Simplified methods of determining true sugar in blood

Title

Creator

Samson, Delfin D.

Language

English

Source

Volume XV (4) April-June 1959

Year

1958

Subject

Blood sugar

Blood sugar -- Analysis

Medicine -- Periodicals

Rights

In Copyright - Educational Use Permitted

Fulltext

SIMPLIFIED METHODS OF DETERMINING TRUE SUGAR IN BLOOD * * Excerpt of a paper read before the first Scientific Meeting of the Philippine Diabetes Medical Society on March 8, 195? in the Auditorium of the College of Education of the University of Sto. Tomas, The experi mental data presented in this paper were obtained in studies supported by grants from the Institute of Nutrition, and the National Research Council of the Philippines. DELFIN D. SAMSON. Pk.C. Department of Biochemistry, Coliege of Medicine University of the Philippines There are quite a number of methods for the quantitative determination of sugar in the blood. Most of these have for their basis the reducing action, of glucose in hot solution on certain metallic ions like cupric and ferricyanide ions. The ex tent of reduction is then measured by colorimetric, titrimetric or gasometric methods. Among the more common are the clas sical procedure of Folin and Wu, the equally celebrated method of Somogyi, Shaffer and Hartmann, and others like those of Nelson and Somogyi, Lewis and Benedict, Folin and Malmros, Hagedorn and Jensen, Bang, and Myer and Bailey. Some of these however, are used less often due to certain objectionable features that make their adoption quite difficult, tn the Philippines the methods most commonly employed arc those of Folin and Wu, (1) and of Somogyi, et. at (2). Both of these are macro methods requiring not less than a milliliter of whole blood for every determination. The Folin and Wu method gives an amount of "sugar" that is slightly high; the normal range being 90 to 120 mg per cent, against the 70 to 100 mg per cent of Somogyi. The higher value ob tained with the Folin and Wu method is attributed to reducing substances other than glucose present in the blood and deter mined in its filtrate with the sugar. These substances may occur in sufficient amount in the blood and increase the "sugar” value considerably. In the Somogyi method, the non-sugar reducing substances, namely glutathione and glucuronic acid. 243 244 ACTA MEDICA PatLlPPINA go down with the proteins of the blood when these are preci pitated during preparation of the filtrate. But the Somogyi method involves a titrimetric procedure which makes it cum bersome and technically difficult. In view of these considerations, those of us in the staff thought of presenting a specific method that is simple, yet, without sacrifice of accuracy. A modified macromethod was introduced (3) in 1952 for the determination of true blood sugar. I. MODIFIED MACROMETHOD FOR TRUE BLOOD SUGAR Principle: The proteins and the non-sugar reducing sub stances of the blood are precipitated with sine hydroxide, the filtrate heated with alkaline copper and the cuprous oxide formed treated with phosphomolybdic acid for color develop ment The color is then compared with the standard similarly prepared. Procedure: (a) Preparation of the Filtrate. Into a clean and dry 50 ml Erlenmeyer flask 7 ml of water are measured, followed by 1 ml of blood, rinsing the pipette with the clear water above the layer of blood. The mixture is shaken by a rotatory motion and subsequently treated drop by drop, shak ing the Rask after every drop, with 1 ml of 10% zine sulfate solation, followed by 1 ml of 0,5 N sodium hydroxide. The mixture is shaken in the same manner til! brown and then filtered. (b) Determination of the Sugar: 2 ml of the filtrate are measured into a Folin and Wu sugar tube, treated with 2 ml of alkaline copper reagent and heated with the standards (0.2 mg and 0.4 mg glucose per 2 ml), and the blank, in a boiling water bath for 6 minutes. The tubes are then cooled in water for 3 minutes and each treated with 2 ml of phosphomolybdic acid reagent. After complete solution of the cuprous oxide is effected, each tube is diluted with water to the 25 ml mark and shaken cautiously. The colors are then compared in a photoelectric colorimeter. BLOOD SUGAR DETERMINATION 245 Calculation: pit x cone, of S x 500 = mg glucose per 100 ml of blood. RS Using the above modification, therefore, we prepare a fil trate in accordance with the method of Somogyi, et of. and determine the sugar by that of Folin and Wu. The results obtained by use of this combined procedure are constantly lower than those of the original Folin and Wu. The differ ences range from 10 to 20 mg per cent. These differences are within the range of differences observed between the Folin and Wu and the Somogyi methods as noted by several investi gators (4). Pressed for a procedure that would make possible the de termination of true sugar from less than a drop