Differential Amperometric Determination of Alcohol in Blood or Urine Using Alcohol Dehydrogenase Marllyn Dix Smlth and Carter L. Olson College of Pharmacy, The Ohio State University, Columbus, OH 43210
Alcohol concentration In whole human blood and urine was measured by enzymatic differential amperometry at tubular carbon electrodes In flowing streams using alcohol dehydrogenase as the enzyme catalyst. 2,6-Dlchlorophenollndopheno1 was used as the redox mediator and electroactive species. The differential mediated method eliminates normal interferences found In blologlcal fluids. Measurements were made on 10-20 HI of sample. The instrumentation Is very simple and Inexpensive. The method yields reproducible results which are in excellent agreement with results obtained using the standard dichromate oxidation method and the alcohol dehydrogenase spectrophotometric method of determining blood or urine alcohol.
The determination of alcohol is probably the most routine and requested test performed by forensic laboratories. Various studies have supported the conclusion that “alcohol has been found to be the largest single factor leading to fatal crashes” ( I ) . The police and judiciary, as well as the public, are greatly interested in the chemical test for alcohol in the blood, breath, or urine of a person who is thought to be inebriated. In 1965, the National Safety Council Committee on Alcohol and Drugs recommended that the presumptive limit for alcoholic impairment be lowered to 0.10% w/v ( 2 ) .As the presumptive level had decreased, the need for improved analytical accuracy has increased. Three techniques are presently used for the analysis of alcohol. They are gas chromatography, dichromate oxidation, and enzymatic oxidation. A new method for the enzymatic determination of alcohol in blood (or urine) is the subject of this paper. The enzymatic oxidation is followed amperometrically. Instead of using a spectrophotometer to measure absorbance change due to NADH concentration changes, the change in current a t a constant applied potential of a coupled enzymatic reaction is measured a t tubular carbon electrodes (TCE). The reaction and measurement sequence is as follows: CH,CHzOH NADH
+
+
NAD’
ADH
DCPIP(oxidized)
NADH
+
CH,CHO
(1)
diaphorase
(fast)
NAD’
+
DCPIP(reduced) (2)
Amperometric measurement ( E =- 0.070 V vs.sec.)
DCPIP (reduced)
* DCPIP (oxidized) (3)
In the presence of nicotinamide adenine dinucleotide (NAD+) and alcohol dehydrogenase (ADH), ethanol is oxidized to acetaldehyde. In this enzymatic oxidation, NAD+ is reduced to NADH. The NADH produced is coupled with the dye, 2,6-dichlorophenolindophenol(DCPIP), using the enzyme, diaphorase, to speed the reaction. When reaction 2 is sufficiently fast, the enzymatic oxidation of ethanol is the rate-determing step and the rate of reduction of the dye will be proportional to the concentration of alcohol in the sample. 1074
ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
EXPERIMENTAL Differential Measuring Apparatus. The instrumentation used in this determination of blood or urine alcohol was the same apparatus used in the differential determination of serum lactate dehydrogenase (3).The electrode cell consists of two tubular carbon electrodes connected via a salt bridge to a common saturated calomel electrode. The electrodes were the same tubular carbon electrodes used in the assay of lactate dehydrogenase. The bridge circuit for measuring differential current was the same as that used in the previous differential amperometric measurements (35 ) . The bridge circuit measures the difference in current passing through the electrodes when a fixed potential was applied. The bridge resistances were both fixed a t 10 kohms. All measurements were made directly with a high impedance recorder (Leeds & Northrup Model L 680). Currents were normally measured using a 1or 2-mV full scale recorder range. The recorder was damped by 50 p F capacitor. A push flow system was used as previously described (3).The delay line as well as the tubing from the mixer to the first TCE were enclosed in a water jacket maintained a t 25.0 0.1 “C. Reagents. All solutions were made with double distilled water. All pH 8.8 buffer solutions contained 0.1M KC1 and 0.1 sodium pyrophosphate adjusted with concentrated HC1. All pH 7 . 3 buffer solutions contained 0.1M KCl and 0.1M sodium dibasic phosphate adjusted with HC1. Reagent solutions contained 0.01% gelatin since gelatin stabilizes alcohol dehydrogenase in solution ( 6 ) . 