ON THE TRUE SUGAR CONTENT OF SKIN AND OF MUSCLE IN DIABETIC AND NON-DIABETIC PERSONS

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1 ON THE TRUE SUGAR CONTENT OF SKIN AND OF MUSCLE IN DIABETIC AND NONDIABETIC PERSONS BY HARRY C. TRIMBLE AND BENJAMIN W. CAREY, JR. (From the Biochemical Laboratory of Harvard Medical School, Boston) (Received for publication, December 22, 1930) Recently Folin, Trimble, and Newman (1) in the course of an investigation of the distribution and recovery of dglucose injected intravenously into animals observed that shortly after the sugar had been introduced the skin contained increased quantities of reducing substances and the concentration approached that in the blood. And when a series of specimens was taken from the same anesthetized animal at regular intervals after the sugar injection it was noted that the reducing content of this tissue declined in such a manner that it always remained beneath but more or less parallel to that in the circulating fluid. In these same experiments the reducing substances in muscles were found t o be elevated also but to a much smaller extent than in the skin. In that research the analytical methods employed were sufficient to measure merely the total reducing substances of the tissues examined and it was not possible to state just how much of this represented true sugar. More recently Hiller, Linder, and Van Slyke (2), Folin and Svedberg (3), Somogyi (), and Benedict (5) by their investigations have established that, by employing only short periods of fermentation with washed yeast, the true sugar of blood or of blood filtrates can be measured and have described various procedures for accomplishing t,his. In the light of this advance it became of interest to know to what extent the elevation of reducing substances in skin and muscle observed by Folin, Trimble, and Newman represented real increase of carbohydrate. And since the earlier research had furnished information only with regard to experimental animals it seemed desirable that upon t.his occasion we should devote our attention to the situation existing in human subjects. 655

2 True Sugar of Skin and Muscle During the preparation of this manuscript for publication there has come to our attention the recent report of Urbach and Sicher (6) upon the sugar content of skin under normal and pathological conditions. These investigators were able to employ a micro procedure in which small cylinders of skin, weighing 0 to 150 mg., were removed from an animal or a patient with an electric cutting appliance and then the total reducing substances were measured by a modified HagedornJensen method. The results which they report for single specimens from normal and diabetic individuals are in general agreement with those which we record below. Of particular interest are some experiments of these workers in which they obtained a series of specimens from the same individual, covering the hours immediately after intake of glucose. The finding that under such circumstances the total reducing substance of the skin tends to rise and then fall parallel with the sugar in the blood is in agreement with that observed by Folin, Trimble, and Newman in their experiments on animals. EXPERIMENTAL Plan of Experiments Specimens of human skin and of muscle were obtained fresh from the operating rooms of The Palmer Memorial and The New England Deaconess Hospitals. Breasts removed by radical technique, limbs and feet amputated from diabetic patients, and skin cut away during abdominal operations provided liberal quantities of material from which portions of tissue well outside of any infected area were excised. Then the sugar was extracted from weighed portions of the material and its concentration de: termined. A sample of blood taken near the conclusion of the operation enabled us to obtain pract,ically simultaneous values for the sugar contents of blood, skin, and muscle under the conditions studied. Methods Extraction of TissuesThe specimen was obtained immediately after its amput,at,ion by one of us present in the operating room at the time. It was carried to a laboratory just outside where portions of skin were quickly dissected free from underlying fat. Usually portions of 50 to 0 gm. were obtained (though muchless

