HX641 00910 QP99.3.B5 Sco8 1914 The content of sugar 1 Reprinted from the American Journal of Physiology Vol. XXXIV— June i, 1914— No. Ill THE CONTENT OF SUGAR IN THE BLOOD UNDER COMMON LABORATORY CONDITIONS By ERNEST LYMAN SCOTT [From the Department of Phyeiclcgy of Columbia Unb'ersity, New York] Submitted in partial fulfilment of the re'^nirements for the degree of Doctor of Philosophy, in the Faculty of Pura Science, Columbia University Reprinted from the American Journal of Physiology Vol. XXXIV — June i, 1914 — No. Ill THE CONTENT OF SUGAR IN THE BLOOD UNDER COMMON LABORATORY CONDITIONS By ERNEST LYMAN SCOTT [From the Department of Physiology of Columbia University, New York] Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Columbia University Digitized by tine Internet Arciiive ir in 2010 wit^^rtundin0 frg^ Open Knowledge Commons (fortne Medical Heritage Library project) Sco2 http://www.archive.org/details/contentofsugarinOOscot Reprinted from the American Journal of Physiology Vol. XXXrV— June i, 1914 — No. Ill THE CONTENT OF SUGAR IN THE BLOOD UNDER COMMON LABORATORY CONDITIONS By ERNEST LYMAN SCOTT [From the Department of Physiology of Columbia University, New York] Received for publication April 18, 191 4 I. — Introduction THE use of variations in the concentration of sugar in the blood as an indication of the response of the animal to experi- mental conditions offers many theoretical advantages over the use of the presence of, or variations in the amount of, sugar in the urine. This is true, first, because changes in either direction may be detected. While sugar is always present in the blood, it is ordinarily present in the urine in minimal quantities only. The urine, therefore, can ordinarily be used to show only an increase in mobilized sugar, while the blood will show either an increase or a decrease. Secondly, profound changes of concentration of sugar may occur in the blood without giving rise to a detectable glyco- suria. This may be due to the short duration of the change, or the change may not be of suffi.cient magnitude to lead to the excretion of sugar by the kidneys or it may possibly be due to a modification of the kidneys themselves. This very sensitiveness may, however, lead to serious difficulties in the handling of the animals before and during any experiment which involves the estimation of sugar in the blood. The third and most fundamental advantage lies in the inti- mate relation which exists, on the one hand, between the blood and the cells which are using the sugar and, on the other, between the blood and the stores of carbohydrate. Presumably it is the greater difficulty of technique which has deterred many investigators from using glycaemia rather than glycosuria, as the criterion of change in the organism. Too often this has detracted greatly from the value of the research. Others have recognized the more fundamental bearing of variations in the 272 Ernest Lyman Scott sugar of the blood but apparently without recognizing the great delicacy of the mechanism with which they were working. The result is that in many cases well-conceived experiments are largely vitiated by inadequate or improper controls. Hirsch and Reinbach and Roily and Opperman ^ have recently called attention to the necessity of controlling, as far as possible, every factor in experi- ments of this nature. The purpose of this paper is threefold: first, to determine, if possible, a set of conditions under which the amount of sugar in the blood of one laboratory animal, the cat, will be approximately constant. Upon such conditions, when once established, experi- mental conditions may be superimposed with reasonable assurance that differences from the constant are due to the new factors; secondly, to study the effects of some of the common conditions to which animals are subjected before being submitted to the experi- mental procedures in order that those which modify the concen- tration of sugar in the blood may be determined; and, lastly, to study, for the same reason, a few of the experimental procedures frequently used in experiments involving the estimation of the sugar in the blood. Abundant evidence from the literature, as well as my own work, shows that the most painstaking attention to all details is demanded if trustworthy results are to be obtained. Although, as will be seen from the above, the experiments to be reported were not primarily planned to throw light upon the problems of the mobilization and use of sugar by the organism, it is thought that some of the results found may have a deep physi- ological significance. An extended discussion of the theoretical or possible significance of my results would, however, be out of place at this time. The attempt is made to discover and catalogue a few of those disturbing factors which are constantly entering into our experiments, unbidden and frequently without our knowledge, and which lead us to false conclusions. Many, perhaps most, of the factors studied by me have been previously investigated for other animals and indeed some of them for the cat. It was never- 1 References to the literature cited will be found in Section VII, arranged alphabetically according to authors. Where more than one article is cited from one author the particular articles to which reference is made is indicated by the small numbers. Content of Sugar in the Blood Under Laboratory Conditions 273 theless thought desirable to correlate the results for a single animal and by a single uniform method. In part because there has been comparatively little work done upon the cat, and in part for the reasons given below, this animal was selected for the research. Long ago Boehm and Hoffman called attention to some of the advantages of cats for laboratory work. They mentioned especially their cleanly habits, their uniform size and the fact that they had found them to be more uniformly healthy than other common animals available for estimation of the sugar of the blood. There are, however, other and perhaps more fundamental advantages. A large amount of work has been done on excised muscles — a type of experiment for which the cat seems to be particularly adapted. Notes published by Lee and by Lee and Harrold show that some of this is directly related to the use of sugar by the organism. Again, some authors, as Macleod and Pearce, have sought to avoid, by decerebration, the extended use of drugs in experiments where prolonged anesthesia is necessary. The same results may be obtained in a more physiological manner, and with less hemorrhage, by cerebral anaemia. Leonard Hill and Stewart with his co-workers have shown that the dog, because of peculiar- ities of the blood-supply to the brain, is not so well adapted for this procedure as is the cat. Pike has confirmed Porter's state- ments that cats are better adapted for experiments involving vaso- motor responses than are dogs. II. — ■ Method of Analysis The preparation of the animal and method of obtaining the blood will be discussed later. Only the chemical processes in- volved will be described here. It is not possible to estimate the sugar by any known method in the presence of protein. Many reagents have been used and many methods proposed for the re- moval of the protein from blood preparatory to the determination of sugar. In 1908 ]\IichaeHs and Rona ^ proposed the use of colloidal iron hydroxide for this purpose. Their method has been well received and is widely used at present. Recently, however, Lesser reports that it is not satisfactory, in the form proposed by the authors for the blood of either frogs or turtles. In the 2 74 Ernest Lyman Scott limited use