(One Figure in Text.) added, in the case of the blood, that of the extraction of the sugar fromn

Transcription

1 A METHOD FOR THE ESTIMATION OF SUGAR IN B1LOOD. BY E. WAYMOUTH REID, Professor of Physiology in University College, Dundee. Grocers' Company Research Scholar. (One Figure in Text.) To the main chemical difficulty of accurate estimation of glucose is added, in the case of the blood, that of the extraction of the sugar fromn a solution rich in proteids yielding on precipitation a coagulum from which the last traces of the substance are as a rule with difficulty washed; indeed the extent to which the proteid precipitate lends itself to easy extraction is after all, provided the method of sugar estimation is reliable, the gauge of the practical value of any method. In the following method the results have been obtained by using (i) Phospho-tungstic acid as the proteid precipitant, (ii) The filter-plate as an extraction apparatus, (iii) The Allihn-Soxhlet gravimetric process for the estimation of the extracted glucose. The phospho-tungstic acid precipitate of the blood-proteids when heated and subsequently cooled, takes on an almost mineral character, can be ground in a mortar to extreme fineness, and when thrown upon a filter-plate of proper construction and sucked dry by a pump after each application of wash-water, can be efficiently extracted with a smaller quantity of water than by any other manipulative method with which I am acquainted. As will appear from an analysis quoted below, glucose is in no way affected by this reagent. Into a beaker of about twenty ounces capacity and of the shape known as " medium " are potured 250 c.c. of a 7 0/0 solution of phosphotungstic acid holding 2 c.c. per 100 of commercial hydrochloric acid. A glass rod provided with a piece of rubber tubing over one end is placed in the beaker and the whole tared. The animal is now bled to the extent of about 50 c.c. of blood from a cannula with a fine nozzle, the contents of the beaker being stirred as the blood flows into the solution, and the gross weigfht taken to obtain the weight of blood used for analysis. The mixture is now raised to boiling in an oil bath to avoid

2 SUGAR IN BLOOD. 317 fracture (an enamelled iron mug holding castor oil is convenient), atnd is well stirred as the temperature rises. The height of the beaker allows ample room for possible frothing. The proteids soon collect into a mass, at first spongy and floating, but later, as the mass cools, hard and lying at the bottom; so hard indeed that care must be taken that the beaker is not mechanically fractured'. Any flocks of coagulated proteid on the side of the beaker are rubbed down with the rubberslhod rod. The whole is cooled, since the phospho-tungstic precipitate of the blood proteids is slightly soluble in hot solution. The supernatant fluid is filtered off into a porcelain evaporating basin, the filter washed into the same basin, the contents nearly neutralised (left acid) with sodic hydrate, and at once set on the water bath to reduce in volume. The proteid mass is emptied into a ten-ounce mortar and ground till it assumes the fineness and appearance of chocolate paste, the pestle washed into the mortar, and the contents of the latter thrown on to a 6 centimetre filter-plate2, lying in a 5j inch funnel fitted to a filter-flask and pump. The beaker and rod are now finally washed on to the mnass lying on the plate and the final mortar and filter paper washings added. The mass is sucked dry, the pump turned off, the fuinnel filled up with fresh water, and the process repeated three times. Cold water is used in preference to hot to keep the proteids out of solution as far as possible. The whole washings measure less than 500 c.c. These washings are now transferred to a large boiling flask (preferably a 2-litre Jena glass flask), nearly neutralised with sodic hydrate, but left acid, boiled down with the flask on the slant to about 50 c.c., and added to the material in the porcelain basin on the water bath. The whole mass may be reduced to about 50 to 70c.c. and is finally cooled, almost exactly neutralised, filtered and made up to from 100 to 150 c.c. for estimation of sugar. As regards the estimation of glucose, it is well known that excess of copper in the mixture of Fehling's fluid and glucose solution increases the reducing power of the sugar, so that in running in sugar solution slowly into a fixed volume of Fehling's fluid, the amount of copper 1 This remark refers particularly to dog's blood: with ox blood the precipitate is softer. If more than 50 c.c. of blood has been inadvertently added to the 7 0/0 phospho. tungstic acid solution so that the clot is not well formed, a few drops of a saturated solution of the acid may be added at this stage. *2 The filter-plates that act most efficiently are those with fine holes and a rubber ring made by Messrs Kaehler and Martini of Berlin.

3 318 E. W. REID. reduced by each successive addition of sugar solution is a dimitnishing quantity, as the dilution of the Fehling's fluid increases. This trouble is of course usually dealt with by making a series of titrations adding to a known volume of Fehling's fluid, all at once, a certain volume of the sugar solution and testing the acidified filtrate of the mixture for copper by potassic ferrocyanide. When of two volumes of sugar solution differing by only A c.c., one gives copper in the filtrate and the other does not, the mean volume is taken as that necessary to exactly reduce the copper in the volume of Fehling's fluid taken. Such a procedure not only involves the use of considerable quantities of sugar solution, but is very tedious. In the Allihn-Soxhlet gravimetric method, into a relatively large fixed volume of Fehling's fluid is discharged at once a relatively small fixed volume of sugar solution not over 1 0/0 strength, so that the excess of copper is kept as nearly constant as possible. The time of boiling in each titration is in all cases two minutes. The copper oxide is then collected, reduced to metallic copper, and weighed. In this process then, the amount of copper, the concentration of its solution, and the time of boiling are constant in all titrations, the variable factor is the amount and concentration of sugar in the mixture dependent upon the concentration of the small fixed volume of solution added. The number of milligrammes of metallic copper reduced by a given numnber of milligrainmes of pure glucose under the conditions has therefore to be experimentally determined at all the different concentrations, from the weakest at which any appreciable reduction occurs, to the strongest within the limits set, i.e. the fixed small volume of 1 0/0 glucose. This has been done for all the sugars and the results are collected under the title of Wein's Tables'. Since this extrenmely simple and reliable method seems to be little known among physiologists it may be of advantage to give the details here Ȧ tube, similar to that in the accompanying figure, is packed with a glass wool plug which is covered with asbestos that has been well boiled out in alkali, acid and water. 1 Dr Ernst Wein. "Tabellen zur quantitativen Bestimmung der Zuckerarten." Stuttgart. Max Waag. 2 These tubes can be obtained from Messrs Bender and Hobein, Gabelsbergerstrasse 76a, Munich.

