A MODIFIED FOLIN AND WU BLOOD SUGAR METHOD.

Transcription

1 A MODIFIED FOLIN AND WU BLOOD SUGAR METHOD. BY VERA E. ROTHBERG AND FRANK A. EVANS. (From the William H. Singer Memorial Research Laboratory, Pittsburgh.) (Received for publication, August 7, 1923.) In the preceding paper data have been presented to emphasize anew the statement of Folin and Wu that in their method for quantitative blood sugar estimation* the standard solution must contain very nearly the same amount of dextrose as the blood filtrate being tested. Examination of these data shows that if approximately correct results on all blood fltrates are to be obtained with this method it would be necessary to have more standard solutions than can be used conveniently. This is necessary, probably in large part although perhaps not entirely, because the curve of color dilution of the color developed in this procedure is irregular and, therefore, if the standard and unknown are not almost the same color at the time the calorimetric readings are made, errors are introduced. An error in a method used for quantitative work is unfortunate under any circumstances, and particularly so in a blood sugar method of widespread popularity in these days of insulin therapy of diabetes when correct values for the blood sugar, especially when low, are so important In this report a minor modification of the Folin and Wu method for blood sugar determination is presented whereby the color developed by the unknown blood filtrate is always the same as that of the standard dextrose solution at the time the calorimeter readings are made; and one standard alone may be used for comparison with many blood filtrates and two, or at most three standards, will make possible correct determinations of the amount of dextrose in any blood filtrate ever encountered. The modification of the Folin and Wu method for quantitative blood sugar determination used in this laboratory consists merely in: (1) Diluting the unknown after the addition of the phos 1 Folin, O., and Wu, H., J. Biol. Chem., 1920, xli,

2 444 FolinWu Blood Sugar Method phomolybdatephosphotungstate sugar reagent and development of color until it is approximately the same shade as the standard, instead of to a constant amount, before calorimetric comparison is made; and (2) introducing this variable factor, the amount of dilution, in the final formula for calculating the amount of dextrose in the unknown. The determination is carried out in tubes FIG. 1. a, Tube used for the Folin and Wu blood sugar determination. b, Tube used for the modified Folin and Wu blood sugar determination. differing from those suggested by Folin and Wu only in that they are longer and graduated in steps of 2.5 cc. from 12.5 to 50.0 cc., Fig. L2 In every other particular the Folin and Wu technique is followed. * Satisfactory tubes of this character may be purchased fromtheemi1 Greiner Company, New York.

3 V. E. Rothberg and F. A. Evans This modification will be practical only if the error in the Folin and Wu method for estimating blood sugar such as was demonstrated in Tables I and II of the preceding paper was due ent,irely, or almost entirely, to irregularity in the curve of color dilution of the color developed in the determination. Experiments presented in the preceding paper demonstrate that this is so, provided the instructions of Folin and Wu are carefully followed. Their statement that the phosphomolybdatephosphotungstate sugar reagent must be waterclear is especially important, because if it has a yellowish tinge an additional source of error may be introduced. Other errors may.also be present; for example, the amount of reduction by more concentrated solutions of dextrose is possibly not proportional to that by weaker solutions; the degree of reoxidation which perhaps takes place in performing the determination may not be the same for strong and weak solutions of dextrose; the amount of water added after development of color may introduce an error; and several other features must be considered. If these errors are present, they are so small that they are within the limits of error for ordinary calorimetric determinations, so that if that due to the use of inferior reagents is eliminated, the only error of consequence remaining is that due to the irregularity of the curve of color dilution of the color being dealt with. Before the modification herein suggested was adopted for routine use, however, several experiments were carried out to prove that it was practical. Experiment I.A specimen of Difco dextrose shown to be pure by polariscopic examination was used to make dextrose solutions of the following strengths: 0.11,0.12,0.13, etc., up to 0.2 mg. of dextrose per cc. of water, the equivalent of 110,120,130, etc., up to 200mg. of dextrose per 100 cc. of blood. The routine Folin and Wu blood sugar determination was carried out on each of these solutions, using 2 cc., exactly as is done with the proteinfree blood filtrate. At the same time the procedure was carried out with these solutions in the new tubes described above, but diluted, after the addition of the phosphomolybdatephosphotungstate sugar reagent and development of color, to the amount at which theoretically they would compare exactly in color with the standard containing 0.1 mg. of dextrose per CC. diluted, after the addition of the reagent and development of color, to 25 cc. All these dilutions were made promptly, at as nearly the same time as possible. Each test was compared in the calorimeter with a standard dextrose solution containing 0.1 mg. of dextrose per cc., the same amount as is in the filtrate from a blood containing 100 mg. of dextrose per 100 cc., set at 20 on the calorimeter scale. The results appear in Table I.