that I have made of the method I have found it fairly- satisfactory but have preferred the phosphotungstic acid method described below. A method which requires but a small amount of blood for the analysis possesses many obvious advantages. Be- cause of this the method recently described by Lewis and Benedict or the micro-chemical methods of Bang or of Michaehs will prove of great value to both the chnician and the experimentalist, pro- vided that they give the same satisfactory results in other hands that have been reported for them by their authors. Dehn and Hartman are now pubhshing a series of researches in which they are developing a method for the use of picric acid as the oxidizing agent in sugar determinations. Because of the greater deUcacy claimed for it the picric acid method may supplant the use of cop- per for this purpose. The method which I have used is very similar to the one used by Pfeffer for the removal of proteins from bacterial cultures prior to the determination of sugar. Reid and, more recently, Oppler have described methods for removing the protein from blood by this reagent. In my own method the blood was drawn directly from the blood-vessels into a beaker weighed with sufficient i per cent am- monium oxalate to make the final concentration of oxalate in the blood about 0.25 per cent. The beaker was constantly shaken while the blood was being drawn. The second weighing was made at once. Any blood on the sides of the beaker was then washed down with distilled water and about 300 cc. of water added. This was done for the double purpose of preventing glycolysis, as suggested by Rona and Dobhn, and of breaking down the corpuscles so that any sugar contained within them might be freed. This was suggested by the work of A. Loeb, Rona and Michaelis,- Rona and Takahashi and others. As soon as the laking appeared complete the solution was washed into a 500 cc. volumetric flask, which was then filled to the mark with distilled water. It was then divided into two equal portions with the aid of a 250 cc. flask, and each portion was washed into a precipitation jar. About 1.2 cc. of a freshly prepared 10 per cent solution of phosphotungstic acid was then added for each gram of blood taken. This addition was made slowly from a dropping funnel while the mixture was being stirred with a mechan- ical stirrer. About twenty minutes was allowed for this precipita- Content of Sugar in the Blood Under Laboratory Conditions 275 tion, but, thanks to the dropping funnels and the stirrers, did not consume much time on the part of the operator. The result was a brown or chocolate colored precipitate, from which a limpid filtrate rapidly separated, that gave none of the common protein reactions. After the precipitation was complete, each portion was washed into a 500 cc. flask, which was then filled to the mark, and filtered through an ordinary filter without suction. An aliquot part of about 350 cc. was taken for analysis. The phosphotungstic acid was removed by the addition of 25 cc. of a saturated solution of barium hydroxide. After this addition, the mixture was allowed to stand for a time at room temperature. It must not be heated at this point, nor should it be allowed to stand much longer than is necessary to complete the reaction. The completion of the reaction was determined by the addition of a few drops of the barium solution to a few cubic centimeters of the clear supernatant fluid. When the reaction was complete it was again filtered and the precipitate was well washed with water. This filtrate was rendered just acid to htmus with sulphuric acid to precipitate the excess of barium, and the barium sulphate was removed by filtration. The final filtrate was evaporated to about 50 cc. in a Jena evaporating dish, and the sugar was estimated by the "Uniform method for sugar analysis" described by Munsen and Walker. Calculations were made from the tables given in Bulletin 107, Edition of 191 2, of the Bureau of Chemistry, United States Department of Agriculture. The Bulletin, in addition to the table, contains a brief description of the method. The accurate control of the estimation of sugar in blood or other solutions containing protein is very difficult. The present method was controlled as follows. A sample of blood was prepared as described above, except that a known quantity of glucose was added to one of the two portions just before the precipitation was begun. From this time on the estimation was completed in the usual manner. Evidently the amount of sugar recovered from the portion to which the addition was made, less the amount added, should be equal to that recovered from the other portion. Refer- ence to Table i will show that this was the case within the Hmits of error permissible for work of this type. This method presupposes that the added sugar exists in the blood in the same condition as 276 Ernest Lyman Scott S Q <^_- 00 10 <^ »^ p o *c ir5 >o OJ t^ fO I ro f— 1 a rt NO j 1 X W d 10 d d d o (^ NO '^ • -f t^ ^' d d d d d 6 ^ OS 0\ NO 00 00 •^ •^ NO c .2 _Q ** 1 ^ d d d d 'C S" ^ a NO NO + 5^ d d d d 1) r^ CO ON ^ NO =^ r^ t^ t^ ^ lO ^ NO t--^ ^ d d d d d 'J |X5 CO r-1 CO 4- 'o <^ d u J? '^ tC ^ Z d d d S .s 00 00 NO •^ «M Ov I--. ^ ,_, t fO NO NO >^ -O ^o d 1 ^ d d d Si "* Tf t— t + NO ,_l a! -<* T3 £; 7 c 0) > 'J t x •^ > a S 5^ .g l-l bO u \ bO en a ^ 1 3 _^ "b C T3 TJ "o rt 6 _o > H s s < Content of Sugar in the Blood Under Laboratory Conditions 277 that naturally present — a presumption which is by no means proved. For this reason, even though the results are satisfactory, one cannot be sure that all of the sugar has been recovered. The degree to which results obtained by any one method are consistent, one with another, gives another means of judging of the accuracy of the method. The reader will have to be the judge of the way in which the present method responds to this test after having studied the tables submitted — especially Table 5. Rona and IMichaelis ^ have compared different methods for the removal of protein from blood and have found a variation in the amount of sugar recovered after the use of the several methods. This, they beheve, is because the glucose does not all exist in the blood in simple solution. There is no present need for postulating the exact condition of the sugar, since any aggregation of the car- bohydrate molecules or any combination of them with either pro- tein or lipoid might easily interfere with the complete recovery of the dextrose by any of the methods available. Somq authors, notably Arthus, and Rosenfeld and Asher, have sought to show, by dialysis, that the sugar exists in the blood in simple solution. Consideration of the law of mass action, however, reveals the limitations of this method of attack. The equilibrium is at once destroyed by the removal of any portion of the sugar which may be in true solution. The disturbed condition will bring about a continuous dissolution of any loose combinations present so long as the removal occurs. In this way it is conceivable that a great deal of sugar may be removed by dialysis which did not originally exist in free solution. From these considerations it follows that before the work of different authors or the results obtained by different methods may be compared, a factor of comparison must be estabhshed. That is, the amount of sugar recoverable from the same blood by each of the methods, under the same conditions, must be found, and the resulting ratios considered in making comparisons. If it is true that the form in which the sugar is present in the blood influences the amount recoverable by the different methods, it follows that the