Table I11 indicate t h a t t h e blank fluorescence (measured against distilled water) increased b y 5 scale units in 20 minutes a n d t h e sample fluorescence (measured against t h e blank) decreased b y 4 units i n t h e same period. Because t h e fluorometer was zeroed against the blank before each measurement, the apparent change in sample fluorescence could be attributed to changes in the fluorescence of the blank. A new blank was prepared after 20 minutes and measuring the sample relative to i t gave the same fluorescence reading that had been observed initially. Effect of Order of Reagent Addition. T h e order of reagent addition turned o u t t o be critical when using propylene glycol as a solvent for Calcein W. It was necessary t o a d d t h e Calcein W solution t o t h e K O H before diluting with water. If this procedure was not followed, fluorescence of t h e blank appeared t o be erratic. S o such effect was observed on sample fluorescence. T h e order of reagent addition was unimportant when aqueous solutions of Calcein JJ’ were used. Effect of Varying KOH Concentration. Varying t h e a m o u n t of K O H used in a sample produced significant changes in fluorescence intensity as shown in Figure 1. T h e shape of t h e curve is quite interesting, showing a distinct minimum in t h e vicinity of 2.0 ml. of 2 N K O H . T h e decreasing fluorescence between 0 and 2 ml. of K O H can be attributed t o decreasing
plexes, or t h a t fluorescence was being observed by reaction of calcium with the different isomers contained in the commercial preparation of Calcein. Therefore, it is essential to stress that Calcein concentration is critical, and the reagent should be added quaiititat,ively from a 1.00-ml. pipet. O
I 0
, ,
,
2
3
,
,
4
“ILLIIITfSI
8
or
, 6
,
7
,
e
,
8
,
10
ACKNOWLEDGMENT
I,
~ O T L ( S * I U Y *,DlO,,DT
Figure 1. Effect of KOH concentration on fluorescence of sample Calcium concentration 2.00 p g . / 2 5 ml.
fluorescence of t h e reagent itself as t h e solution was made increasingly alkaline. Beyond 2 ml. of KOH, the chelate fluorescence developed to its maximum value, causing a n increase in fluorescence, and a region of the curve where fluorescence is relatively independent of base concentration. Although it is this region that was selected for the analysis, the base used was added quantitatively from a 5.00-ml. pipet. Effect of Varying Calcein Concentration. Varying t h e concentrations of Calcein, while holding all other concentrations constant, produced a plot of fluorescence us. Calcein concentration t h a t showed two maxima which were reproducible between different runs. Although the interpretation of this behavior is difficult, i t appears either t h a t t h e calcium and Calcein mere forming several com-
The authors acknowledge the cooperation of the clinical laboratories of the J. C. Blair Hospital, Huntingdon, Pa., and the hltoona Hospital, Altoona, Pa. LITERATURE CITED
(1) Clark, E. P., Collip, J. B., J. Biol. Chem. 6 3 , 461 (1925). ( 2 ) Kirkbright, G. F., Stephen, IV. I., Anal. Cham. Ada 27, 294 (1962). (3) Phillips, Robert E., G. K. Turner
rlssociates, Palo Alto, Calif., private communication, 1963. (4) Roxe, J. H., Kahn, B. S., J . Biol. Chem. 81, 1 (1929). (5) Wallach, D., Ilelano, E., Soderling, J., Surgenor, D., ANAL. CHEM. 31, 456 (1959). (6) Welcher, F. J. “The ilnalytical Uses of Ethylenediaminetetraacetic Acid,” pp. 124-8, Van Nostrand, Princeton,
N. J., 1957. RECEIVEDfor review October 16, 1962. Accepted May 20, 1963. This research was supported under the National Science Foundation Undergraduate Science Education Program, grant KO. h73F-G21698. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1963.
Automatic Amperometric Measurement of Reaction Rates Enzymatic Determination of Glucose in Serum, Plasma, a n d Whole Blood HARRY
1.