2,6-Dichlorophenolindophenol(DCPIP) was selected as the mediator for NADH coupled reaction because it reacts rapidly with NADH in the presence of diaphorase and the reduced form of the dye can be oxidized at a relatively low anodic voltage. Also, the oxidized form of the dye is stable in solution a t p H 8.8 when stored a t 0-5 OC. At a potential of +70 mV (vs. SCE), the ratio of the faradaic current due to the oxidation of reduced DCPIP to the residual current due to the oxidation of endogenous amines and impurities in the reagent is maximized. At this potential, the current is linear with reduced dye Concentration. Sodium 2,6-dichlorophenolindophenol obtained from Eastman Organic Chemicals was used. The dye was dissolved in buffer solution pH 8.8 and filtered. The filtrate was evaporated to dryness. A quantity of water equal to the initial solution volume was added to the dry dye-buffer crystals. The concentration of this stock s o h tion of dye was determined spectrophotometrically using the molar absorptivity reported to be of 19,000 a t 600 nm (7). This stock solution was stable for at least a month when stored a t 0-5 “C. Diaphorase and NAD+ were obtained from Worthington Biochemical Company. Alcohol dehydrogenase was obtained from Sigma Chemical Company, Stock No. A-7011. Both absolute and 95% ethanol were used in preparing the alcohol standards. All chemicals were reagent grade and were used without further purification. Human blood and urine samples were obtained from the Columbus Police Department and Ohio State Patrol Crime Laboratory. In the test samples, the reagent solution contained 2.78 mM NAD+, 100 ppm diaphorase, and 60 p M DCPIP. The concentration of ADH solid protein in the reagent solution was varied from 3.24 to 8.44 ppm for each set of samples. The resulting reaction rates varied from 10 to 40 nA/ppm ethanol. Obviously, the greater the concentration of ADH, the more sensitive the method was. However, the reaction must not proceed so fast that the diaphorase coupled reaction becomes rate-determining. Procedure. The reagent was prepared as follows: NAD’, diaphorase, ADH, and dye were added to pH 8.8 buffer and allowed to set for 12 hours at 0-5 OC. The purpose of this “aging” is to allow time for the reduced dye present in the reagent to be reoxidized by oxygen in the solution. The presence of reduced dye was caused by trace amounts of ethanol in ADH, ethanol and reduced dye in the DCPIP, and NADH (approximately 1%)in NAD+. Bubbling oxygen through the reagent hastened the process, but the ag-
+
itation and foaming were undesirable. ADH substantially free of ethanol from horse liver was also used, but it yielded no real advantage over the less expensive crystallized and lyophilized ADH from yeast. Immediately before running samples, approximately 3 ppm of additional ADH crystals were added to the reagent solution. The pumping rate was adjusted so that the time between electrodes was 60 seconds. The exact differential time was determined by observing the time it took for a bubble to go from one electrode to the other. Concentration-activity curves of each component were determined by varying the amount of NAD+, diaphorase, dye, or ethanol in the reagent and pumping it into the reagent line. Since concentrated solutions of ethanol were used in the reagent except in the ethanol study, the “aging” of the reagent due to trace contaminants was unnecessary in these studies. These concentration-activity curves were run a t pH 8.8 and 25.0 O C . The ADH solutions were prepared from a stock solution of ADH crystals dissolved in pH 7.3 buffer and stored a t 1 O C . The stock solution was then further diluted (1:lOOO) with pH 8.8 buffer and that solution was kept in ice as the experiment proceeded. A potential of +70 mV vs. saturated calomel electrode (SCE) was applied to each electrode. Initially, a buffer solution was pumped into the reagent line and buffer into the sample line. This establishes a base-line differential current. Next, the electrodes were calibrated. Four known ethanol standards were pumped through the sample line. A known concentration of alcohol was run as a standard between groups of ten unknown samples. After all samples were analyzed, the electrodes were again calibrated with four known concentrations of ethanol. The electrodes were then flushed with buffer. When the system was not in use, buffer was left in the electrode and a +70 mV potential was applied. With this procedure, the electrode was ready for use even if several days had elapsed since the last run. Since the ADH enzyme method is so sensitive to alcohol, very dilute solutions of samples of urine or blood were required. Ten or 20 fi1 was a sufficient sample volume size. The limiting factor in the accuracy of this method was the sampling technique and equipment. A Gilson Pipetman pipet and a Lab Industry Diluter were used in all correlation experiments. The electrodes were cleaned about twice a month according to the following procedure. With no potential applied to electrodes, Clorox was pumped into both reagent and sample line for 5 minutes. Then distilled water was pumped through the lines for 1 hour. A potential of +70 mV (vs. SCE) was applied to the electrodes, and buffer solution was pumped through the lines for 15 minutes. This potential was applied for a t least 6 hours prior to using the electrodes to allow time for the residual current to fall below 10 nA. In the following studies, ethanol was given in ppm. One ppm is equivalent to 0.165mM and 7.87 X 10-5 % w/v ethanol assuming 95% ethanol has a density of 0.787 a t 23 “C (8).T o determine the amount of ethanol present in wt % in each sample, the differential amperometric current for that sample is divided by the average calibration slope for that set of 10 samples, the dilution factor, and 1/7.87 X The dilution factor was predetermined by dividing the weight of liquid expelled from the pipet by the sum of the weights of diluent and pipet liquid. This factor was reproducible with different samples of blood or urine.