4 FolinWu Blood Sugar Method Experiment g.dextrose solutions of the following strengths were prepared: 0.22, 0.24, 0.26, etc., up to 0.4 mg. of dextrose per cc. of water, the equivalent of 220,240,260, etc., up to 400 mg. of dextrose per 100 cc. of blood. Procedures similar to those outlined for Experiment 1 were carried out on these dextrose solutions and the comparison in the calorimeter was made against a standard dextrose solution containing 0.2 mg. of dextrose per cc., the same amount as is in the filtrate from a blood containing 200 mg. of dextrose per 100 cc., set at 10 on the calorimeter scale. The results appear in Table II. TABLE I. Equivalent dextrose loo;m&l;;i solutions mg in Per of tested Determinations according to original Folin and Wu method. Contenta of tubes diluted to 8 volume of 25 co. Average Dextrose,f colorim recovered eter readings. Per Error. Solutions read against the 100 mg. standard eet at 20 on the oolorimetm scela mg. per cent cc Solutions read against the 200 mg. standard set at 10 on the calorimeter scale Determinations according to the modified method. Contents of tubes diluted to volume at which the color of the unknown shouldmatch, theoretically, that of stsndsrd. rohlme to which Average l&xtrose contents of colorim recovered of tubes were eter readings. Per 100 cc. diluted. ElTor. Solutions read against the 100 mg. standard set at 20 on the calorimeter scale. w. per cent o Because of the results obtained in Experiments 1 and 2 we expected to find that one standard solution would suffice for determinations on all blood filtrates; that the color developed by the test on the blood filtrate from a blood containing 400 mg. of dextrose per 100 cc. when diluted to 50 cc. would match that developed by the standard dextrose solution equivalent to the filtrate from a blood containing 100 mg. of dextrose per 100 cc., when diluted to 12.5 cc. In other words, we expected to find

5 V. E. Rothberg and F. A. Evans 447 that the color developed by a dextrose solution four times as strong as the standard solution, when diluted to 50 cc., would match that of the standard solution diluted to 12.5 cc. Such, however, was found not to be the case. Under these conditions a plus error of about 8 per cent is introduced so that the color developed by the stronger solution must be diluted to about 55 cc. before it matches that of the 100 mg. standard diluted to 12.5 cc. Apparently, therefore, the possible sources of error mentioned above, other than the irregularity of the curve of color mu TABLE 1 Determinations according to original Folin and Wu method. Contents of tubes diluted to B volume of 25 cc. Average 0 f c&rimeter : readings. Dextrose recovered Per 100 DC. Error. 3olutions read against the 200 mg. standard set at 10 on the oolorimeter scale WI. p&r cent cc II. 3eterminations according to the modified method. Contents of tubes diluted to volumeat which the color of the unknown &;uld match, theoretically, that of stand olume to which contents Average of o&rim Dextrose recovered of tubes eter were diluted. readings. IOKO. Error. 3olutions read against the 203 mg. standard set at 10 on the calorimeter wale m7. per cent dilution of the color developed, do come into play. When the unknown does not differ more than 100 per cent from thestandard, however, the error introduced by these factors is too small to be demonstrated by ordinary calorimetric determinations, but when there is a difference much greater than 100 per cent an appreciable error creeps in. When the standard dextrose solution equivalent to the filtrate from a blood containing 100 mg. of dextrose per 100 cc. is used for comparison by this revised method with the

6 448 FobWu Blood Sugar Method filtrate from a blood containing 400 mg. of dextrose per 100 cc. the error is less than when the determination on a similar filtrate diluted to 25 cc. is read against a 200 mg. standard, diluted to the same amount as ordinarily done with the method of Folin and Wu. And, inasmuch as absolute values for the sugar of the blood when in such great concentration are not necessary in routine clinical work, one standard may be used with this revised method for all blood filtrates and fairly satisfactory results be obtained. If results closely approaching absolute values for all blood filtrates are desired, however, standard solutions differing from the sugar concentration in the unknown not more than 100 per cent must be used. To perform the test according to the modification above suggested the Folin and Wu blood sugar determination on the standard dextrose solution (or solutions) chosen and on the unknown blood lilt.rates is carried out in tubes described above. After the addition of the phosphomolybdatephosphotungstate sugar reagent and development of color, the test on the st,andard solution is diluted to 25 cc. and those on the unknowns are diluted to the cubic centimeter mark on the tube at which the color intcnsity most closely resembles that of the standard. The amount of dilution of each unknown is recorded and they are then compared in the calorimeter with the standard preparation. If the dilutions of the unknowns in the tubes have been made carefully, they should read on the calorimeter scale very nearly that, figure at which the standard preparation was set. When, as here obtains, the dilution of the unknown is not the same as the standard solution, the formula presented by Folin and Wu for calculating the final results by their method, namely rsg X 100 = mg. of dextrose per 100 cc. of blood, must be multiplied by the fraction dilution of unknown dilution of standard (25 CC~ We have, therefore, 20 dilution of unknown x 100 x reading 25