PARDUE
Department of Chemistry, Purdue University, West Lafayette, Ind.
b An amperometric method for the continuous measurement of reaction rates is described. The method is applied to the determination of glucose in serum, plasma, and whole blood. Hydrogen peroxide produced by the enzymatic oxidation of glucose reacts rapidly with iodide in the presence of molybdate catalyst to produce iodine. The rate of increase in iodine concentration is measured using a polarized rotating platinum electrode. Rate measurements are completed near zero reaction time where the rate of formation of iodine 1240
ANALYTICAL CHEMISTRY
is proportional to glucose concentration. Glucose is determined in 0.02 ml. of blood sample with measurement times ranging from 10 to 100 seconds. Recoveries of 50 mg. of glucose added per 100 ml. of samde ranae from 95 to 104% with an ave;age of
100%.
G
osrD.qv cstalyzes the oxidation of glucose to gluconic acid by oxygen Hydrogen peroxide ~ ~ r o d u c cby ~ l t l i i i retlctiori is c:i.sily tlctected and serves as a convenient means of following the course of the reaction. 1,wnqR
Sunderman and Sunderman (6) have given a comprehensive listing of papers basedon this reaction. The methodmost commonly used to detect hydrogen peroside is to react i t with an organic dye in the presence of horseradish peroxidase. The colored product is measured spect,rophotometrically Several methods for glucose have been described in which the amount of colored product is me3:isured after the reaction has approached completion or has been stopped after a ~ircdc.trr~r~~ir~ect res[-tion tirile. Tliaht: procedures provide good selectivity but are tedious and time-consuming and
the peroxidase enzyme is expensive. Also fluoride used as a preservative in blood samples interferes with the peroxidase-catalyzed oxidation of the organic dye by peroqide. Procedures employing automatic. equipment to measure the rate of for.mation of colored product near zero rextion time have greatly simplified procedures and have reduced the total measurement time but still employ the peroxidase enzyme (1,s). The authors mentioned above have developed a method in which the hydrogen peroxide oxid zes methanol to formaldehyde in the presence of catalase (6). The amount of f'irmaldehyde produced during a 1-hour reaction time is determined by its color reaction v i t h chromotropic acid. A41though the method eliminates the need for the peroxidase enzyme, the procedure is tedious and time-conxming. The reaction sequence is not readily adaptable for continuous measurement and automation would be difficult. Kecently Malmstadc and Pardue deecribed a reaction schl?me in which the hydrogen peroxide produced by the oxidation of glucose cxidizes iodide t o iodine in the presenve of molybdate catalyst (4). l h i s scheme was used for the development of automatic potentiometric (5) and specti~ophotometric( 2 ) procedures for glucose in blood serum and plasma based on the detection of iodine formed near zero reaction time. These procedures ha.i.e the combined advantages of the selectivity of the enzyme procedure, a n automated measurement step, short measurement times, and elimination of the horseradish peroxidase. Also the nxthod is free of interference from many anticoagulants and preservatives used in blood. I n this work the same reaction sequence is used but the iodine produced is detected amperometrically. The current between a pair of polarized platinum electrodes immersed in the reaction mixture increases lineiwly with iodine concentration. An automatic measurement device provides direct readout of the time required for the current to increase over a small predetermined interval. T h e reciprccal of readout time is a linear function of glucose concentration being proportional to it when the measurement is completed before a n appreciable fractioii of the glucose has reacted (5). The characteristics of' the new method are similar to those of tl-e potentiometric ( 4 ) and spectrophotometric ( 2 ) methods. It has the advantage of simpler initial setup over both these IT ethods. Unlike the potentiometric method i t does not require n stable reference solution which must be prepared ortce daily. The sensitivity of the aniper(~rnetricnietliod is n h i t the same as tlrc: potentiometric nicthod but sliglitly gimter than the spectrophotometric method.