of blood, we scanned the literature and came upon the ultramicro methods of Natetson (5). The procedure for blood sugar described by thia author calls for delicately made micropipettes and especial ly constricted small sugar tubes which are not available locally. Furthermore, it employs tungstic acid reagent for precipitation of the proteins, and alkaline copper and phosphomolybdic acid reagents for color development. These reagents are essentially similar to those of Folin, and Wu and, therefore, a method that does not give the true sugar value of the blood. With our macro modification in mind (34 for true sugar, we patterned an ultrainiero modification (6) after that of Natelson. We substituted a Sahli 20 cnun ultramicro pipette, 1-ml serological pipettes and ordinary small pyrex test tubes in place of those recommended by Natelson. The results produced were very close to those obtained by our modified macro method. II. MODIFIED ULTRAMICRO METHOD FOR TRUE BLOOD SUGAR Principle: Hemolyzed blood is treated with zinc hydrox ide, the filtrate heated with alkaline copper and the cuprous oxide formed treated with phosphomolybdic acid for color de velopment. The intensity of color is then compared with the standard in a photoelectric colorimeter. Missing pages (pages 246-247) 248 ACTA MED1CA PH1LIPPINA Table 1 indicates values on aliqoots of one sample of blood making a total of 20 analyses. The difference between means is not statistically significant. Table 2 TRUE BLOOD SUGAR VALUES AS DETERMINED BY THE MACRO AND MODIFIED ULTRAMICRO METHODS IN PAIRED SAMPLES Milligrami! giacwe per 100 biL human blood Samples Macro ■ethodr Departcaent of Medicine Modified Ultramlctw method; Department of Bjocbemlalry 1 172,0 1695 2 151.6 162.1 160.0 168,8 4 128.0 121.6 5 133.0 1302 6 136.0 107.5 7 142.0 150.0 a 200.0 202.0 y 121,0 122.5 IV 121.0 1122 11 128,0 120.0 12 164.0 165.2 13 140.0 139.1 14 120.0 126.3 IB 130.0 132.6 16 140.0 140.8 17 137.0 129.1 18 170.0 176.2 19 165.0 166.2 20 76.0 92,0 21 112.0 128.0 22 240.0 252.0 23 97.0 106.0 24 140.0 208.8 25 128.0 129.1 26 105.0 108.3 27 118.0 145.8 28 110.0 118.7 2ft 160.0 120X1 80 78.0 80.0 31 136.0 173.0 82 102.0 134.0 S3 92.0 04,8 34 80.0 55,5 35 64.0 50.0 36 1560 120,1 37 73.0 51,0 88 47.0 37-0 88 92.0 69.2 BLOOD SUGAR DETERMINATION 24$ 40 41 95 TABLE If (Cotothwed) «.o 83.3 65.0 62.5 65.0 68.8 89.0 70.8 92.0 79.2 46.0 75.0 78.0 98.0 86.0 100.0 60.0 68.0 22.0 40.0 60.0 70.0 86.0 88.0 48.0 64.0 72.0 93.8 53.0 79.1 55.0 91.7 97.0 U2-6 58.0 91.7 «4.0 102.1 420 89^ 71.0 80.0 86.0 84.0 116.0 68.0 7L0 72.0. 68.0 64.0 97.0 66.0 68.0 68.0 57.0 6 LG 97.0 82.7 03.0 61.5 60.0 78.8 53.0 76.9 64.0 84.6 53.0 88.4 60.0 92.3 81.0 107.7 67.0 623 55.0 58.3 290.0 2M.6 62.0 425 40.0 72.9 83.0 95.8 64.0 78.6 76.0 72.0 86.0 78.0 71.0 68.0 65.0 76.0 42.0 50.0 83.0 84.0 66.0 72.0 81.0 80.0 57.0 74.0 46.0 92.9 43.0 76.1 80.0 69.2 95.0 96.1 2W ACTA M01CA PHttIPMNA TABLE H (Continoad) W « 99 1» 191 102 in IM LOS 106 Ml W 1» 110 96.Q 50.0 62.0 50.0 122.0 165.0 73.0 73.0 56.0 78.0 88.0 148.0 148.0 206.0 62.0 94.2 67.3 60.0 921 no.o 128.3 66.0 75.4 45.3 60.4 66.0 13«A 148.1 1BU 74.0 Mom difference ................-.......................................... ■ 4.04 StaadMd deviation of difference ........................... = 62.04 Standard error of mean difference ...................... = 5.92 = 0.68 Table 2 indicates results of analyses of human blood by two separate departments. The two methods employed give values not markedly different and whatever difference was observed was not statistically significant. SUMMARY AND CONCLUSION Simplified macro and ultramicro methods for determining true sugar in 'the blood are hereby presented. Values obtained by the modified macro method compare favorably with those of the macro method of Somogyi, Shaffer and Hartmann. The same is true of the results obtained by the modified ultramicro method compared with those of Nelson and Somogyi. The dif ferences in 'both cases are not statistically significant REFERENCES 4. FOLIN, O., and WU. H.: A Simplified and Improved Method for the Determination of Blood Sugar. J. Biol. Chern., 41:867 (1920). 2 HAWK, P. B., OSER, B. U and SUMMEESON, W. HL: Practical Physiological Chemistry. 13th ed . p, 571, The Blakiston Company. Inc. (1954) Philadelphia BLOOD SUGAR DETERMINATION 251 3. SAMSON. D. D., find CORDERO. M; Minor Modifictionu of Some Standard Clinical Laboratory Methods, Acta Mod. Phil.. 6:261 (1962). 4. SOSKIN, S.: Carbohydrate Metabolism. Univ. Chicago Press (1946) p. 76. 5. NATELSON, &: Routine Use of Ultramicro Methods in Clinical Laboratory, Am. J. Clin. PathoL, 21:1153 (1951). «. SAMSON, D. D., MANIPOL, Y„ and AGARAO, R, T.: Comparison of the Macro Method and the Modified Ultramicro Method of Blood Sugar Determination, Acta Med. Phil., 14:43 (1957).

pages

243-251

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