7 V. E. Rothberg and F. A. Evans 449 or simplifying, 80 X dilution of unknown reading = mg. of dextrose per 100 cc. of blood. If the amount of dilution necessary to make the color developed by t.he unknown blood filtrates match that of the standard is considered in relation to the strength and dilution of the standard used, a rough idea of the amount of dextrose in the bloods in question may be obtained. For instance, if the standard dextrose solution, equivalent to t,he filtrate from a blood containing 100 mg. of dextrose per 100 cc., diluted to 25 cc., is used, the values are as follows: Dilution of unknown solution. Dextrose per 100 cc. Dilution of unknown of blood. II solution. cc. mg. cc. mg If the standard dextrose solution, equivalent to the filtrate from a blood containing 200 mg. of dextrose per 100 cc., diluted to 25 cc., is used, the values are as follows: CC. D&c;,,,,100 mg loo 15.o cc. Dilution of unknown solution. cc Dext;;; o;dloo cc. P. mg Folin and Wu point out clearly that in their method for blood sugar determinations it is necessary, after the addition of the

8 FolinWu Blood Sugar Method phosphomolybdatephosphotungstate sugar reagent and development of the color, to carry out the subsequent dilution on the standard solution and on the unknown blood filtrate at the same time. Experiments in this laboratory demonstrated the importance of this instruction, for if the dilutions of the standard a,nd unknown are made 5 minutes apart an appreciable error is introduced. This observation is of importance in relation to the modification of the Folin and Wu blood sugar method here presented, because the dilution of the test on the unknown blood filtrate is not made all at once, but in two, or more, steps. If one keeps in mind the necessity for dilution of the standard and unknown at approximately the same time, and works rapidly, it is possible to dilute at least six unknowns to match the standard promptly enough to eliminate this source of error. The method of procedure which has been devised with this point in mind is as follows: After the addition of the phosphomolybdatephosphotungstate sugar reagent and development of colorbut before dilutionthe tests on unknown blood filtrates are compared with that on the standard dextrose solution. One can tell whether the color developed in the unknowns is less than, about the same as, or more than that of the standard dextrose solution; and depending on this observat,ion the unknowns are immediately diluted to a mark of less than 25 cc. to the 25 cc. mark, or to a mark of more than 25 cc. The standard is then diluted to 25 cc., the contents of each tube are thoroughly mixed, and they are compared again. Any further dimon of the tests on the unknown blood filtrates which are necessary to make them about the same color as the standard is carried out promptly. If it is expected that any of the proteinfree filtrates being tested are from bloods containing more than 200 mg. of dextrose per 100 cc., it is wise to carry out at the same time a preparation not only on a standard dextrose solution containing 0.1 mg. per cc. of solution, but also on one containing 0.2 mg. per cc. of solution; for, as stated above, accurate results cannot be obtained by comparing the test on such a blood filtrate with the weaker standard diluted to 12.5 cc. With a, little practise on the procedure outlined, it is possible to match the color of the test on unknown blood filtrates with that of the standard dextrose solution diluted to 25 cc. very rapidly, and in most instances in no more than two steps.

9 V. E. Rothberg and F. A. Evans 451 Since, in the procedure suggested, the total dilution of the test on unknown blood filtrates is not carried out all at once, but by the addition of smaller amounts of water several times, it is necessary in mixing the contents to invert the tube more often than in the original method. It seemed possible that, under these circumstances, enough fluid would be lost on the stopper and around the mouth of the tube to introduce an appreciable error. This, however, was not found to be so. A tube filled with water inverted fifteen times, the stopper being removed after each third inversion, showed no diminution in the volume of its contents that could be made out on gross inspection. This number of inversions is sufficient to mix thoroughly with its contents water added to a tube five different times, more than is ever necessary in carrying out the procedure presented above. SUMMARY. If correct values are to be obtained with the Folin and Wu blood sugar determination it is essential, for the oolorimetric comparison, to have the color finally developed by the blood filtrate being tested very nearly the same as that developed by the standard dextrose solution with which it is to be compared. This necessitates for accurate results the use of more standard solutions than is convenient. In this paper the details of a technique are presented in which the color developed by the unknown blood filtrate with the Folin and Wu method is the same as that of the standard dextrose solution when the calorimetric readings are made; and one standard may be used for many bloods, and two or at most three make possible accurate determinations of the dextrose in any blood every encountered. The modification here presented consists simply in diluting the unknown in the testtube after the addition of the phosphomolybdatephosphotungstate sugar reagent and development of color until it is approximately the same color as the standard, and including this variable factor in the formula for calculating the final results.

10 A MODIFIED FOLIN AND WU BLOOD SUGAR METHOD Vera E. Rothberg and Frank A. Evans J. Biol. Chem. 1923, 58: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#reflist1