Selective, and Sensitive Reactions,” pp. 289-91, Academic Press, New York, 1949. (8) Harlow, G. D., AXAL.CHEM.34, 1482 (1962). (9) Heilhron, I. h3., “Dictionary of Orgamc Compounds,” Vol. I, p. 247, Oxford University Press, New York, 1953. (10) Kirshna, S., Das, B., @ant. J . Indian Chem. Soc. 4, 367 (1927). i l l ) Koenim. C.. Ber. 11. 615 11878). ( l 2 i Marpl;,’ L. ’W.) Fritz, J. S., ANAL. CHEM.34, 796 (1962). (13) Marvel, C. S., Johnson, R. S., J . Org. Chem. 13, 822 (1948).
LITERATURE CITED
( 1 ) Xckerman, Lev., IYD. ENG.CHEM. A i 41.. ED. 18, 243 (1946). ( 2 ) i\llen, P., Jr., J . Org. Chem. 7, 23 ( 1942 j.
( 3 ) Burkhard, 11. K., Sellers, D. E., I)eCou, F., Lambert, J. L., Ibid., 24, i b , (1969). (4) Cundiff, It. H., Markunas, P. C., hsar,. CHEM.28, 792 (1956). ( , ? j I h i d . , 34, 584 (1962). ( 6 ) I)al Xogare, S., “Organic Analysis,” i-01. I, p. 378, Interscience, New York,
-
>-
1953.
( 7 ) Feiyel, F., “Chemistry of Specific,
(14) Ponjini, S., Farm. Sci. e. Tec. (Pavia) 2, 198 (1947); C . A . 42, 3701. (15) Samuelson, Olof, “Ion Exchangers in Analytical Chemistry,” Wiley, New York, 1953. (16) Smiles, S., Bere, C., “Organic Synthesis,” Vol. I, p. 7, Wiley, New York, 1946. (17) Tan der Heijde, H. B., Dahmen, E. A,, Anal. Chim.Acta 16, 378 (1957). (18) Whitmore, F. C., Hamilton, F. H., “Organic Synthesis,” Vol. I, p. 492, Wiley, New York, 1946. RECEIVEDfor review March 4, 1963. Resubmitted June 29, 1964. Accepted September 23, 1964.
A n Automatic Spectrophotometric Method for the Specific Enzymatic Determination of Galactose in Whole Blood and Plasma CHRISTOPHER S. FRINGS and HARRY
1.
PARDUE
Department o f Chemistry, Purdue University, lafayette, Ind. An automatic enzymatic method is described for the determination of galactose in blood. The method is based on the catalysis by galactose oxidase of the oxidation of galactose. Hydrogen peroxide produced by the enzymatic reaction oxidizes o-dionisidine in the presence of peroxidase to a species which absorbs a t 440 mp. The formation of the colored species is detected spectrophotometrically. Commercially available automatic control equipment provides direct readout of the time required for a predetermined decrease in per cent transmittance to occur. The reciprocal of the measured time interval when plotted against the galactose concentration provides a linear working curve. Automatic results for aqueous samples show relative standard deviotions within 2% over a range of 33-500 p.p.m. galactose. Results for galactose in blood between 99 and 1500 mg. per 100 ml. show similar limits of precision and accuracy.
T
XEED for good methods for determining galactose quantitatively in blood for the purpose of diagnosing galactosemia has been discussed in recent papers (1, 2 ) . It has been suggested that a laboratory test for the disease should be available in every laboratory ( 1 ) . h symptom of galactosemia is the presence of galactose in the blood. Difficulties associated with conventional methods for galactose in blood have been discussed ( 3 ) . The present work was undertaken to provide a simple, rapid, and selective method for this analysis. This has been accomplished utilizing the enzyme galactose oxidase.
