower detection limit. When determining samples of widely differing thyroid hormone content, no memory effect may occur in the sampler system. For this reason, the very generous rinsing time of 40 sec was chosen, so that even samples with a 104 fold hormone surplus will not have any effect on the next measurement.
ACKNOWLEDGMENT The authors thank Ch. Jorde for the layout and testing of the numerous printed circuit boards which were necessary for the construction of the instrument. Received for review July 23, 1973. Accepted November 15, 1973.
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