amount recoverable by one and the same method may be expected to vary with the variation of the condition of the sugar or of any of its combinations which occurs as a result of 278 Ernest Lyman Scott experimental procedures. Thus, the difficulty of interpretation of results all of which are obtained by the same method is also in- creased. We have at present no means of knowing that the sugar exists in the blood in the same state under different experimental conditions to which the animal has been exposed. Thus a rise or fall in recoverable sugar following any experimental change to which the animal may have been subjected, may be due to a change in the condition of the sugar in the blood with no variation in the absolute amount. These possibilities of misinterpretation must be kept in mind in studying the following results. III. — The Effect of Some of the Preliminary Conditions UPON THE Concentration of Sugar in the Blood Under this head will come only those factors which, apart from the actual experimental conditions, interfere with the concentration of sugar. , The changes in environment undergone by an animal on enter- ing the laboratory cannot be presumed to be without influence upon the point in question. Hence, if uniform results are to be expected, sufficient time must be allowed for all of the animals to establish themselves in equilibrium with their new surroundings. Of the many factors that might play a part in bringing about varia- tions, two seemed especially liable to do this. These were, first, the changes in the character of the diet and feeding habits, and, secondly, the mental excitement incident to the new conditions. Time must be allowed the animals to establish themselves upon their new diet and to become accustomed to their new environ- ment. Of the two, very probably the latter is the more productive of variations. A week seemed none too long a time to allow to the animals for this purpose, and hence was taken as the minimum limit in the usual routine. The few animals which were killed after a shorter period in the laboratory will be specially mentioned in the tables. In Table 2 it is shown that the physical condition may be a disturbing factor. Here it is seen that the concentration of sugar may be high, as in numbers 58, 74 and 106, or be in essential Content of Sugar in the Blood Under Laboratory Conditions 279 8a « fM CJ en l-H 3 Jb -0 ^ OJ -i-" ° ^ >» £ (U C/D Ui 3 C -ri t/i ^ *--! C c3 a; a ^ rt >»«• U -^g s ,rt 'H4 fe.S 1 a. tn § " tn J3 (U > '^ 81^ -0" rt fe --3" bO c3 1 < '•S} h-1 P^ W w hJ ro 1-4 .; 5 C2 ^ '■-1 Ov t^ OS 00 lO ■4-i —C CN "* 11^ «*-• ^ D ' + 1 1 + 1 + JS-a & 3 ^ tj 3 ■73 — > -S ^ " 1 _ Jg2 rt _0 :^ SO 3 00 10 ^ ?r, i ^^ ^-H 3 to d d d d d d "i ni u -" > i u "-o s 5 — OS U-! V— ( LO <>) so 10 CO CO TjH ^■^ 1— 1 <-< 1 2 c 3 vO 00 r^ .^ r^ r^i <>I re ^ CN CO 1 "^^ d d d d d d d + + 1 1 + 1 + -a ^5? 2i 10 t^ C^I 00 so r^ .^ . P i* r^ so so 00 •^< c y > S 3 p d d d d d d d ^ to jj I-. 00 CO r^ ^ so -0 bC 1 O; (M OS r-~ OS '*. 1 10 ^ ^' CO CO CO 8 - ro ro CN CN CO CN 'a a 00 •0 CO OS 00 ro ^ Os_ '*. 00 sl^ vd t~-^ iO CN OS 0^" 06 CM i:^ t--. lO t~-. 00 '"' jj r> (N to (N 1 OS ^. O; 10 C0_ m ^ rq r4 CN CN CN CO V3 1 ^ ^ S li^ ^ ^ ^ ^ 00 _ ^ UO so CO 10 SO t^ OS 28o Ernest Lyman Scott harmony with that of normal animals as shown by numbers 60 and 61; again in one case, number 95, which had apparently been running for a long time, the concentration was low. From this it is evident that one of the conditions for concordant results is the rigid exclusion of all animals which are not, so far as can be de- termined, in good health. While, as was shown above, a certain minimal stay in the laboratory should be allowed all animals before the sample is taken, too long a preparatory period is not desirable. The animals in general do not do as well in confinement as when free, and become especially liable to infection. For this reason, too, in experiments of long duration great care must be taken to protect the animal from all forms of infection and other influences which, aside from the purely experimental conditions, might lead to a changed physical condition. The length of the period intervening between the last feeding and the collection of the blood may be an important factor. Bang and others have fed animals varying amounts of different carbohydrates in solution, and have followed the resulting changes in the concentration of sugar in the blood. Boe agrees with Bang that the hyperglycaemia induced in rabbits by this means has disappeared by the end of the third hour. Fischer and Wishart report a return to normal, in dogs which have ingested fifty grams of glucose in solution, by the end of the second hour. There is no doubt but that such experiments are of great value in determining the changes in glycaemia which take place under the conditions of the experiment. However, conclusions as to the conditions follow- ing an ordinary meal must be drawn with caution, since the time relations following the ingestion of protein and fat, or of these with starch, are not necessarily the same. This is true not only because of the different quantities of carbohydrate taken into the body under the different conditions, but also because of the difference in rates of absorption dependent upon the necessity for digestion in the usual meal and the interference arising from the other elements of the meal. In any case, it was thought best to allow sufficient time for any passing disturbance to disappear. With two exceptions the animals were allowed to live from sixteen to twenty-four hours after the last meal before the sample of blood was taken. In a number of cases the alimentary canals were Content of Sugar in the Blood Under Laboratory Conditions 281 examined and found empty as far as the ileocoecal valve. Each of the two exceptions noted above were killed three hours after a meal, one of meat, the other of bread and meat. The one which had received meat alone, number 77, yielded 0.066 per cent sugar, which is, as will be seen by comparing with Table 5, in approximate agreement with the standard. The other cat, number 76, yielded a concentration of 0.086 per cent. This should, of course, be com- pared with the results shown in Table 4. When this is done it is seen that it is well within the limits of variation. Hence no signifi- cance can be attached to the variation in a single experiment from the average — 0.078 per cent — which is found for the correspond- ing series. There seems to be some difference of opinion with regard to the effect of the character of the diet. Seehg finds less disturbance of the concentration of sugar in the blood of dogs given ether when the diet has consisted of bread for several days than when it has consisted largely of meat. As this point is of so much importance to the experimentahst, some attention was given to it. A diet consisting only of bread was found to be impractical for cats, so that they were given stale bread and cooked beef hearts, approxi- mately pound for pound, together with the water in which the hearts were cooked. Even on this diet the animals did not do so well and were more subject to respiratory infection than those receiving the diet to be described later. It was not usually possi- ble to keep them in a satisfactory condition on this diet for a longer time than two weeks. Aside from this, or perhaps because of it, a constancy of results for the quantity of sugar in the blood could not be obtained which approached that with the other diet. The results obtained are summarized in Table 4. It will be noticed that the variation between the extremes — 0.056 per cent and 