HE
Galactose oxidase catalyzes the oxidation of galactose to the dialdose producing an equivalent amount of hydrogen peroxide. h method for galactose in aqueous solutions utilizing the iodineiodide redox couple to detect the formation of peroxide was described recently ( 3 ) . Attempts to adapt this method for galactose in blood were only moderately succeqsful. The coupled enzyme reaction using horseradish peroxidase and o-dianisidine to detect the hydrogen peroxide was superior to iodide in this reaction. The present method is baqed on the measurement at 440 rnp of the rate of formation of the colored product resulting from the coupled reaction. Automatic control equipment measures the time required for the transmittance to decrease over a small predetermined interval. The reciprocal of the measured time is a linear function of galactose concentration. Results for aqueous galactose solutions between 33 and 500 p.1j.m. and galactose in blood between 100 and 1500 mg. per 100 ml. are precise and accurate to within 2%. Measurement times range from a few seconds a t higher concentrations to about 5 minutes a t the lower concentrations reported here. EXPERIMENTAL
Reagents. A11 solutions are prepared in water which has been passed through a mixed cation anion exchange resin bed. All solutions are stored at 4’ C. GALACTOSESTANDARDS. Standard galactose solutions are prepared by dilution of a 2000 p.p.m. solution prepared by dissolving 2.000 grams of D ( + ) galactose (Sigma Chemical Company,
St. Louis, Mo.) in water and diluting to 1 liter. PHOSPHATE BUFFER. Potassium dihydrogen phosphate (1.4 grams) is dissolved in 900 ml. of water. The solution is adjusted to pH 7.0 and 0.75N KaOH and diluted to 1 liter. GALACTOSEOXIDASE. Five milligrams of galactose oxidase dry powder (Worthington Biochemical Corp., Freehold, N. J., 5 to 7 units per milligram) is triturated in a mortar, diluted to 50 ml. with buffer solution and filtered through Whatman No. 1 filter paper. This gives an enzyme concentration of about 0.5 Worthington unit per milliliter. This solution was stable for several days a t 4” C. COLORREAGENT. Thirty milligrams of o-dianisidine (Eastman, Rochester, N. Y.) in 3 ml. of methanol and 12 m g . of horseradish peroxidase (Worthington Biochemical Corp., Freehold, N . J.) are dissolved in 500 ml. of buffer. DEPROTEINIZATION REAGENTS. A zinc sulfate solution is prepared by dissolving 5 grams of ZnS01.7H,0 in 100 ml. of water. =\ barium hydroxide solution is prepared by dissolving 4.5 grams of 13a(OH)?.8H20in 100 ml. of water. These solutions are titrated to the phenolphthalein end pqint. The more concentrated solution is diluted with water until the acid and base strengths of the two are the same. Instrumentation. Changes in transmittance are observed using a regulated Bausch & Lomb Spectronic 20 spectrophotometer. l‘he unregulated model is not satisfactory. l’he instrument meter is disconnected and external hookup leads are connected to the amplifier output leads (points G and H on t h e instrument diagram). I t should be noted t h a t both points G and H are about 100 volts above instrument ground. Caution must be exercised in handling these leads and in connecting them to measurement VOL. 3 6 , NO. 13, DECEMBER 1 9 6 4
2477
equipment. No difficulty will be encountered if no connection is made to the instrument ground. The hookup lead from point G is connected through an 8.2-K and a 1-K resistor in series to the lead connected to point H. The divided output signal is measured across the 1-K resistor. -1bias voltage variable between 0 and 1 volt is introduced between the divider circuit and the measurement system so that a small fraction of the total change in transmittance can be observed. The positive end of the bias is connected to the end of the 1-K resistor nearest point G on the Spectronic 20. Connections to measurement equipment are made between the negative terminal of the bias (Point A ) and the end of the 1-K resistor nearest point H (Point B ) . Yo other modifications of the instrument are required. Data were obtained both by potentiometric recording of response curves and by automatic measurement of the time required for the transmittance to change over a small predetermined interval. A Sargent SR recorder is used for recording response curves. A Sargent Model Q Concentration Comparator with Model Q RR rate adapter (E. H. Sargent and Co., Chicago, Ill.) is used for automatic measurements. Reagents and samples are handled with automatic washout pipets and syringes. Procedure.
PREPARATIONOF
EQUIPMENT.RECORDED CURVES. All instruments are permitted to warm up for 5 minutes before use. T h e wavelength of the Spectronic 20 is set at 440 mp. The positive and negative recorder inputs are connected to points A and B , respectively. The recorder sensitivity is set a t 100 niv. full scale, the chart speed is set a t 1 inch per minute. With no cell in the spectrophotometer the dark current is adjusted by bypassing the bias voltage and adjusting the dark current knob until the recorder reads zero. The 100% T point is set with a blank solution in the light path by adjusting
Table I.