RESULTS AND DISCUSSION Although initial reaction velocity with small amounts of ethanol has its maximum velocity a t pH 8.2 ( 9 ) ,most alcohol enzymatic assays are run a t pH 8.8 (10, 11). Therefore, for this comparative study, pH 8.8 was used. At pH 8.8, any reduced DCPIP present in the reagent can be oxidized slowly by oxygen. This is important since reduced dye initially present in the reagent adds to the background current. This reoxidation is much slower a t lower pH’s. Also a t pH 8.8, there is no potentiation of the rate of ADH reaction with whole blood samples, At pH 7.3, where the ADH enzyme is more stable (12), whole blood samples give potentiated rates of ADH reaction. A concentration-activity curve of each component was determined. The final enzyme dilution is given with each reagent concentration study. All concentrations specified in the following figures are those concentrations before
i
t , # # . , . , . . , I 260 2
DCPIP l v M l 0 ETHANOL IM)
0
Figure 1. Rate dependence of 2,6-dichlorophenol indophenol and
ethanol reagent concentrations .W
,
,
,
,
,
,
,
,
,
Figure 2. Rate dependence of diaphorase and NAD’ reagent con-
centrations stream splitting. Generally, the splitting factor was 1:l. The differential amperometric response was linear with respect to ADH dilution. Using 52.5 ppb of ADH in the sample line, the ethanol concentration-activity curve was determined as shown in Figure 1. The other component concentrations are: NAD+, 1.39mM; diaphorase, 50 ppm: DCPIP, 0.081mM. Several of the reagent components precipitate out of solution when the ethanol concentration in the reagent solution was above 0.5M. The concentration-activity curve for NAD+ was determined as shown in Figure 2. A 52.5 ppb ADH solution was used in the sample line. The concentrations of diaphorase and DCPIP in the reagent lines were the same as in the ethanol study. Ethanol concentration in the reagent line is 0.5M. The diaphorase concentration-activity curve was similarly determined as shown in Figure 2. Again 52.5 ppb ADH solution was used in the sample line. NAD+, DCPIP, and ethanol concentrations were the same as in the previous studies. The 2,6-dichlorophenolindophenolconcentration-activity curve was also determined as shown in Figure 1. The ADH solution in the sample line was 52.5 ppb. NAD+, ethanol, and diaphorase were 1.39mM, 0.5M, 50 ppm, respectively. Maximum reagent concentrations were used for diaphorase (100 ppm) and DCPIP (60pM). However, submaximal amounts of NAD+ (2.8mM) were used to limit the rate of the alcohol dehydrogenase reaction. Arrows indicate the actual concentration of each reagent used in the correlation studies. The decrease in maximum activity from NAD+ curve to DCPIP curve was due to the decrease in activity of the ADH stock solution with time. Although the same dilution of ADH was used in each study, the stock solution was losing activity with time. ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
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Table I. Comparison of Amperometric Method to Spectrophotometric Enzymatic Method and Dichromate Oxidation Method
I
Test
U
NO.
C.C.
1 2 3 4 5 6
0.99 0.98 0.99 0.99 0.99 1.00
0
Slope
Intercept
0.95 1.03 1.00 0.98 1.04 1.03
0.001 -0.006 0.017 0.004 - 0.002 -0.002
No. of samples
21 24 11
20
Type sample
Ub
U U U U,BC u, B
12 34 Correlation coefficient. * Human urine. Whole human blood.
Table 11. Specificity of the Enzymatic Amperometric Method Alcohol
Typical differential amperometric data for human whole blood and urine containing alcohol
Figure 3.
0.6
Ethanol Methanol iz -Propanol Isopropyl alcohol 12
-Butanol
77-Pentanol Propylene glycol
0.2
0.3
% ETHANOL IENZYMATIC SPECTROPHOTOMETRIC
0..
