I A Spectrophotometric Method for

I and biologically orienMshldents

Terri L. Daines and Karen W. Morse

Utah State Universnty Logan84322

Colorimetric or spectrophotometric methods hased upon the reaction of glucose with primary aromatic amines in glacial acetic acid have been utilized clinicallv with differ-

Determination of Glucose in Blood Serum A freshman laboratory experiment for medically

ent degrees of s u ~ c e s s . ~ ~ ~ Perhaps the most su&essful is the selective reaction of glucose with o-toluidine, a primary ar- omatic amine, to give a stahle green colored ~ o ~ ~ l e x . ~ T h e reaction adheres to the Beer-Lamhert law over a wide range of concentrations, and is hased on clinical tests for the quantitative determination of glucose in serum and ce- rebrospinal We have devised a laboratory experi- ment for students in General Chemistry hased upon this clinical test.

The reaction that is used in the experiment is

I CH?OH

glucose

the determination. (For the student interested in further information regarding clinical glucose methods, glucose tol- erance tests, interpretation of the tests, etc., see footnotes 1, 2, and 4.) We have found over a testing period of one year that the experimental results have been consistent in that excellent standard curves are obtained and indeed, in every case, the expected changes descrihed ahove were oh- served (see the figure). Another advantage is that although i t is a t times difficult to present a biologically or medically related experiment to a lower-level chemistry class and still require a fairly high level of experimental sophistication both in terms of technique and instrumentation, we have found that the experiment descrihed fulfills the latter ex- pectations. The students performing the experiment gener- ally have had no previous lahoratory experiment hut have the equivalent of a quarter of introductory organic chemis- try or are taking the introductory organic course concur- rently with the lahoratory.

Videotapes prepared in cooperation with the Utah State University television studio were helpful in instructing stu- dents on the use of the spectrophotometer as well as in cor-

NH? _ H-C-OH rect volumetric techniques.

I + H,O Experimental

HO-C-H I Reaaents I ~ - - -

o-toluidine H-C-OH Glucose Standard, concentration = 150 mg glueose/100 ml.

I Ortho-Toluidine Color Reagent: 950 ml glacial acetic acid with 50

H-C-OH ml o-toluidine and 3 g thiourea? "Dolce," Glucose Tolerance Test

I Beverage, Hopping Bottling Company, Mt. View. Calif. 94040 or 5-6 sugar packets in orange juice or punch to make the sugar pal-

CH?OH atahle. Nausea can result with some people by intake of a coneen- blue-green complex trated sugar solution.

Blood samples are drawn from a student volunteer3 and the glucose content of the hlood serum is examined after a pe- riod of fasting (-9 hr), 40 min after drinking a concentrat- ed 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

126 / Journal of Chemical Education

' 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 commer- cially 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., (Edi-

tors), "Clinical Chemistry: Principles and Technics,': Harper and Row, Harerstown. Md.. 1974. DD. 1271-1305. Ib) Re~ort of 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 Meet- ing, 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 Ortho- Toluidine 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 imme- diately centrifuged and the serum carefully separated from the blood cells. The serum may be stored in the refrigerator for ap- proximately 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) fast- ing sample (8) sample obtained 40 min after drinking glucose solu- tion (9) sample obtained 1 hr, 40 min after drinking glucose solu- tion.

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 and 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 allow- ing 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 solu- tion). Zero the spectrometer a t 635 nm using the reagent hlank (the distilled water sample). Read the absorbance of eaeh of the

, . I / * I ti!-<. 4 0 MIN rn

W U Zm. 6 '

m Cc

5 0 100 150 CONCENTRATION (mg/lOOml)

Concentration versus absorbance: glucose standard curve plus blood serum samples.

*STANDARD FASTING / . 40 MIN.

samples (the standard solutions and the unknown serum samples) within 1 hr as the final colored solution is stable for this long. Zero the spectrophotometer between each reading using the reagent hlank (the distilled water). Find the glucose concentration of the serum samples from the calibration curve.

Acknowledgment

W e wish t o thank t h e s tudents of Chemistry 144 who have carried ou t this experiment a n d to Robert Moore for his suggestions on source material and help in collecting t h e serum samples. Gratefully acknowledged is support re- ceived from a Research and Training F u n d through t h e Provost's Office a n d also funds from t h e College of Science.

Volume 53, Number 2, February 1976 / 127