0.104 per cent — is equal to 86 per cent of the smaller number and that 83 per cent vary from the average of the series by more than 10 per cent. Evidently this is not a satisfactory diet where a constant concentration of sugar is the end sought. The other diet was cooked beef hearts with the bread omitted. This was found to be more satisfactory. While, as seen from Table 5, the extreme variation is between 0.096 per cent and 0.056 per cent and is thus almost as great as the variation of the previous 282 Ernest Lyman Scott series, only 25 per cent of the animals vary from the mean by more than 10 per cent. Cf. Table 9. Why the average for the animals allowed carbohydrate food in addition to the meat should be higher than that for those given meat alone, is a question difficult of answer. According to the ideas generally held, the character of the food is immaterial beyond the first few hours after the meal. Further, if the difference is due directly to the differences of diet, one would not look for the ex- treme variations which were found. There are other possibihties, however. Reference to Table 2 shows that animals with some types of infection seem to have a relatively higher content of sugar than the standard animals. As above noted, animals fed on the bread and meat diet are more prone to infection, and it is possible that in some cases incipient disease was overlooked. Again these ani- mals were more restless and quarrelsome than those on the meat diet, and this would tend toward higher results. Rose thinks that carbohydrate feeding does not materially increase the amount of sugar in the blood of rabbits. It is, though, well to note in this connection that the rabbit is a herbivore, and as such may have better provision for handling carbohydrates than the cat, which is by nature a strict carnivore. One experiment reported by Roily and Opperman ^ indicates that it is immaterial whether the protein given to the dog is derived from animal or vegetable sources. Jacobsen's ^ results are also of interest here. It has long been known that the more intense emotions are a frequent cause of glycosuria. This was early spoken of by Rayer and somewhat later by Frerichs. Recently Cannon and some of his co-workers have laid especial stress upon this form of glyco- suria; and have shown that for cats at least it is of purely psycho- logical origin. Pavy ^ speaks of the necessity for "tranquillity" on the part of the animal while the sample is being drawn, and Eckhard emphasizes the fact that rabbits must not be tied in the holder for work involving glycosuria. Naunym very early reported an increased amount of sugar in the blood of animals which had been bound. Among the later writers Jacobsen,^ Hirsch and Reinbach and Loewy and Rosenberg ^ have discussed in detail many of the difficulties in the way of the use of rabbits for experi- ments, of this type. Presumably this difiiculty hes, in large part at Content of Sugar in the Blood Under Laboratory Conditions 283 least, in the nervous disposition of these animals and their prone- ness to excitement. Roily and Opperman^'^ discard them as entirely unsuited for such work. Seelig finds no glycosuria in one dog which had been bound for two and one-half hours. Roily and Opperman ^ also think that dogs may be safely used for such ex- periments, while Loewy and Rosenberg on the other hand find the concentration of sugar in the blood of both dogs and of rabbits increased by sensory stimulation, though, it is true, the increase in the dogs was not so marked. Boehm and Hoffmann first demonstrated glycosuria in cats as a result of binding them on a holder and so called it "Fesselung Diabetes." This result has been interpreted as being due to various factors, as mental excitement, loss of heat, and muscular exertion. It has been shown in Cannon's laboratory that the first factor alone is sufficient. He therefore suggests the term "emo- tional glycosuria." The general fact that hyperglycaemia and frequently glycosuria follow excitement in all laboratory animals, with the possible exception of the dog, and in man is widely ac- cepted. The only reason for adding to the already extended literature of the subject is to find to what extent the handling of an animal which is necessary for obtaining a sample of blood or in preparing it for an experiment may disturb the standard conditions. In my experiments, all animals in which excitement was evident were discarded, except as noted in the tables. A few in which excitement was evident were killed, and the results proved the necessity of Pavy's rule of complete tranquilhty if consistent find- ings are desired. Two animals, numbers 108 and no, were held, as if given ether by a cone, though ether was not actually given in either case. A third was placed in a bell jar for about the length of time that would ha,ve been required for etherization, had ether been given. Others were subjected to other conditions which are apt to occur in the laboratory and which produced sHght excite- ment, as indicated by crying or otherwise. The results, with a brief description of the conditions in each case, are given in Table 3. It will be seen that in every case there is a noticeable rise in the amount of sugar contained in the blood. These results show that the animal must be, as Pavy says, tranquil, not alone at the time that the sample is drawn, but for some time before. From 284 Ernest Lyman Scott u w Ui fi >. , ^ ?^ ■M >-» ^ t-t c/3 )-• G c ■g 2 15 ■4-1 bC _3 >2 43 s a; -g bc bC.S P C CO ^ in G 2 bo c S G (fi 3 -0I5 "^ OJ S , 3 c/3 3 i; '-I cr -a g 3j bC T3 a G tfi ci 'IT 0^ a bo -a a 1— 1 (U c 1— 1 c75 "u G W vO '-' < -S a X fO c ,„ 2^- d C t« "^ ^ ro I>1 ro so 00 r-i T3 i^ ^ '"' -t ro CN ^ so Oi *-■ CV iS ^ >- 3^>. S.2 ^ CO ro OS SO •rf l>5g j^ t^ s rv-^ -f CO § J3 "—I '— ' >— 1 i"" d d d d d d d d U*" >*-. c T3 „ rt CO * ^ ^ OS r^ <-N) tn ^ 1 J:! C *^ ro t^ ^^ d _3 ro c^q CO 10 ^— ( ^-H d d d d d d d d < + + + + + + + + -a ^s^ ^ rN 00 00 ro OS CN so . p t^ On r^ ^ CN CO Gm, recov '•— ^ '-H d d d d d d d d ii a-^S C^ 't r4 00 rt^ UO T3 M t-~ rq >o so '^ Ov 00 0-^ ^ Ch d ^ CO d On' d 10* C> • "^ tN r<0 fo ro CN CN CN r:i^ ^ m -a c & ci rf s t^ ID ro vO "-O SO SO "1 0_ q rO sq OS 00 CO "S ^~' MO ^ 10 ,_,( 10 SO S^ ^ t^ vO vO SO "* 10 ^-^ LO OS T3 "1 »-H ro ^ T— * '— ' CN oi ro CN ro ro CN CN CN X fe ^ S S ^ [i^ fl^ P^ tN '^ ^ C-) 9S »o 10 t^ OS OS -I Content of Sugar in the Blood Under Laboratory Conditions 285 what has been said, it will be seen that those experiments in the past in which the blood has been drawn from an artery or vein without anesthetics have a very doubtful value, since it is hardly probable that an animal will undergo such an operation and remain in perfect tranquillity. The necessary restraint is of itself suffi- cient to influence the results as indicated by animals number 108 and no. Of the disturbing effect of anesthetics more will be said later. There remains then only the possibility of rapidly killing the animal with the least possible excitement and the rapid with- drawal of the sample of blood after death. This was long ago appreciated by Pavy, who killed his animals by pithing with a Bernard needle and then collected the blood from the heart or from the thoracic cavity after severing the large blood vessels. Sudden decapitation and collection of the blood from the severed neck vessels seemed to offer some advantages over Pavy's method, and was used throughout the present work. The interval during which the animal was held' before decapitation seldom exceeded three seconds, while about fifteen to twenty seconds more were required for the collection of the