I
L
L
l
ROTATING ELECTROE-
STATIONARY ELECTRODE
Figure 1 . Experimental setup for the amperometric determination of glucose
The method has been adapted to the determination of glucose in blood, serum, and plasma. Two-tenth milliliter of sample are diluted and deproteinized to give a final volume of 10 ml. Then 1.00 ml. of the deproteinized solution is added rapidly t o the cell containing 1.00 ml. of a composite reagent which contains all catalysts and reactants except glucose. The instrument is activated by closing the start switch momentarily after which the measurement is completed automatically and rate data are read from a dial within a few seconds to 2 minutes depending upon the glucose content of the sample. The relative standard deviation computed on the basis of from three to five results for each series is within 2%. The relative error for the recovery of 50 mg. of glucose added per 100 ml. of sample is within 3%. EXPERIMENTAL
Instrumentation. T h e experimental setup used in this work is shown i n Figure 1. The reaction takes place in a 15-mm. 0.d. test tube partially submerged i n water controlled at 25" i 0.1' C. The electrodes are prepared b y sealing 22 gauge platinum wire into 3-mm. o.d. glass tubing with about l / 4 inch of the wire exposed. I n the case of the rotating electrode, the 3-mm. tubing is sealed onto 6-mm. o.d. tubing which fits the stirring chuck of unit B of the Sargent Model Q Concentration Comparator. Electrical contact is made b y a copper wire dipping into mercury inside the electrode. This electrode is rotated at 2000 r.p.m. and in addition to detecbing the formation of iodine provides rapid and efficient stirring of the sample solution. The polarizing source con&ts of a 1.5-volt 13 battery in series with it 100-ii 1H 1t,rriticirneter. Eleutroly~isc: u r w i t resulting from the simultaneous ositlatjion of iodide at the stationary electrode and
reduction of iodine a t the rotating clectrode is measured by the voltage drop across a precision resistor in series with the polarizing source and electrodes. A 1000-ohm resistor was used in this work. The measurement and control system is similar to that described earlier ( 4 ) consisting of the Model Q Comparator, an auxiliary relay system, and a timer reading to the nearest one-tenth sccond. A single modification of the original relay system is made. The connections between pins B and H of the concentration comparator and relay contacts b and h (Figure 4 of reference 4 ) are broken and the latter are connected to a shielded two-conductor cable for connection across the bias voltage Eb as shown in Figure 1. S o modification of the concentration comparator. is required. The bias voltage source is constructed from a mercury battery, precision resistors, and a ten-turn Helipot t o give a voltage variable betm-een 0 and 10 millivolts. Samples and reagent,s are handled with 1-cc. hypodermic syringes. Ilowever, the sample handling equipment described earlier ( 5 ) could be used. Solutions are removed from the sample tube by a n aspirator tube. Reagents. All solutions are prepared in water which has been passed through a mixed cation-anion exchange resin bed. T h e stability of all solutions containing potassium iodide is dependent upon t h e care with which t h e solutions a r e prepared and m a y vary from one water supply t o another. BUFFER CATALYST. Eighty-two grams of potassium dihydrogen phosphate, 42 grams of potassium monohydrogen phosphate, and 13 grams of ammonium molybdate [ (XSH~)&IO~O~~. 4H201 are dissolved in water and diluted to 1 liter. This solution is stable indefinitely. POTASSIUM IODIDE. Eighty-three grams of potassium iodide are dissolved in water and diluted to 1 liter. This solution is stable for several weeks when stored at 5' C. GLUCOSE OXIDASE. Six-hundredth gram of glucose oxidase (Sigma, Type 11, purified) is dissolved in water and diluted to 50 ml. This solution is stable for 2 weeks when stored at 5" C. GLUCOSESTAKDARDS. Standard glucose solutions are prepared b y dilution of a 1000 p.p.m. solution prepared by dissolving 1.000 gram of C.P. reagent in wat'er and diluting to 1 liter. These standards are stored at 5" C. DEPROTEIKIZINGREAGENTS.Zinc sulfate septahydrate, 2% solut'ion in water. Barium hydroxide ochhydrate, 1.870 solution in water. ,4 portion of the zinc sulfate solution is titrated slowly with the barium hydroxide solution using phenolphthalein as indicator. The appropriate dilution is carried out to make the acid-base strengths of the solutions equivalent. COJIPOSITE REAGENT.Fifty milliliters of composite reagent are prepared by riiixiiig 20 ml. each of t.he I)ufTet,catalyst and pot>assiuniioditlc sol\itioris and 10 ml. of the enzyme solution. VOL. 35, NO. 9 , AUGUST 1963
1241
Table I. Automatic Amperometric Results for Glucose in Serum, Plasma, and Whole Blood
Samplea KO. no. detns. 1 2
Glucose foundb (mg./
4 3 3 4 5 3
3 4 5 6
3 5 3 3 4 3 5 3 3 Samples 1 to 5 10 are oxalated 12 13 14 15 16 17 18 19 20
100
ml.)