Automatic Results for Aqueous Galactose Solutions
Final galacto5ej P.P.m. Taken Found 50 49.5 66.6 66.6 200 203 263 266 333 333
s o . of
runs 2 3 4 3 3
Table
II.
the 100% T knob until the recorder reads full scale with the bias voltage set a t 520 mv. .4UTOMATIC MEASUREMEXTS. The Concentration Comparator and rate adapter are connected as described in the instructions. The red and black leads to the instrument are connected to points A and B , respectively. The sensitivity switch is set in the 0.100-volt position and the reagent selector switch in position 1. With no light on the phototube and the bias voltage bypassed, the zero adjust on the comparator is set a t 5.00 (zero volts). The dark current knob on the Spectronic 20 is adjusted until the comparator reads zero. A blank is then placed in the light path, the bias voltage is set a t 590 mv. and the 100% T knob is adjusted until the comparator again reads zero. The bias volt,age is then set at 620 mv. and the comparator zero adjust a t 4.00 (20 mv.). The instrument is ready for automatic measurements. I n each case above, the blank solution consists of 1 ml. each of galactose oxidase, c o l o ~reagent, and water. The above settings permit rate measurements to be made between 100 and 85y0 T. The response curves remain essentially linear during this interval. DEPROTEINIZATIOS OF BLOOD. Onehalf milliliter of sample is drawn into a wash-out pipet and flushed out with water to give a total volume of 4.00 ml. Then 0.50 ml. of Ba(OH)z solution is added and the solution is mixed thoroughly. Next 0.50 ml. of ZnSOl solution is added and the mixture is mixed thoroughly. The mixture is centrifuged for 3 minutes. MEASUREMENT STEP. All samples and reagents are equilibrated to 35' C. before measurement. One milliliter each of the color reagent and sample are added to the cell followed by 1 ml. of the galactose oxidase solution which is injected vigorously to provide mixing. For automatic resulk the start switch on the rate adapter is pressed and the reaction time is read from the time
Rel. Reciprocal time, std. dev., set.-' X lo2
70
Error, 70
1.10 1.63 8.20 11.5 15.2
...
1.0 0.0 1.5 1.1 0.0
1.8 1.7 1.1 0.0
Automatic Results for Galactose in Plasma and Whole Blood
Sample
yo,of
N0.Q 1 2 3
runs
Galactose mg./100 Taken 150 450 600
ml. blood Found
Rel. std. dev.,
%
Error, yG 1 33 0 33 0 50
1.4 3 152 0.4 4 448.5 1.o 4 603 1.1 0 80 4 3 750 756 0.9 1 00 5 4 100 99 1.3 1 67 6 3 300 305 1.0 0 0 7 3 600 600 Samples 5 to 7 are whole blood. These samples had 12 Samples 1 t o 4 are plasma. EDTA as an anticoagulant. Oxalated and citrated samples gave similar results
2478
ANALYTICAL CHEMISTRY
after the measurement is completed automatically. For recorded curves the time required for the signal t o decrease by 20 mv. after the plot becomes linear is read from the chart. Sample concentration is read from a plot of reciprocal time us. galactose concentration constructed using 200 and 400 p.p.m. standards. RESULTS A N D DISCUSSION
Quantitative Data. Plots of reaction rate (reciprocal time) us. galactose concentration between 33 and 500 p.p.m. approach linearity. Therefore, a calibration curve can be constructed from two standards. Typical quantitative data for aqueous galactose solutions are given in Table I. The relative standard deviat,ion and accuracy of t h e method is within 2% throughout this concentration range. Since blood and plasma samples undergo a thirty-fold dilution from start' to finish, the data in Table I1 represent blood galactose between 150 and 999 mg. per 100 ml. The data in Table I1 are typical of many results obtained for galactose in blood. These data represent recoveries of galactose added to blood prior to deproteinization. Precision and accuracy are again observed to be within 2%. Comparable data were obtained when solutions containing gahctose were permitted to stand for periods up to 1 hour before and after deprot,einization. For galactose in blood below 99 mg. per 100 ml., measurement times become increasingly longer. Response Curves. There is a significant interval between the point of mixing reagents and the time when the reaction rate reaches the value limited by the galactose concentration. The rate measurement is made only after this limiting rate has been reached. For t'he lowest concentrations, this may amount to up to 5 minutes. T h e times given in Tables I and I1 represent only measurement times and not total analysis times. Effects of pH, temperat'ure, and interferences discussed earlier (3) apply here. ACKNOWLEDGMENT
We are indebt'ed to Dr. Eliott and Miss Marie Mart,in of Home Hospital, Lafayette, Ind., for supplying blood samples. LITERATURE CITED
(1) Brewer, G. T., Tarlov, A. R., Am. J . Clin. Puthol. 39, 579 (1963).
(2) Donnell, G. N., Collado, M., Kock, R., J . Pediut. 5 8 , 836 (1961). (3) Pardue, H. L., Frings, C. S., J . Electroanul. Chem. 7 , 398 (1964). RECEIVEDfor review June 2, 1964. iiccepted September 11, 1964. Work supported in part by a grant from the Indiana Elks Association to Purdue Vniversity.