blood. As a further precaution against excitement, an attendant, from whom the animals were accustomed to receive food, brought them to the laboratory and assisted throughout the preparation of the animal and the collec- tion of the blood. That the amount of blood drawn relative to the total amount in the body may affect the concentration of sugar in the sample seems to have been overlooked by previous investigators. In studying this relationship a number of standard experiments were tabulated in the order of the increasing amounts of blood drawn, when this was expressed in grams per kilo of body weight. It was then found that the respective concentrations of sugar were arranged in the reverse order: that is, the more blood drawn per kilo of body weight, the lower is its concentration of sugar. This is wholly independent of the actual amount of blood drawn, as is shown below. The phenomenon is somewhat surprising since we know that hemorrhage under certain conditions causes hyperglycaemia. The relationship of which we are speaking, however, is not to be confused with the so-called "hemorrhage hyperglycaemia" as that term is commonly used. 286 Ernest Lyman Scott In the curve shown in figure i the data are derived from Table 5. Unfortunately the body weights of the animals used in the earher experiments were not recorded, hence these experiments are not available for our present use. The amounts of blood drawn 0.100 0,09C 0.08C2 0.07C 0.060 0.050 96 91 89" •50 49 10 20 •107 63 •69 •48 59 •47 50 Figure 1 40 per kilo of body weight are plotted on the x-axis. The concentra- tions of sugar in the blood, expressed in grams per cent, were plotted on the ^'-axis. Provisionally the line represented by the equation x/a + y/h = i is taken as representing the relation found. In the equation x and y are variables; x represents the amounts Content of Sugar in the Blood Under Laboratory Conditions 287 of blood drawn per kilo of body weight, and y the concentrations of sugar in the blood, a and h are constants whose values have not been exactly determined, but which approximate 133 and 0.084 respectively. If this relationship between the amount of blood drawn and its concentration of sugar is constant from one animal to another, and if the above formula is its true expression, ;y is a constant in the equation y = h {%' — x) / a -{- y' ; where a and h have the values as- signed to them above, x' and y' represent respectively the amount of blood drawn per kilo of body weight and its concentration of sugar in any particular experiment, x may have any arbitrary value. Under these circumstances y represents the concentration which would have been found had x grams of blood per kilo of body weight been drawn. This calculation has been made for a number of the experiments, x being taken as equal to 30. The results, when recorded in the tables, are found in the column headed values calculated for 30 grams per kilo body weight. Since there have not been enough estimations made to v/arrant the assignment of exact values to a and h, the optimum position of the curve was ascertained by trial and the values were found mechanically after having plotted the results on coordinate paper as for figure i . The results for the standard animals, calculated in the manner just described, are given in Table 5, column 10. A comparison of columns 7 and 10 of this table shows, first, that the mean for the series has not been modified; secondly, that the difference between the highest and the lowest results for the eleven animals compared is greatly reduced. Again the number of individuals which vary from the mean by more than 10 per cent is reduced from 4 to i, or from 36 per cent to 9 per cent of the whole number of animals compared. Thus it v/ill be seen that in general the calculated values approach still closer to a constant than do those derived directly from the analysis. In order to test this relationship still further the blood was drawn from four animals in a series of samples in each of which the sugar was determined. The results obtained confirm those ob- tained by the former method and are given in Table 6. A study of this table reveals that in every case, with the exception of the third sample in experiment 95, the concentration of sugar in any sample 288 Ernest Lyman Scott in the series is lower than for any sample in the same series pre- viously drawn. These results do, however, indicate that the curve representing the relation is not a straight line, but that it falls more rapidly at first than it does later. It was suggested that this relationship is due only to an error in analytical technique and so is of no physiological significance. For instance, it might be that a greater percentage of the sugar is recovered when only small amounts of blood are used. While it is true that such an error might lead to somewhat similar results, the TABLE 4 The Effect of a Diet of Bread and Meat upon the Concentration of Sugar in the Blood of Cats No. of Exp. Sex Days on diet Body wt. k. Blood drawn gm. Blood per k. body wt. gm. Gm. % glucose recov- ered Var. from stan- dard mean- table 5 Values cal. for 30 gm. blood per k. body wt. Absolute %of mean Concen- tration gm.% Var. % of stan- dard 45 F 24 2.50 93.29 37.12 0.100 + 0.031 45 0.105 + 52 46 M 12 2.94 101.42 34.50 0.070 + 0.001 1 0.073 + 6 53 M 24 2.98 83.62 28.06 0.104 + 0.035 51 0.102 + 48 54 F 24 2.75 79.55 28.93 0.060 - 0.009 13 0.059 - 14 55 M 24 2.50 72.66 29.06 0.056 -0.013 19 0.056 - 19 56 M 18 2.94 65.22 22.18 0.080 + 0.011 16 0.075 + 9 Mean 0.078 + 0.009 13 0.078 + 13 criticism cannot be valid for three reasons. First, it has been found that when the sugar in unequal samples of the same blood was determined, slightly smaller concentrations were found in the smaller samples. This was probably due to some slight negative error which has a tendency to be constant. This error would be multiphed by a larger factor when the absolute amounts recovered were computed to percentages and so lead to the smaller concentra- tions found in the smaller samples of blood. Thus it will be seen that in so far as this error would have any tendency, it would be to Content of Sugar in the Blood Under Laboratory Conditions 289 hide the relationship found rather than to simulate it. Secondly, the effect is the same whether the variation in the amount of blood per kilo of body weight is brought about by drawing different amounts of blood from animals of approximately the same weight or by draw- ing the same amounts of blood from animals of different weights. Compare experiments 59 with 91, and 69 with 89 in Table 5. TABLE 5 The Concentration of Sugar in Cat's Blood under the Conditions which Were Selected as Standard Blood Gm.