METHOD)
Correlation between amperometric and spectrophotometric assays of human whole blood and urine containing alcohol
Figure 4.
As previously mentioned, the analysis for ethanol was more difficult than for the enzyme ADH since trace amounts of contaminants were in the reagent components. T o obtain a stable base line, it is important that the background current a t each electrode from the reagent alone be kept as low as possible. I t was typically less than 10 nA when the standard procedure of adding a known amount of ADH, typically 3 ppm to the “aged” reagent was used. This method required calibration with known ethanol concentrations before, during, and after a series of samples were run. The constant calibration was to determine the exact activity of the ADH enzyme present in the reagent with time. A typical calibration curve gives a straight line. The rate of loss of activity of ADH was linear with time and typically amounted to about 2.3%per hour. The rate is proportional to the concentration of enzyme present. Additions of urine or serum containing no alcohol to known dilutions of alcohol gave the same slope as known dilutions of alcohol without urine or serum a t pH 8.8. Urine and blood samples obtained from the Columbus Crime Laboratory were analyzed using the differential amperometric technique. The final flow stream concentration was a 1:lOOO dilution of urine or blood (10.0 p1 of sample diluted to 10.0 ml). At the Columbus Police Department Crime Laboratory, blood or urine alcohol was determined by the dichromate oxidation procedure. This procedure consists of distilling a known quantity of sample, oxidizing the distillate, and observing the color change of the chromic acid in a photometer a t 600 mp (13).A sample of the 1076
ANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
Relative spec~iicity
100
0 10.4 0.22 1.5 0.22 0
experimental data for the differential amperometric method is shown in Figure 3. Similar to the LDH study ( 5 ) , about 4 minutes were required for a new sample to clear through both electrodes and reach a steady-state value. Spikes in the data were caused by bubbles passing through the cell. Bubbles were used to separate the samples. Noise on the steady-state plateaus were less than 0.5% full scale. A correlation of the differential amperometric data to the Columbus Crime Lab data is given in Table I (tests number 1 through 4). Blood and urine samples obtained from the Ohio State Patrol Crime Laboratory were also analyzed using the differential amperometric method. The Ohio State Patrol Crime Laboratory used the standard enzymatic method for the determination of urine or blood alcohol using the Sigma premeasured reagent kit. This enzymatic oxidation of ethanol also used alcohol dehydrogenase. The increase in absorbance due to NADH production was followed spectrophotometrically a t 340 nm. Since this is an equilibrium reaction, semicarbizide which reacts with acetaldehyde was added to “pull” the reaction to NADH formation (12). A correlation of the differential amperometric data to the Ohio State Patrol Crime Lab data is given in Table I (tests number 5 and 6). The correlation of test number 6 is also graphically given in Figure 4. The specificity of this differential amperometric assay for ethanol was determined. The same conditions and reagent concentrations were used as in a normal urine or blood sample run a t pH 8.8. The results are given in Table 11. The method using initial reaction rates as described in this paper shows less interferences from other alcohols than the equilibrium enzyme methods (14) where the slower reacting alcohols have more time to react.
CONCLUSIONS The differential amperometric method provides an inexpensive, easily automated method for measuring urine or whole blood alcohol concentrations. The method had the additional advantage of cycling NADH produced which prevents the reaction from coming to equilibrium. The reagent can be made several days prior to use since the diaphorase, NAD+, and DCPIP are stable in solution a t 0-5
"C for up to 1 month. In the review article on alcohol testing (Z),Mason and Dubowski stated that impairment of the nervous system correlates best with arterial concentration of alcohol. They also suggested that arterial blood be obtained from a deep cut in the finger tip, which is 65-85% arterial blood in composition. Because of the small sample requirements, this technique could be conveniently used with the differential amperometric enzymatic method. The differential amperometric method has high sensitivity, is specific for ethanol, and can be successfully applied to routine analysis for samples of 10 t o 20 pl.
ACKNOWLEDGMENT We are grateful for the support and cooperation of the Columbus Police Department Crime Laboratory Division and the Ohio State Patrol Crime Laboratory Division in providing whole blood and urine samples.