3 H. C. Trimble and B. TV. Carey, Jr. was employed satisfactorily upon several occasions when the amount available was small). The specimen was weighed (to gm. only) and then plunged into an approximately equal weight of boiling water contained in a beaker. Heat was applied at once and maintained until the liquid again boiled. Ordinarily the tissue was cut into small strips and sometimes was passed through a food chopper before dropping it into the hot water. It was not observed that these variations in the treatment had any effect upon the concentration of sugar found. While one individual cared for the specimens of skin another cut off sections of muscle (pectoralis major and gastrocnemius being used most frequently), weighed them, and likewise plunged them into boiling water. It was considered important that the interval elapsing between the amputation of the tissue and the stoppage of enzyme action by heat should be as small as possible. In our experiments this was usually 5 and never more than minutes. The specimens were then removed to our laboratory for extraction and analysis. In accomplishing this it was our custom to employ three or four successive treatments for periods of to 15 minutes with portions of boiling water sufficient completely to cover the tissue. The extracts were poured off through a filter of cheesecloth (previously washed and extracted with boiling water) upon which the bulk of the tissue was collected. Then by drawing toget.her the upper edges of the cloth and squeezing gently it was possible to press out most of the liquid from the tissue and thus insure almost complete change of the extract.ing fluid at the end of each treatment. The nearly dry residue was then readily returned to the extraction beaker for further treatment. The specimens used were always cut into strips or ground up by passing through a food chopper while in the first extraction liquid (if this had not already been accomplished before they were plunged into the boiling water). Also at this time the chopped tissues were further macerated by rubbing in a mortar with about 15 gm. of washed quartz sand. Finally the combined extracts were mixed, cooled, and their total weight determined. At this stage any fat which had solidified was collected, weighed, and then an appropriate correction was applied to the weight of the tissue originally taken for analysis.

4 658 True Sugar of Skin and Muscle Removal of ProteinIn the case of blood the procedure of Folin and Wu (7) was employed. As in our previous research the extracts of muscle and of skin were freed from protein and clarified by precipitation with tungstic acid, followed by treatment with Lloyd s alkaloidal reagent (8). In detail the procedure has been as follows: We customarily found it convenient to take volumes (0 cc.) of extract and to add 1 volume ( cc.) each of per cent sodium tungstate and of $ N sulfuric acid. After shaking vigorously and filtering, 20 cc. of the filtrate were mixed with 5 cc. of 0.1 N sulfuric acid and about 1.5 gm. of Lloyd s alkaloidal reagents were added. This was agitated gently for 2 minutes and then filtered. Measurement of Total Reducing SubstancesThis last filtrate was analyzed for total reducing substance with the modified copper reagent of Folin and Wu (9). Usually 2 cc. of this filtrate contained a quantity of reducing subst ance appropriate for colorimetric comparisons. At times, however, it was necessary to make dilutions of this material before analysis or to employ a more dilute sugar standard. Anhydrous glucose from the United States Bureau of Standards served for the preparation of all standard solutions. In computing the concentration of reducing substances per 0 gm. of fresh tissue the concentrations found in the filtrate analyzed were multiplied by factors which compensated for the dilutions during protein removal and treatment with Lloyd s reagent. This product when multiplied by the total volume of extract and divided by the weight of tissue taken gave the concentration of total reducing substance (expressed as mg. per cent) in the fresh specimen. Measurement of NonFermentable Reducing SubstanceFor this we have employed fermentation by yeast by means of a procedure essentially like that of Benedict ((5) p. 65). In detail our process has been as follows: Preparation of Washed YeastIn each of a series of centrifuge tubes about onesixteenth of a cake of Fleischmann s compressed yeast was placed and cc. of distilled water added. With a stirring rod the yeast was rubbed and stirred until a uniform suspension resulted. Then the tubes were centrifuged and the supernatant fluid decant.ed. A fresh port,ion of distilled wat,er was added and the washing process twice repeated. Treated in this