% Var. from standard mean, table 5 Values cal. for 30 gm. blood per k. body wt. No. of Sex Days on Body wt. Blood dra^vn per k. body wt. glucose re- Exp. diet k. gm. gm. covered %of mean Concen- Var. % Absolute tration gm. % of stan- dard 12 M 1 — 97.86 — 0.062 - 0.007 10 — — 13 M 1 — 94.36 — 0.076 + 0.007 10 — — 15 — 1 — 129.73 — 0.063 - 0.006 9 — — 16 — 1 — 60.94 — 0.063 - 0.006 9 — — 23 — 5 — 116.33 — 0.070 + 0.001 1 — — 47 F 9 2.00 84.07 42.04 0.056 - 0.013 19 0.064 - 7 48 F 14 2.66 86.10 32.37 0.061 - 0.008 12 0.063 - 9 49 F 16 2.46 66.28 26.94 0.072 + 0.003 4 0.070 + 1 50 M 16 4.96 125.31 25.26 0.074 + 0.005 7 0.071 + 3 59 I\I 14 3.16 122.95 38.91 0.059 - 0.010 15 0.064 - 7 68 F 18 3.25 55.80 17.17 0.096 + 0.027 39 0.088 + 28 69 F 16 2.19 66.77 30.49 0.066 - 0.003 4 0.066 - 4 89 F 6 3.65 65.84 18.04 0.075 + 0.006 9 0.068 - 1 91 M 25 3.05 39.56 12.97 0.074 + 0.005 7 0.062 + 10 103 M 8 2.23 62.61 28.08 0.070 + 0.001 1 0.069 ± 107 F 10 2.25 68.35 30.38 0.073 + 0.004 6 0.073 + 6 Mean — — 2.93 83.93 27.38 0.069 — — 0.069 — 290 Ernest Lyman Scott Thirdly, if the figures given in Table 6 are compared, it will be seen that the progressive decrease in concentration is entirely inde- pendent of the absolute amount of blood drawn. No experiments have yet been made to determine the physio- logical significance of this decrease in the concentration of sugar when a larger proportion of the total blood is drawn at one time. The most plausible explanation which occurs to me is that it is due to the leaching of the tissue fluids into the blood vessels which occurs during severe hemorrhage. Evidence as to whether such a leaching does occur could be obtained by making simultaneous estimations of the sugar and the hemoglobin in the blood. Varia- tions of the viscosity of the blood might also be used to throw light upon this question. Professor Burton-Opitz has found that blood drawn 25 to 30 minutes after a severe hemorrhage has a lower viscosity than that drawn before the hemorrhage. More than this, he assures me ^ that from his experience he would expect that the last of a large amount of blood drawn at one time would have a noticeably lower viscosity than would the first portions. This harmonizes directly with the theory advanced above. Increased concentrations caused by the usual methods of with- drawal of blood have without doubt been the reason why the effect of the relative amount of hemorrhage upon the concentration of sugar in the blood has been so long overlooked. Factors are introduced by these methods which cause a greater or less discharge of the stores of glycogen, and so any small diminution in the concentrations of sugar in the blood is hidden. Pavy's method of collection should give the same results as mine, provided all the blood which has flowed from the vessels up to the time of collection is analyzed. However, I have been unable to find all of the necessary data in any of his tables. Schenck reports a very small difference between the first and second of two consecutive samples. In two experi- ments, the concentration of sugar in the second sample was less than in the first, and in one experiment, greater. Anderson, in two different experiments, finds almost the same concentration of sugar in each of three consecutive samples of blood. The later samples have, however, a shghtly greater concentration of sugar than the earher ones. Pavy ^ reports an increased amount of 1 Personal communication. Content of Sugar in the Blood Under Laboratory Conditions 291 sugar in the later samples of bullock's blood whether the animals were killed by the Jewish or by the pole-axe method. In none of the above were the samples so obtained that the discharge of stored glycogen would have been prevented. Though probably sex of itself has no influence upon the concen- tration of sugar in the blood (cf. Bang), it is quite possible that the greater excitability of the male cats which Cannon ^ has found to exist may operate through the mechanism for emotional glyco- suria to simulate such an influence. If this were true, it would be especially noticeable in experiments which involve much handling of the animal or its confinement in apparatus. While for animals kept under standard conditions I have found the mean for females slightly higher than that for males, the difference is small. More- over, the nature of the individual variations. Tables 4 and 5, make one hesitate to attach any significance to this slight difference. It is quite possible that the results would have been otherwise for a series of animals confined in some apparatus, as a respiration chamber, for a period of time. The only evidence that I have on this is drawn from two animals which were confined in a small cage and exposed to a lowered temperature for a period of two hours. A small female cat, number 92, Table 3, resented the treat- ment and yielded a concentration of 0.149 P^r cent of sugar, while a large male, number 93, was apparently tranquil throughout the period, and yielded a result even lower than usual, 0.049 P^r cent. It seems to me that the stress should be laid upon the nature of the particular individual, rather than in blindly choosing animals of either sex. The sex of almost all the animals used is indicated in the tables. The results for some of the longer series are sum- marized in Table 7. It is very doubtful whether there is any direct relationship between the body weight of the animal and the concentration of sugar in the blood, provided that the animals used are otherwise comparable. It must be borne in mind that variations in weight may be brought about by an abnormal physical condition, e.g., tuberculosis, and conversely these variations may be used as a means of detecting such abnormal conditions. A study of Tables 4 and 5 or of Table 8, in which the results bearing upon this point are summarized, reveals the fact that if the animals are divided 292 Ernest Lyman Scott < ^ m 0^ J3 o\ :i: ^ t-- ■<* r^ .0-^ CO ^5 CD ON,o "r; -t g^ r<- s^ 'C ?^ 00 T! ON Wf^ CN CO 00 •0 10 10 t--^ -d c ^ •* St^ rvi -u t^ 1-- IT 10 q d •P ^ >^-" r^ q s s:^ rvi d (N T) 0^ CN On' CN 00 d S" SJ 3 4J 0-. «f§ CN 1 «■ ■* 1 =1 c 2 lO +j (3 tn *-> cS Ov.^ "+ 03,0 >0 CN a ■'^ On 1^ x-o y CO ro l-lpg ^ ^ 1^ rt fi W) tc [/) -a >« c c -O ,, , -4-3 en L^ 3 cJ 6 s c <; U ^ ^ 0) 1 a _o d 0) (U S 0) 5 '^ ^ 1 ^ u 1 . § 1 1) in 4) On 3 ^ - ^ ^ ^ "emal 0.166 H T3 0) r^j lU 3^21 W f^ 1) =^ 1 6 1 1 rt fa f^ lU CN 3^-1 < -u 4. rt ^ £ CN 00 -a pL c -a 1 t^ K ^ l^ rt s oq <■ a. ^ P t-- rt fa <=i 0. OS 0: NC NO fA C -c * ro to Ov "0 CO -+ CN CO + + + + + + + + u< '-' ^ . rt g v« ^ "S^ -s g ro ro ■* NO CN) -SJ a. 3 J^ + + 1 1 1 10 + CN 1 1 ^_o 'rt 3 tj 1 K CO tN Cn) J^ CO >o CO uo _3 T! (^ 00 CN 10 On 13 t= rt '— ' '— " '— ' *— ' r^O ■•—1 *— • 6^ d d d d d d d d Ci ^ C/3 3 >oi On rO CO ^ •^ g ■" 5 c ON CN 10 On ro CN CO ro "-1 ^ + + + + + + + + ;-^ c^ > ^ ^f-, ro CnI r^4 't CN ^ 1^ tn + + 1 ' 1 10 + CN 1 1 4J g|^ ro r~] NO fO CN u ■^ 8 <^ 00 >o On, d S-H g T— 1 »-H c^ U d d d d d d d d -^i 00 <^ >o 4 ^ 1 -* ^. '*. 1 0' t— Ov' 1 6: ^ ^ 1 J^ 0\ 10 CO •^ CO Si g J2 + p2^ t^ 1 1 c ^« '—1 ro CN •- c rt g-g.a c.S S (J •- Mj3 t« . On +J "* CN "1 I On <» 1 f^ fO tN r^" cvi CN -, c D *^ • tn - 4 1 S ^ S 1 S % % 1 c (3 ^^1 vO Tt< 00 ^ NO t i< M3 1^ t^ <5 r^ 00 00 ^ -C g Content of Sugar in the Blood Under Laboratory Conditions 299 Seelig reports that ether gives much less trouble in this way with dogs which have been fed on bread for some time than with those which have been on a meat diet. Macleod also has sought to avoid the disturbing effect of ether in the same way. The results which I have obtained with cats fed on the bread and meat diet described on page 281 are shown in Table 11. A compari- son of these results with those given in the preceding table shows that while there is still so much variation that they would be un- satisfactory as a basis for experimental results, they are more uniform than those obtained with the meat diet. Also the cumula- tive ejffect of the ether is not so great. A definite relation between the ease with which the equiHbrium of the mobile carbohydrates of the body is disturbed and the type of diet given the animal would be of considerable theoretical in- terest. Such a difference must imply a difference either in the chemical form of the carbohydrate or in the tissues in which it is stored. This theoretical interest, together with the opportunity which might be offered the experimentaUst of reducing the varia- tions to a minimum, would warrant sufficient work to establish either the existence or