M. F. Mason and K. M.Dubowski, Clin. Chem., 20,26 (1974). M. D. Smith and C. L. Olson, Anal. Chem., 46, 1544 (1974). W. D. Mason and C. L. Olson, Anal. Chem., 42,488 (1970). W. J. Blaedel and C. L. Olson, Anal. Chem., 36, 343 (1964). E. Racker, J. Biol. Chem., 184, 313 (1950). R . E. Basford and F. M. Huennekens. J. Am. Chem. SOC., 77, 3873 (1955). "Handbook of Chemistry and Physics", 45th ed., Chemical Rubber Co., Cleveland, OH, 1964, p F-3. R. K. Bonnichsen and H. Theorell, Scand. J. Clin. Lab. Invest., 3, 58 (1951). Worthington Enzyme Manual, Worthington Biochemical Corp.. Freehold, NJ (1972). Sigma Chemical Company: assay procedure for alcohol dehydrogenase. S. Takemori, E. Furuya, H. Suzuki, and M. Katagiri, Nature (London), 215, 417 (1967). J. Bauer, W. Bray, Tore, and Ackermann, "Bray's Clinical Laboratory Methods", 6th ed., 1957, p 511. Sigma Technical Bulletin No. 330-UV, Sigma Chemical Company, St. Louis, MO.
LITERATURE CITED
RECEIVEDfor review October 9, 1974. Accepted February
(1) Alcohol and Highway Safety A report to the Congress from the Secretary of Transportation, 1968. p 8.
18, 1975. This study was supported in part by the National Institutes of Health Grant GM-15821.
Bromination of Nucleotides: Coulometric Determination of Cytosine, Thymine, and Uracil Compounds James E. O'Reilly Department of Chemistry, University of Kentucky, Lexington, KY 40506
The technique of coulometric bromination at constant current with biamperometric end-point detection has been applied to the quantitative analysis of several biologically important pyrimidine compounds-cytosine, thymine, and uracil-and their nucleosides and nucleotides. Sub-micromole quantities of these compounds can be determined with precisions on the order of 0.3 to 1 % relative standard deviation and accuracies, relative to spectrophotometric values, within f 2 %. The pyrimidine nucleotides can be determined, with only minor or negligible error, in the presence of large (>lOO-fold) excess of adenine or its nucleosides and nucleotides. The effects of varying experimental conditlonspH, generating current magnitude, etc.-are discussed. The coulometric method is compared with the usual UV spectrophotometric assay of nucleotide compounds.
During the course of a study involving metal-ion binding to nucleotides, it became apparent that there was a need for a more precise and accurate method for the analytical determination of biological purines, pyrimidines, and their nucleosides and nucleotides. Presently, the most popular method for nucleotide assay appears to be UV spectrophotometry, employing various spectral reference values (1-3). While this method is relatively rapid and sensitive, the accuracy and precision attainable are rather modest (&2 t o 4%). It appeared that a coulometric method could fulfill the desired analytical objectives; in particular, coulometric titration with electrogenerated bromine appeared to be suitable, since bromine is known to react rapidly and quantita-
tively with organic compounds not unlike purines and pyrimidines ( 4 ) . This report presents the results of the application of the coulometric titration technique to the analytical determination of the various sub-components of nucleic acids: purines, pyrimidines, nucleosides, and nucleotides. Perhaps the primary advantage of coulometric titration is the excellent precision and accuracy possible under appropriate conditions. Accuracies and precisions better than 0.5% are fairly common (4-7) and values of 0.01% or better are obtainable with care (8). Other advantages of coulometric titrimetry are that it involves only the fundamental quantities current and time (or, the coulomb); thus, it is a relatively "absolute" method and free from the uncertainties associated with the use of standard substances. Further background information on amperometric and coulometric titrations is available in the monographs by Stock ( 4 ) and Lingane ( 5 ) and in the biennial reviews now authored by Stock (6) and by Davis (7).
EXPERIMENTAL Apparatus. T h e constant current source used was a ChrisFeld Microcoulometric Quantalyzer M o d e l 6. T h e biamperometric endp o i n t detection system was constructed f r o m common electronic components a n d was similar in construction t o others t h a t have been described (9, 10). T h e p o t e n t i a l applied t o t h e indicator electrodes was m o n i t o r e d w i t h a H e a t h EUW-24 VTVM; a Simpson M o d e l 374 microammeter m o n i t o r e d t h e c u r r e n t t h r o u g h the indicator electrodes. W h e n indicator current vs. t i m e plots were desired, a K e i t h l e y M o d e l 16 D i g i t a l M u l t i m e t e r was used in conj u n c t i o n w i t h a H e a t h E U - 2 0 5 stripchart recorder. T h e coulometric t i t r a t i o n cell employed was similar in design t o t h a t of Evans (10). I t was necessary t o isolate t h e generating cathANALYTICAL CHEMISTRY, VOL. 47, NO. 7, JUNE 1975
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