For qualitative testing, put a cubic centimeter of Fehling's solution into a test-tube (or if the copper and the alkaline sodio-potassium tartrate solutions are kept separate, a cubic centimeter of each), and dilute with distilled water to 5 c.c. Boil, and if, after the lapse of a couple of minutes, the solution remain unchanged, it is fit for testing. If it becomes turbid or a red sediment falls, it is spoiled, and a new solution should be obtained.38 A cubic centimeter of the suspected urine is then measured out and added drop by drop to the solution kept hot. If there is much sugar, the first drop will throw down a yellow precipitate of suboxide of copper, which becomes rapidly red. If no reaction takes place after adding the entire cubic centimeter of urine, the addition should be continued until 4 c.c. are added, when, if, after the mixture has cooled, there be no response, it may be concluded that the urine is free from sugar. By operating with a cubic centimeter of the test-fluid and the same quantity of urine or multiples thereof, we may roughly estimate the proportion of sugar. Thus, if the cubic centimeter of undiluted urine just decolorizes the cubic centimeter of Fehling's solution, sugar is present in the proportion of one-half of 1 per cent.; or if a half cubic centimeter of the urine removes all the color, the quantity is 1 per cent. If the urine is highly charged with sugar, it may be diluted, and the degree of dilution being remembered, a rough quantitative estimation may be similarly made.
38 Should this not be possible, a little more soda may be added and the fluid filtered, when it is again ready for use.
If the urine contains very minute quantities of sugar, the reaction is less satisfactory. The copper is reduced, but the suboxide is so small in quantity that it is obscured by the excess of copper solution, and a mixture results which is greenish or greenish-yellow or yellow or milky, and on standing a small yellow sediment falls to the bottom. Now, it dare not be said that it is sugar which produces such reaction. It may be sugar, but it may also be uric acid. Uric acid is really more frequently a source of error than is commonly supposed. I have myself seen the reaction due to it so vivid that I did not suspect it could be due to any reducing agent excepting sugar; but, noting the next day a copious sediment of uric acid which had fallen during the night, a testing of the supernatant fluid then revealed no reaction whatever. Such a urine, after being treated by the lead process to get rid of the uric acid, fails also to respond. But this process is very tedious,39 and cannot be conveniently carried out by the busy practitioner. The same thing is, however, accomplished by treating the urine with hydrochloric acid, which in twenty-four hours precipitates all of the uric acid. Simple precipitation by lead acetate solution and filtration does not answer, because all of the uric acid is not thus removed. Other substances, as hippuric acid, urates, hypoxanthin, etc., are said to act similarly, but they produce no practical interference with the test. On the other hand, a small amount of sugar may be present and yet fail to show the reaction, because the cuprous oxide is held in solution by certain substances. Such are ammonia and nitrogenous matters, including albumen, creatinin, pepsin, peptones, urinary coloring matters, etc. The latter probably produce their effect through the ammonia which is given off while heating them in the presence of an alkali. Hence all albumen should be precipitated and filtered out of urines suspected to contain sugar, and the heat applied should not be too great. Finally, excess of glucose will also hold in solution cuprous oxide, so that the suspected urine should not be added in too large a quantity at a time, but rather drop by drop.
39 The details of this process will be found in the writer's work on the Practical Examination of Urine, 5th ed., 1883, p. 63.
But qualitative testing is not sufficient during the treatment of a case of diabetes. The percentage of sugar and the quantity discharged in twenty-four hours should be determined occasionally. The process is done as follows: Place 10 cubic centimeters of Fehling's solution in a porcelain capsule, and dilute it with 40 c.c. of distilled water. Fill a Mohr's burette with the urine, which, if it contain more than 1 per cent. of sugar, should be diluted with nine times its bulk of distilled water. Slowly heat the contents of the capsule to boiling, and then allow a little of the diluted urine to run in from the burette; continue the cautious addition of urine and the gentle heating until the blue color is completely removed from the Fehling's solution. To determine the exact moment at which this takes place requires a little experience, but its recognition is facilitated by carefully tilting the capsule after each addition and stirring, so that its clear white surface may be seen through the edge of the fluid and contrasted with the latter. The number of cubic centimeters of urine used should now be read off from the burette, the number of c.c. of undiluted urine calculated therefrom, and each c.c. multiplied by .005 grm. The result indicates the quantity of sugar in grams in the urine employed, whence the percentage of sugar is determined, and also the twenty-four hours' quantity, the amount of urine passed in that period being known.