5 H. C, Trimble and B. W. Carey, Jr. 659 way the fourth aqueous extract of the yeast gave a negligible blank with the FolinWu copper reagent. If the material employed was not fresh, or if it had stood in a refrigerat.or after having been washed a day or more previously, satisfactory blanks were not obtained. Fermentation of the Tissue Extract cc. of the filtrate obtained after t,he treatment with Lloyd s reagent (see above) were added to a centrifuge tube containing washed yeast. The contents were thoroughly mixed until a uniform suspension resulted and then placed in a beaker of water maintained at 370 for 15 minutes. The mixtures were centrifuged to throw down the yeast cells. 2 cc. of the supernatant fluid were analyzed for reducing substance, by means of the revised FolinWu copper method. A sugar standard containing onefifth of the glucose in the weak standard of Folin and Wu was frequently necessary. In every instance an aqueous extract of the yeast employed was analyzed at the same time and in no case was it found that the washed yeast used was contributing any appreciable portion of the nonfermentable residue found. The concentration of nonfermentable material extracted from the tissues was computed in a manner similar to that mentioned above for the total reducing substance. The values for the true sugar present in 0 gm. of blood or of t issues were calculated by subtracting t,he nonfermentable fract,ion from the total of reducing substances found before fermentation. During the course of these experiments accurat ely measured quantities of pure glucose in varying amounts were introduced into different extracts before precipitation of the protein. The sugar added was accounted for quantitatively by the increase of total reducing substance. Added glucose caused no change in the nonfermentable reducing material. Results The results obtained are presented in Table I. In the case of the nondiabetic subjects it may be observed that in every instance the total reducing substance of the blood was elevated slightly above the normal levelwhich is accounted for by t he anesthesia employed. In a number of these cases t,he nonfermentable material of the blood was not measured. However, our experience with similar material indicates that this fraction usually

6 660 True Sugar of Skin and Muscle TABLE I Sinzultaneous Measurements of Sugar Content of Human Blood, Skin, and Muscle Reducing substances are given in mg. per cent. Blood Skin Muscle Insulin dosage ~ Subject Mrs. By 1521 Pt 136 Tr 12 Miss Cy 122 Mrs. Sn 120 Mr. Sr 119 Pt 8 Averages Mrs. Ye 360 m1t 30 Mr. Ln5 339 Mrs. Ba 298 In 295 Mr. Vt! 266 Mrs. Md 260 Mr. Ce 228 Mrs. Ss 196 Mr. Ln/l 186 ( vq 170 Mrs. Hk 15 Mr. Rl 129 Sn 120 Nondiabetic Diabetic cases* cases _. 28 Averages * Gasether anesthesia was employed in all of the nondiabetic cases. 7 All values were calculated in terms of glucose. $ Novocainespinal anesthesia was used for all of the diabetic cases except nitrous oxide for Mrs. In and Mr. Ce. 0 First operation. 11 Second operation _

7 II. C. Trimble and B. W. Carey, Jr. 661 constitutes in the neighborhood of 15 mg. per cent when the FolinWu copper method is used. The true sugar of the whole blood in these nondiabetic cases therefore may be estimated without serious error by subtracting this quantity from the total reducing substance reported. This would give an approximate average value of 1 mg. per cent for our cases. The total reducing substance in the skin of nondiabetic subjects averaged somewhat more than onehalf of that found in the blood, and when allowance for the nonfermentable portion is made that ratio is lowered only slightly. In muscle the total of reducing substances approached that in the skin, but the proportion which was fermentable was smaller. This indicates, therefore, that the true sugar of nondiabetic muscle is considerably below both that in the skin and the blood. Had our analyses been restricted to that portion of the circulating fluid which is in most int,imate contact with the tissues then the ratios obtained would be even less favorable to skin and muscle than those mentioned, since the plasma is known to contain but little of the nonfermentable reducing mat.erial. With the diabetic individuals it is readily evident that the total reducing material of the skin was increased very definitely and decidedly above nondiabetic levels, while the nonfermentable portion was augmented but little. The data obtained from the diabetic cases are recorded with the values of the total reducing substance found in the blood arranged in a descending order. In general it will be noted that this, with few exceptions, likewise arranges the true sugar content of the skin in gradually decreasing concentrations. The true sugar of the skin appears to average about 65 to 75 per cent of that in t,he blood. That this relationship between blood and skin sugar likewise holds true for a given individual when observed at different times& shown by the data recorded for Mr. Ln and for Mr. Vt. In these two cases we were fortunate in being able to obtain two successive specimens at intervals several weeks apart. In each instance clinical treatment had lowered the blood sugar level and this was reflected in the tissue sugar content as well. In the muscles of the diabetic subjects the tot al of reducing substances was greater than that in the nondiabetic subjects, while that fraction which was fermentable averaged approxi