Rel. std. Redev., covery,c yo yo
62 85 143 93 87 76
1.3 0.4 2.4 3.3 0.0 1.8
97 181 193 176 57 174 94 151 83 are sera.
1.7 0.7 1.8 0.3 0.7 2.2 1.7 2.4 0.7
102 98 97 104 101 9.5
98 103 102 101 103 98 96 98 101 Samples 6 to
plasma with fluoride added. Samples 11 to 15 are citrated plasma with thymol added. Samples 16 t o 20 are citrated whole blood with thymol added. * Found from a calibration curve of a plot of reciprocal time us. glucose concentration. Typical measured times for standards containing 50, 100, 200, and 300 mg. of glucose per 100 mI. diluted 50fold with water are 86.4, 34.9, 15.7, and 10.0 seconds, respectively. Recovery of 50 mg. of glucose per 100 ml. added t o each sample.
Procedure. PREPARATIONOF EQUIPMENT. Units A and B of t h e concentration comparator are connected as described i n t h e instruction manual for t h e instrument. Plugs P1, P2, a n d P3 of the relay system (Figure 4 of reference 4) are connected t o matching sockets i n t h e back of unit A . T h e timer is connected t o P5 of t h e relay system. Other connections are made as shown in Figure 1. The bias voItage Eb is set at 1.00 mv. and the zero adjust helipot on the concentration comparator is set at 5.50 (1.00 mv. prebias) with the range selector switch set at PNP. The comparator is turned on and the reagent selector switch placed in position 2. The polarization voltage is set at 130 millivolts. DEPROTEINIZATION OF SERUM ASD PLASMA. Two-tenth milliliter of sample is drawn into the tip of a wash out pipet and flushed out with water to a total volume of 8.00 ml. Then 1.00 ml. of barium hydroxide is added and the solution is mixed thoroughly. Then 1.00 ml. of zinc sulfate is added and the mixture is mixed thoroughly. The mixture is centrifuged for 3 minutes and placed in the water bath to reach the analysis temperature. Standards containing 10 and 20 p.p.m.
1242
0
ANAlYLlCAl CHEMISTRY
glucose are adjusted to 25.0' C. These standards represent blood glucose concentrations of 50 and 100 mg. per 100 ml., respectively. MEASUREMENT STEP. The reaction vessel is rinsed once with deionized water. Then 1 milliliter of composite reagent is added followed by 1 milliliter of deproteinized sample. The start switch is closed momentarily and then the rate measurement is completed automatically. After the reaction time is recorded and the timer is reset to zero, the instrument is ready for the next sample. CALCULATIONS. The sample concentration in milligrams of glucose per 100 ml. of serum or plasma is read directly from a curve obtained by plotting reciprocal of readout time us. concentration for the standards. RESULTS AND DISCUSSION
Interferences. T h e selectivity of glucose oxidase for glucose has been repeatedly demonstrated. Sunderman and Sunderman (6) have investigated a wide variety of compounds and found that only d-maltose and ascorbic acid interfere. Maltose interfered by being partially hydrolyzed to glucose during the incubation period inherent in their procedure. This interference is absent in the method described here. Since the present method measures the rate of formation of iodine and is independent of the total amount produced, small amounts of ascorbic acid which use up iodine do not interfere so long as the premeasurement time (4) is not excessively long. Amounts of blood ascorbic acid below about 10 mg. per 100 ml. do not interfere. T o determine effects of other oxidants or reductants not anticipated in deproteinized samples on the iodine-iodide couple, filtrates from several samples were added to reaction mixtures containing all components (including a small amount of iodine) except glucose oxidase. In some cases small amounts of iodine were produced rapidly followed by a gradual decrease in iodine concentration. However, in all cases observed, the amounts of iodine produced and the rate of decrease in iodine were small compared to the amounts and rate 0.' iodine production resulting from the enzymatic oxidation of glucose, and cause no significant interference. I n other experiments, i t was demonstrated that recoveries of glucose added to blood filtrates from which glucose had been removed by incubation with glucose oxidase were complete within
=w&.