non-existence of such a relation. This is especially true, since Seehg's results agree with those given above in indicating the hopeful outcome of such a research. The results given in Table 12 were obtained from animals which were to be used by a class of medical students. These animals were killed by decapitation as usual, but without special prepara- tion. The first eight were anesthetized by ether in a bell jar in the usual manner by the students. As soon as muscular relaxation had occurred, they were removed from the jar and decapitated at once. The last five were used for demonstration purposes and had been under ether for periods varying from an hour to three or four hours, during which time the operation indicated had been done. Since these animals were primarily used for other purposes, I was unable to record the full data. The results are, however, given in the hope that they will prove of some value, indicative as they are of the results which may be expected under ordinary laboratory conditions. Chloroform does not seem to offer any advantages over ether (Harley) and has the disadvantage of a greater toxicity. A few experiments of my own, hkewise, give no indication of any 300 Ernest Lyman Scott H %i U •o -^ ^ + + + + + + + CM ^-H + + <2 -- + I + + I + tN t^ -J< »-( IT; (^ O ~; O O Tf< ~ '* ^ o ;^. o "0 o oo — ^ CN > o + + + + + + + + + + + + + + + I I u tN'-iOv'^iOLO'-iO Tf — ■^ — ■— U-, l^CC -.-lY-l-HCNrsl — •-HtN £ 2 p .5 =« u c .n (U •^ t*ji! <1> Oi *J S B^ + + -* -H_ .^^ CO c-i CN r-i •^' — I CM t^ C^ piH tn § tn Ph ^ Content of Sugar in the Blood Under Laboratory Conditions 301 TABLE 12 Effect of Ether on Concentration of Sugar in Blood of Cats which Have Received no Especial Preparation No. of Exp. Sex Body wt. k. Amt. of blood gm. Concen- tration sugar gm. % Per cent of variation from mean of Remarks This series Stand, series Bread and meat series 37 M — 80.05 0.106 - 8 + 54 + 38 38 F — 75.25 0.104 -10 + 51 + 33 39 M — 97.25 0.151 + 31 + 119 + 94 40 F — 52.60 0.094 -18 + 36 + 21 41 M — , 80.60 0.123 + 7 + 78 + 58 42 M — 63.25 0.133 + 16 + 93 + 71 43 F 49.45 0.126 + 10 + 83 + 62 44 F 62.05 0.084 -27 + 22 + 8 Mean — — — 0.115 — + 67 + 47 251 — — 91.25 0.129 -40 + 87 + 65 Pleural puncture, treacheotomy 571 M 3.00 80.89 0.134 -38 + 94 + 72 Decortication 621 841 M M 3.25 2.80 55.66 79.92 0.239 0.298 + 11 + 39 + 246 + 332 + 206 + 282 Decortication less ether than no. 57 851 Mean M 2.75 72.80 0.274 0.215 + 27 + 300 + 212 + 251 + 176 Respiration stopped under ether 1 These animals were used for class demonstration and were under ether for at least one hour and in addition were subjected to the operations indicated. advantage to be derived from its use, since the results are essen- tially similar to those obtained by the use of ether (Table 13). Because of its stimulating action on the cat, no one would think of making use of morphine in drawing blood from this particular animal. It is, however, of interest to note that Luzzatto finds glycosuria following the use of morphine in rabbits. This finding is confirmed by Araki, who also reports similar results for 302 Ernest Lyman Scott TABLE 13 The Relation between Chloroform and the Concentration of Sugar in THE Blood of Animals Prepared in the Standard Manner No. of Exp. Sex Body wt.k. Time in min. from applica- tion of chloro- form to Amt. of blood drawn gm. Concen- tration sugar gm. % % var. from mean Remarks Muse, re- laxation Death This series Stand, series 87 88 97 Mean M M F 3.79 3.50 2.75 5.0 2.0 1.5 5.17 2.67 1.57 78.05 67.94 62.96 0.105 0.098 0.142 0.115 - 9 -15 + 23 + 52 + 42 + 105 + 67 Unusually quiet before and during anesthetization dogs, though he found no sugar in the urine of frogs after morphine. On the other hand, Hirsch and Reinbach think that morphine is without effect on the concentration of sugar in the blood of rabbits. Jacobsen found an undoubted increase in the amount of sugar in the blood of rabbits to which sufficient chloral had been given to produce narcosis. Some investigators have collected blood from one of the large vessels under the local anesthesia produced by cocaine (Fisher and Wishart). For some types of experiment, such a method is particularly desirable, provided the equiHbrium of the mobile sugar is not disturbed by the drug in such a manner that the proper allowances cannot be made. Araki found lactic acid in the urine of frogs and of rabbits after the injection of cocaine. One of the four rabbits injected also secreted sugar with the urine. In the present work four cats were injected beneath the skin of the back with large doses of cocain hydrochloride dissolved in N/8 sodium chloride solution. These animals were all killed in the early stages of the apparent reaction to the drug. (See Table 14 for details.) With one exception each of the concentrations of sugar found was well below the standard concentration. The mean concentration for the series is 86 per cent of the standard mean. Any attempt to explain this finding woulP be premature, since a longer series Content of Sugar in the Blood Under Laboratory Conditions 303 m _g XJ tn OJ 1/3 ^ 2I 2 !3 £ ag u rt ci D >^ ui 0^ cu 00 t/3 QJ Os d ^ 3 1/1 t4 ^ < S "O ?? OS '^ s cSi rs ro ^-H 1 1 1 + 1 c S *-; d .^ > E « ^ t^ t^ Os Os £S 1 + 1 1 + c c.S_, b^ ro 10 00 OS 10 ■* r^ •O Com trat suga bio gm. d d d d ^ ■ "0^ c . OS 00 10 Amt. bloo draw gm CO 0C5 1 00 10 t^ c n a 1) 4) •- . Tim. etwe ijecti and eath min 00 V— t so SO 1 -O.S T3 ■ c _2i . t^ "0 t^ t^ S"3 g 1 <^;|- d d d d 1 ^-« 00 uo r^ 00 lO 00_ ^ sq 1 CN CN CM CN 1 G _^ CN ro so 00 1 Q X Pi:^ 1^ t. S 1 ^ c c-l^ & 00 1^ ._ CN a •z °^ OS ° 304 Ernest Lyman Scott might yield results which would essentially modify the situation. Also too little is known at present of the other factors of metab- olism during intoxication by cocaine to warrant such an attempt. In following the progress of an experiment it is frequently desirable to determine the changes in the concentration of the sugar in the blood at frequent intervals. Unfortunately there is, however, a very serious objection to this procedure. Claude Bernard found that the concentration of sugar is increased by a previous hemorrhage, and his finding has been repeatedly con- firmed. Recently fairly exhaustive studies have been made by several authors. Among others Anderson, Jacobsen, Rose and Schenck have studied this effect in rabbits. Anderson found an increased concentration of sugar five minutes after the hemorrhage, but did not determine whether it was present after a still shorter interval. The consensus of opinion is that the concentration reaches its maximum about thirty minutes after the hemorrhage and that it remains high from three to four hours. Undoubtedly emotional disturbances have frequently contrib- uted a large share to the so-called hemorrhage hyperglycaemia. However this may be, there is no doubt that quite apart from any disturbance due to emotion or to anesthetics, hemorrhage does introduce a modification for which proper controls must be made. Some authors have sought to avoid the introduction of the factor of hemorrhage by the use of a quantity of blood so small that it might be considered as negligible, but this so greatly increases the probable error from analytical technique that the method has a questionable value, at least for most methods of analysis. Further- more the objection to repeated handling of the animal and the consequent excitement are not met by the change in analytical method, and demand exceptional skill on the part of the experi- menter. The literature covering this subject is so ample and, taken as a whole, so conclusive that it was not thought