The Fermentation Test.—A very simple and easy method of determining the proportion of sugar is by Roberts's fermentation method, which, although not so precise as the volumetric process, is still sufficiently so for clinical purposes. A small piece of German yeast or a teaspoonful of liquid yeast is added to about four ounces (120 c.c.) of the urine, which is kept lightly stopped, at a temperature of 20° to 30° C. (68° to 80° F.), for about twelve hours; at the end of this time the sugar will have been converted into alcohol and carbonic acid. The latter will have passed off, and the urine lost in weight because of the destruction of sugar; while the difference between the specific gravity before and after the fermentation indicates the number of grains of sugar per fluidounce. Thus, suppose the specific gravity before fermentation to have been 1040, and afterward 1025; there will have been 15 grains of sugar to the fluidounce, whence, again, the twenty-four hours' quantity can be calculated. If the metric system is used, each degree of specific gravity lost will correspond to .2196 grams of sugar in every 100 c.c. of urine.
The specific gravity of the fermented urine should be compared with that of the urine soon after it is passed, because saccharine urine under suitable circumstances undergoes fermentation without the addition of yeast; and, the specific gravity being thus lowered spontaneously, the reduction in the urine fermented by yeast would appear less than it actually is. At the same time, care should be taken that the urine is of the same temperature when the specific gravity is taken before and after fermentation.
The Picric Acid and Potash Test.—Although attention was called in 1865 by C. D. Braun,40 a German chemist, to a reaction between grape-sugar and picric acid, as the result of which the latter is converted into picramic acid, very little attention seems to have been paid to this announcement. Quite ignorant of it, George Johnson rediscovered this reaction in 1882, and published it in 1883.41 It is applicable to both qualitative and quantitative purposes. In order to make use of it, a standard comparison-solution is made as follows: Take 1 fluidrachm of a solution of grape-sugar, 1 grain to the fluidounce; mix it in a long test-tube with half a drachm of liquor potassæ (U. S. P. or B. P.) and ten minims of a saturated solution of picric acid; dilute the mixture to 4 fluidrachms with distilled water, to facilitate which a tube used for the purpose may be marked at 4 fluidrachms. Raise the mixture to the boiling-point, and continue the boiling for sixty seconds, to ensure complete reaction between the sugar and picric acid. During the boiling the pale-yellow color of the liquid is changed to a vivid claret-red. Cool the liquid by cautiously immersing the tube in cold water, and if it is not then at the level of the 4-drachm mark, raise it to this by adding distilled water. The standard color thus obtained is that which results from the decomposition of picric acid by a grain of sugar to the ounce, four times diluted, or by a solution of sugar containing one-quarter of a grain per ounce. But the picramic solution rapidly becomes pale on exposure, so it becomes necessary to make a more permanent solution to use as a standard. This may be accomplished by combining liquor ferri perchloridi drachm j, liquor ammonii acetatis drachms iv, acidum aceticum (glacial) drachms iv, and water enough to make ounces iiss. The color of this is identical with that of the picric acid reduced by a one-grain solution diluted four times, and, according to Johnson, it will retain its color unchanged for at least six months. At the same time, whenever a new solution is made it should be compared with that of the one-quarter grain per ounce solution of sugar, boiled with picric acid and potash.
40 "Ueber die Umwandlung der Pikrinsaüre in Pikramminsaüre, und Ueber die Nachweisung der Traubenzucker," Zeitschrift für Chemie, 1865.
41 British Medical Journal, March, 1883.