8 662 True Sugar of Skin and Muscle mately the same in both series. The net result indicates a slight but definite increase in the true sugar of the diabetic muscle. In the nondiabetic muscles the true sugar content averaged about onefourth of that of the blood. With the diabetic cases the observed true sugar values did not follow the changes in the blood with as much regularity as with the skin. However the tendency to approach the relation existing between nondiabetic blood and muscle is evident. A majority of the diabetic patients whom we have had an opportunity to study had received insulin several hours before the tissues were removed. It seemed desirable to ascertain whether this factor might have been responsible for the surprisingly low values for the true sugar of muscle obtained in such cases. Therefore we have been able to obtain a few specimens where insulin had not been administered at all, or where it had been omitted for a period sufficient for its action to have been exhausted. This group comprises about onethird of the diabet.ic cases recorded in Table I. The individual instances may be located by reference to the data on insulin dosage which are given there. If attention is focused upon t,hose who had not received insulin for at least 17 hours previous to the surgical operation at which their tissues were removed it will be noted that only in one instance (Mrs. Ba) did we encounter any value for muscle sugar which could be classified as definitely higher than in those individuals with corresponding blood sugar levels where recently administered insulin could still be exerting an influence. On the other hand, if the muscle sugar values of the insulinized and noninsulinized groups are averaged and compared the values are, respectively, 51 and 52 mg. per cent. The series of observations presented is rather brief and the similarity just mentioned may be merely a coincidence. However it furnishes but little support for an attempt to ascribe the low values for muscle sugar recorded above solely to the externally administered insulin. SUMMARY AND CONCLUSIONS Data upon the total reducing substance, the nonfermentable material, and the true sugar content of specimens of blood, skin, and muscle from nondiabetic and from diabetic patients are presented. In the nondiabetic group the true sugar content of

9 H. C. Trimble and B. W. Carey, Jr. skin and of muscle averaged respectively 56 and 28 mg. per cent. For some cases of diabetes the corresponding averages were 1 and 51 mg. per cent when the average true sugar of t.he whole blood was 226 mg. per cent. Thus it is shown that elevation of the sugar concentration in the blood is accompanied by a marked absolute increase of the quantity in the skin while the elevation in muscle is much smaller. The sugar content of muscles from diabetics who had received insulin at short intervals previously averaged almost the same as in the group where externally administered insulin was no longer believed to be exerting an effect. To various members of the staffs of the hospitals mentioned and especially to Drs. L. S. McKittrick and Shields Warren, we express our appreciation for their cooperation in enabling us to obtain the material for this study. We are glad to acknowledge also a grant from a Fund for Research upon Diabetes given by Mr. Francis P. Garvan to Dr. Elliott P. Jo&n. BIBLIOGRAPHY 1. Folin, O., Trimble, H. C., and Newman, L. H., J. Biol. Chem., 76, 263 (1927). 2. Hillrr, A., Linder, G. C., and Van Slyke, D.D., J. Biol. Chem., 6, 625 (1925). 3. Folin, O., and Svedberg, A., J. Bid. Chem., 70, 05 (1926).. Somogyi, M., J. Bid. Chem., 75, 33 (1927). 5. Benedict, S. R., J. Biol. Chem., 76, 57 (1928). 6. Urbach, E., and Sicher, G., Arch. Dermat. u. Syph., 167, 160 (1929). 7. Folin, O., and Wu, H., J. Biol. Chem., 38, 86 (1919). 8. Folin, O., and Berglund, H., J. Biol. Chem., 51, 209 (1922). 9. Folin, O., J. Bid. Chem., 82, 91 (1929).

10 ON THE TRUE SUGAR CONTENT OF SKIN AND OF MUSCLE IN DIABETIC AND NONDIABETIC PERSONS Harry C. Trimble and Benjamin W. Carey, Jr. J. Biol. Chem. 1931, 90: 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