Since most glucose oxidase preparations available a t present contain catalase and since catalase could compete with iodide for hydrogen peroxide, the effect of this enzyme on the present method T+as examined. The catalase content of the enzyme preparation used
was reported to be about 2 x 102 units per milligram and was found by spectrophotometric assay to be 2.5 x lo2units per mg. It was demonstrated that sodium azide at a concentration of 2 x 10-31W in the reaction mixture completely inhibits the decomposition of peroxide by catalase added with the glucose oxidase. This amount of azide does not affect the enzymatic oxidation of glucose at the level of enzyme activity used here. Since the results obtained for glucose in many aqueous solutions and in blood filtrates were the same in the presence and absence of azide, it may be concluded that the catalase present in the enzyme preparation used does not compete effectively with iodide for hydrogen peroxide and therefore does not interfere with the method. Experimental Conditions. T h e deproteinization procedure described is a modification of t h e Somogyi method empirically adjusted so t h a t t h e reaction rate for a glucose standard is the same when i t is carried through t h e deproteinization procedure as when i t is diluted to t h e same extent with water. This increases t h e reliability of t h e method and avoids the necessity for carrying t h e standards through the deproteinization step. Under the conditions described, working curves constructed from a plot of glucose concentration us. the reciprocal of reaction time are linear for samples ranging from 25 to 300 mg. of glucose per 100 ml. (100 fold dilution before final measurement). However, as the data under b of Table I show, the plot has a nonzero intercept. This results from the fact that an appreciable fraction of the glucose is used u p during the measurement step (4). This requires that two standards be used to establish the working curve from which unknown concentrations are determined. If the amount of enzyme used in the measurement step is reduced fivefold and the dropping resistor R in Figure 1 is increased fivefold, then the working curve passes through zero. This has the advantage that only one standard is needed for calibration and unknown concentrations can be evaluated from a simple proportionality (4). The disadvantage of this procedure is that the rate of production of iodine by the oxidation of glucose is so low that the interaction of blood filtrates with the iodineiodide system becomes significant. The result is a decrease in reliability of the method. Therefore, the use of a higher reaction rate with a two-point calibration curve is preferred for best results. Analytical Data. Table I shows typical d a t a for the determination of glucose in serum, plasma, and whole blood. The relative standard deviation of each series of results is in t h e range of 2%. Recoveries of 50 mg. of glucose added per 100 ml. of blood
sample range from 95 t o 104% with a n average value of 100 Results b y t h e automatic amperclmetric enzyme method ranged from 8 to 147, lower t h a n results obtained b y the SelsonSomogyi copper reduction method. This is consistent with other observations (5,6). These data were obtsined using one tenth of the filtrate obtained from a t ~ o - t e n t h milliliter sample so t h a t volumetric errors could be kept
low and so that sufficient filtrate would be available for comparison of the new method with the Somogyi method. Since the measurement step involves only 0.02 ml. of original sample, analyses can easily be carried out on as little as 0 04 ml. of blood. LITERATURE CITED
(1) Blaedel, W. J., Hicks, G. P., AhZL. CHEW34, 386 (1962).
( 2 ) Malmstadt, H. V., Hadjiioannou, S.
I.,Ibzd., P. 452. (3) Malmstadt, ET. V., Hicks, G. P., Zbzd., 32, 394 (1960).
(4) ;Clalmstadt, H. V., Pardue, H. L., Ibzd., 33, 1040 (1961).
H. Pardue, L., Clzn. Chem. 8 , 606 (1962). (6) Sunderman, F. W., Jr., Sunderman, F. W., Am. J . Clin. Pathol. 36, 75 (1961). (j)Rfalmstadt,
RECEIVED for review Sovember 16, 1962 Accepted May 24, 1963.