4 SUGAR IN BLOOD. After a wash with alcohol and ether the whole is dried at 1100 C., cooled in a desiccator, and tared. Into a porcelain basin are placed 60 c.c. of Fehling's fluid, to this 60 c.c. of water added, and the whole caused to boil briskly: 25 c.c. of the sugar solution, concentration of which does not exceed 1 0/0, is now discharged from a pipette into the boiling Fehling's fluid and the mixture boiled for two minutes (a sand glass is convenient). Filtration is now effected by the pump through the asbestos filter-tube, a funnel being temporarily fitted by a cork to the wide end. This filtration must of course be effected as rapidlv as is consistent with the complete collection of the cuprous SBESTOS oxide on the asbestos to avoid any chance of re- 4...G LASSWOOL solution on cooling. The basin and rod are thoroughly washed into the tube with freshly boiled hot water, and after treatment with alcohol and ether, the tube again dried. Hydrogen, FIG. 1. Re washed in acid permnanganate of potash solution duced to eths. and dried in a sulphuric acid bead-tower, is now passed through the cuprous oxide and the reduction to copper effected in a few minutes by heating. Cooling of course takes place in a current of hydrogen to prevent re-oxidation. The tube is finally weighed and from the number of milligrammes of copper the weighlt of glucose read off in the Table. Two estimations can be made in an hour, and the method is far easier to work than the gravimetric method by electrolytic deposition on platinum. The maximum error in duplicate analyses should be one milligramine of copper, which corresponds to about half that weight of glucose, though as will be seen from the Tables the ratio varies at different concentrations. I quote two analyses at different concentrations to indicate how accurate this method may be with pure glucose in solution in water. 0/0 glucose solution mgs. copper from mgs. glucose taken. 25 c.c. of solution. per Table. mean. 01 found..~ * *4996. '1225 (59 8 = 30' *1228. ~~

5 320 E. W. REID. To prove that phospho-tungstic acid does not affect the estimation of glucose by tlle method I quote the following analysis: (1) *4213 gr. glucose dissolved in 150 c.c. of 10 0/0 phospho-tungstic acid and 3 c.c. of commercial hydrochloric acid added. Boiled. Cooled anid nearly neutralised with sodic hydrate, but left acid. Digested on water bath for 3 hrs., cooled, neutralised and made up to 200 c.c. 25 c.c. gave mgs. of copper = 52'6 mgs. of gltucose. i.e. the 200 c.c. estimated to contain * 4208 gr. glucose. Next as regards the recovery from blood of sugar added thereto. 49'51 grs. of freshly defibrinated dog's blood were extracted by the process described above. Final volume of extract 100 c.c. 25 c.c. gave 3068 mgs. copper (mean 30'7) = 16'4 mgs. glucose. Reducing power of total extract of 49'51 grs. blood = '0656 grs. glucose. To 50'10 grs. of same blood added '0824 grs. pure glucose. Total glucose as per previous estimate = *14878 grs. Extracted as above. Final extract measured 100 c.c. 25 c.c. extract gave 7128 mgs. copper (mean 72) = 368 ings. glucose. total extract held * 1472 grs. glucose. Loss * gr., i.e /0. (2) grs. defibrinated ox blood (shed i hr. previously) extracted. Extract measured 125 c.c. 25 c.c. gave 178} mgs. copper (mean 18'2) = 10'1 mgs. glutcose. Reducing power of total extract of grs. blood = '0505 grs. glucose. To 51'48 grs. of same blood added '0938 gi's. pure gluicose. Total glucose as per previous estimate *1440 grs. glucose. Extracted as above. Final extract measured 125 c.c. 25 c.c. extract gave 54 '6 mgs. copper (mean 55l1) = 28-4 glucose. Total extract held '1420 glucose. Loss *002 grs. glucose, i.e. 1'39 0/0. (3) grs. defibrinated ox blood. Extract measured 125 c.c. 25 c.c. gave 10 mgs. copper = 6 1 mgs. glucose. Reducing power of total extract of 45'79 grs. blood = *0305 grs. glucose. To 44'86 gr. same blood added '1334 gr. pure glucose. Total glucose as per previous estimate '1633.

6 SUGAR IN BLOOD. 321 Extracted as above. Final extract measured 125 c.c. 25 c.c. extract gave i5ngs. copper (mean 63 8) = 327 mngs. glucose. Total extract held *1635 grs. glucose, i.e. no loss. Fouir analyses of blood fromn the carotids of dogs gave by thie above p1rocess the following percentages of glucose (reckoning the total reducing power as glucose): *1550/o, 131 /o, *167 0/0 and i1440/o. In conclusion I may add that in addition to its accuracy, the above tnethod is to be recommended for the ease and comparative celerity with which it can be conducted. Jiuly 28, 1896.