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A Spectrophotometric Method for Determination of Glucose in Blood Serum
Terri L. Daines and Karen W. Morse Utah State Universnty Logan84322
A freshman laboratory experiment for medically and biologically orienMshldents
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Colorimetric or spectrophotometric methods hased upon the determination. (For the student interested in further the reaction of glucose with primary aromatic amines in information regarding clinical glucose methods, glucose tolglacial acetic acid have been utilized clinicallv with differerance tests, interpretation of the tests, etc., see footnotes ent degrees of s u ~ c e s sPerhaps . ~ ~ ~ the most su&essful is the 1, 2, and 4.) We have found over a testing period of one selective reaction of glucose with o-toluidine, a primary aryear that the experimental results have been consistent in omatic amine, to give a stahle green colored ~ o ~ ~ l e x . ~ T hthat e excellent standard curves are obtained and indeed, in reaction adheres to the Beer-Lamhert law over a wide every case, the expected changes descrihed ahove were ohrange of concentrations, and is hased on clinical tests for served (see the figure). Another advantage is that although the quantitative determination of glucose in serum and cei t is a t times difficult to present a biologically or medically rebrospinal We have devised a laboratory experirelated experiment to a lower-level chemistry class and still ment for students in General Chemistry hased upon this require a fairly high level of experimental sophistication clinical test. both in terms of technique and instrumentation, we have The reaction that is used in the experiment is found that the experiment descrihed fulfills the latter expectations. The students performing the experiment generally have had no previous lahoratory experiment hut have the equivalent of a quarter of introductory organic chemistry or are taking the introductory organic course concurrently with the lahoratory. Videotapes prepared in cooperation with the Utah State University television studio were helpful in instructing students on the use of the spectrophotometer as well as in corH-C-OH rect volumetric techniques. NH? H,O Experimental HO-C-H
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o-toluidine
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H-C-OH
I I CH?OH
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H-C-OH
CH?OH glucose
blue-green complex Blood samples are drawn from a student volunteer3 and the glucose content of the hlood serum is examined after a period of fasting (-9 hr), 40 min after drinking a concentrated glucose solution, and 1 hr and 40 min after drinking the solution. Immediately after the samples have heen drawn a t the ahove intervals, the hlood cells are separated from the serum by centrifugation and the cells removed from the serum to prevent further metabolism of the glucose. The serum may he stored in the refrigerator until ready to he used. The drawing of the blood samples may be done in any medical lahoratory as a technical laboratory or a hospital. Since separation of the serum from the hlood cells may he carried out immediately a t such facilities and since the serum is stable for davs when refriaerated. the samples may he collected in advance of the actual laboratory time. Caution: Individuals who are diabetic or have a history of diabetes should not he allowed to act as donors of blood samples. Since a normal individual donating the sample will have fasted for a period of approximately 9 hr, the glucose level in the hlood will initially come within the range 70-120 mgI100 ml. Intake of a concentrated glucose solution causes the hlood ducose level to rise ahove the normal range. After a period of time the glucose level returns to the normal range or close to it, depending on the exact time of
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126 / Journal of Chemical Education
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Reaaents ~
Glucose Standard, concentration = 150 mg glueose/100 ml. Ortho-Toluidine Color Reagent: 950 ml glacial acetic acid with 50 ml o-toluidine and 3 g thiourea? "Dolce," Glucose Tolerance Test Beverage, Hopping Bottling Company, Mt. View. Calif. 94040 or 5-6 sugar packets in orange juice or punch to make the sugar palatahle. Nausea can result with some people by intake of a coneen-
trated sugar solution.
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Dubowski, K. M., Clin. Chem., 8, 215 (1962) and references therein. 2Hyviiinen, A,, and Nikkili4, E. A,, Clin. Chim. Aeto, 7, 140 (1962) and references therein. Since a glucose tolerance test in humans may not be possible or practical, the experimental method may be alternately illustrated by substituting for the laboratory human serum samples commercially available lyophilized control serum of varying glucose levels. Such commercial serum is available from Dade Division American Hospital Supply Corporation, Miami, Florida 33152 as Moni-Trol I or from Hyland Div. Travelon Laboratories, Inc., Los Angeles, California 90052. The authors thank the referee of this paper for this suggestion. ' (a) Henry, R. J., Cannon, D. C., and Winkelman, J. W., (Editors), "Clinical Chemistry: Principles and Technics,': Harper and Row, Harerstown. Md.. 1974. DD. 1271-1305. Ib) R e ~ o r tof the committie on ~t&stics of the ~ m e r Diabetes . ASSO;.,June 14, 1968: Diabetes, 18, 299, 1969. ( e ) The Nonreproducibility of An Abnormal 2-hour Glucose Tolerance Test, R. G. Troaler, M.D. and M. C. Lancaster, M.D., Clinical Sciences Division, U.S.A.F. School of Aerospace Medicine, AMD (AFSC) Brooks Air Force Base, Texas 78235. Abstract of paper presented at 26th National Meeting, AACC, Las Vegas, Nevada, August 18-23,1974. Hyland Glucose Test (Ortho-Toluidine Method) is supplied as a complete test package containing Glucose Standard and OrthoToluidine Color Reagent and carries Hyland List No. 030-121.
Procedure Blood samples are drawn after a 9-hr period of fasting, 40 min after drinking a concentrated glucose solution (e.g. "Dolce"), and 1 hr and 40 min after drinking the solution. The samples are immediately centrifuged and the serum carefully separated from the blood cells. The serum may be stored in the refrigerator for approximately 3 da (stopper tightly) without change in the glucose Content. Five standard solutions are prepared by pipetting the glucose standard solution (2-10 ml in increments of 2 ml) into a 10-ml graduated cylinder and diluting to 10 ml with distilled water. The resulting solutions range in concentration from 0.3-1.5 mglml. Samples 6 9 consist of the following: (6) distilled water (7) fasting sample (8)sample obtained 40 min after drinking glucose solution (9) sample obtained 1 hr, 40 min after drinking glucose solution. Label nine clean and dry spectrophotometer tuhes from 1-9. Pipet 0.50 ml distilled water into tube number 6. Into sample tubes 1-5 and 7-9 pipet 0.45 ml of distilled water. Pipet 0.05 ml of samples number 1-5 and 7-9 into their respective tuhes. Pipet 3.5 ml of ortho-toluidine color reagent into each tube (1-9) and mix well. Caution: Do not pipet by mouth since this reagent contains elacial acetic acid a n d oan cause burns. Handle carefullv. Place all tubes in a boiling water bath for 10 min. Remove tubes from water bath and cool solutions to room temperature by allowing tuhes to stand in running tap water for about 5 min and then a t room temperature for about 5 min. Swirl eaeh occasionally to avoid temperature gradients (differences in temperature within the solution). Zero the spectrometer a t 635 nm using the reagent hlank (the distilled water sample). Read the absorbance of eaeh of the
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*STANDARD FASTING 40 MIN. * I ti!-