Studies of the Acetylation of Steroids Using 1-Carbon-14-Acetic Anhydride OSCAR V. DOMINGUEiZ,' J. RODMAN SEELY,? and JACK GORSKI3 Department o f Biologiccrl Chemistry, University of Utah College of Medicine, Salt lake City, Utah
b The acetylation reaction has proved useful in isotope derivative methods for the determination of steroids. This report deals with a study of the general application of the acetylation reaction to steroids, using 1 -C14-acetic anhydride. The data indicate that the composition (ratio of acetic anhydride to pyridine) of the acetylating reagent is of importance in determining the course of the reaction from the standpoint of products and completeness of reaction. There i s an appreciable difference in the rlEactivity of the various acetylable groupings of the steroids. The relative 'order of reactivity appears to be: 3-phenol 21-OH 3P-OH :> 6P-OH
> 20a-OH >
20P-OH
>
:>
16a-OH
> > >
secondary 17P-OH secondary 17aOH. The concentration of steroid over the range 0.6 to 100 jig. per 0.1 ml. of acetylating reagent does not seem to influence the relative degree of acetylation. A ratio of 1 :5 acetic anhydride: pyridine appears to be best suited for applicability to a wide variety of steroids to achieve a single product and complete reaction.
steroids with multiple acetylable groups requires knowledge of the relative ease with which hydroxyl groups at the various positions will be acetylated by a commonly used reagent such as a mixture of acetic anhydride and pyridine. h knowledge of the relative acetylability of the various hydroxyl groups in different concentrations of pyridine can be useful in two ways. Optimal conditions can be selected for the complete acetylation of a given compound in developing a quantitative microanalytical method. On the other hand, conditions can be chosen which will lead to partial acetylation of hydroxyl groups in certain positions; the acetates formed from a n unknown steroid will then give a clue to the number of hydroxyls present and their probable location. This paper reports experiments using Ac20-1-C14 for the acetylation of steroids aimed at determining which groups on the steroid molecule are acetylable using varied compositions of acetylating reagent, the relative reactivity of the various acetylable groups, and the condi-
Table
T
of endogenous steroids in extractti of biological material by radioactive acetic anhydride ( - 4 ~ ~ 0has ) already been used for the microestimation of certain steroids (1, 3, 10, 12-14), but the general application to HE
Present address, Am3rican Medical Center, Denver, Colo. Postdoctoral Fellow of the Helen Hay Whitney Foundation at the time of these studies. Present address, Department of Pediatrics, University c d Washington School of Medicine, Seattle, Wash. 3 Present address, Department of Physiology, University of Illinois, Urbana, Ill. 1
1.
AcmnxrIoPi
Exp. no.
Soh. no.
I
1-B
EXPERIMENTAL
Materials and Methods. STEROIDS. Steroids having various combinations of hydroxyl groups were selected. Crystalline samples of t h e steroids \\-ere tested for purity b y chromatography on paper, sulfuric acid spectra, and color tests (6,6, 18). ACETIC ~ N H Y D R I D E - ~ -One C ~ ~milli. curie of X c ~ 0 - l - C ~(4.6 ~ mc./mmole), obtained commercially, was diluted t o 4 ml. with nonradioactive vacuum redistilled ACZOand stored in a desiccator over calcium chloride. These experiments were carried out using t h e same lot of ~ c ~ 0 - 1 - ~ 1 4 . PYRIDINE. Vacuum redistilled pyridine was stored in a desiccator over calcium chloride.
Ratio of Acetic Anhydride to Pyridine and Volumes Used for Acetylation of Steroids in Experiments 1 -A and 1-B
11 1-A
tions which will give complete acetylation and a single product from a single compound and be generally applicable. The results here reported have already been used by West, Damast, and Pearson (16): Gorski and E r b (8),and W e s t (17 ) to identify steroids by selective acetylation.
I11 IV V I I1 I11
IV V
Ratio Ac?O!pyridine 1:l 1:3 1:7 1:15 1:31 1:l 1:3 1:7 1:15 1:31
Ac,O, ml. 0.4 0.4 0.4 0.4 0.4
0.4 0.2 0.1 0.05
0.025
Volume pyridine. ml. 0.4 1.2
2.8
6.0
12.4 0.4 0.6 0.7 0.i5
0.776
Volume per samnlc Total used for ketyla&n mixture, Mixture, Acto ml . X present, X 0.8 1.6
3.2
6.4 12.8 0.8 0.8 0.8 0.8 0.8
50 100 200 400 800 50 50 50 50 50
VOL. 35, NO. 9, A U G U S T 1963
25
25 25 25
25 25 12.5 6.25 3.12 1.56
0
1243