necessary to add to it. Changes in the concentration of the sugar in the blood may be used as a measure of the effect of a substance which has been in- jected into the animal. In such experiments it is usually presumed that the effect of the injection aside from the drug is nil. My own experiments are too few in number to allow of general conclusions. Content of Sugar in the Blood Under Laboratory Conditions 305 But in harmony with the rest of my work they indicate the neces- sity of complete control of all factors in the experiment. Long ago Bock and Hoffmann showed that large amounts of salt solution caused glycosuria when injected intravenously. But while drugs are frequently dissolved in a solution of sodium chloride for injec- tion, the effect of the salt solution is essentially different from that obtained by Bock and Hoffmann, since usually very much smaller amounts are injected. In my own experiments with cocaine cer- tainly no factor was introduced which increased the concentration of sugar enough to conceal the results due to the cocaine alone, with the possible exception of one animal. (See Table 14.) Especial care was taken in making these injections of cocaine to avoid excit- ing the animal. The same care was exercised in an animal which was injected with 5 cc. of M/8 sodium chloride. This cat was killed five hours later, and the blood yielded a concentration of sugar of 0.0697 per cent, a result almost exactly the same as the standard. Another animal injected through an opening in a small box in which it was confined with the same amount of sodium chloride solution became much excited. This animal, after the lapse of a similar interval, yielded a concentration of 0.098 per cent — a much higher result than the standard. Again, many experiments of this nature involve the confinement of the animal within some form of apparatus. The exact results obtained in animal calorimetry are ample evidence of the availability of this type of research. On the other hand, great care is necessary to avoid exciting the animal. This is illustrated in the two animals exposed to cold as described on page 291. While they were exposed to similar external conditions, one became very restless and yielded a concentration of 0.149 P^^ cent of sugar; the other remained exceptionally quiet and yielded a concentration of only 0.049 per cent. The nature of this experiment, together with the results, would suggest the possibihty that the unpleasant conditions in- volved constitute at least one of the factors leading to the mobiliza- tion of the sugar in Lusk's method of ridding the body of glycogen by shivering. In another experiment four cats were ccnfined in a respiratory chamber and subjected to a temperature of about 32° C. and a relative humidity of about 88 per cent. The results are shown in 3o6 Ernest Lyman Scott Table 15. Animals were selected for these experiments which might be expected to remain quiet throughout the experiment. No. 112 was the only one which proved disappointing in this regard. The results show an exceptionally low average for the concentration of sugar, and this individual is the only one of the four which reached the level of the standard cats. TABLE 15 The Effect of the Confinement of Cats in a Warm, Moist Chamber UPON THE Concentration of Sugar in the Blood No. of Exp. Sex Body wt. k. Amt. of blood drawn gm. Hrs. in chamber Temp. mean C. Rel. hu- midity mean Concen- tration sugar in blood gm. % %of var. from stand, mean Cal. for 30 gm. blood per k. body wt. Concen- tration sugar % var. from stand. gm. % average Ill F 3.65 83.38 6 30.6 .83 0.053 -23 0.049 -29 1121 F 1.62 55.57 6 30.7 .89 0.064 - 7 0.067 -3 113 F 3.78 69.87 6 33.1 .90 0.065 - 6 0.058 -16 114 F 2.90 65.12 6 32.9 .90 0.059 - 14 0.054 -22 Mean — — — 31.8 .88 0.060 - 13 0.057 - 16 1 Excited when removed from the chamber. There seems then to be no reason for attributing changes in the concentration of the sugar in the blood following the injection of small amounts of salt, or the confinement of the animal in appa- ratus to these conditions of themselves. Excitement induced by these conditions may, however, give rise to high concentrations, even five hours after the time of irritation. VI. — Summary and Conclusions I. Glycaemia offers a more satisfactory indication of the condi- tion of mobile sugar than does glycosuria; first, because either an increase or a decrease in the amount of sugar may be demon- strated, while normal urine can show only an increase; secondly, Content of Sugar in the Blood Under Laboratory Conditions 307 because profound changes in glycaemia may occur in response to conditions which do not produce glycosuria; thirdly, the blood is in much more direct relation to the living cells than is the urine. 2. The concentration of sugar in the blood as estimated by dif- ferent methods varies. This may be due, in part at least, to the form in which the sugar is present in the blood. It follows that results obtained by different methods of analysis cannot properly be com.pared until they have been reduced to common terms. Also the possibility is introduced of an apparent variation in con- centration of sugar, even when the method of estimation is constant, which is due to a change in the form in which the sugar is present rather than to a change in the actual amount of sugar present. 3. If consistent results are to be expected, the animals must be uniformly healthy, and must be killed without pain or excitement. Sex or weight, apart from correlated conditions, are probably with- out special influence upon the concentration of sugar in the blood. 4. The normal concentration of sugar may very probably vary with the varying environment of the animal or with changes in its physical state. However, if the environment is uniform and if the animals are killed while in the same physiological condition, con- stant results should be expected. Practically such an ideal result is not possible, but has been approached with some success. 5. The concentration of sugar in the blood decreases as the amount of blood drawn per kilo of body weight of the animal increases. So far sufficient data have not been obtained to es- tablish the mathematical expression for this relation. 6. When ether or chloroform was administered, the concentra- tion of sugar was increased considerably and varied between rather wide limits, whether the diet consisted of meat alone, or of bread and meat, the latter diet giving somewhat smaller variations than the former. After either diet there was a greater concentra- tion after the drug had been administered for thirty minutes than after it had been administered for three minutes or less. 7. The concentration of sugar in the blood after subcutaneous injection of cocaine is more constant than that found after inhala- tion of ether or chloroform and is lower than that found in animals similarly treated but to which cocaine has not been given. 8. It may be shown from the literature that hyperglycaemia 3o8 Ernest Lyman Scott follows hemorrhage. From this it follows that caution must be exercised in drawing conclusions from experiments which involve the analysis of successive samples of blood. 9. The excitement which is apt to attend hypodermic injections or confinement in apparatus may lead to high results and conse- quently to false conclusions, With care, however, such effects may be avoided so that this type of experiment is permissible. VII. — Literature Anderson, Nils: Ueber das Verhalten des Blutzuckers beim Aderlass, Biochemische Zeitschrift, 1908, xii, 1-7. Araki, T. : Ueber die Bildung von Milchsaure und Glycose im Organismus bei Sauerstoffmangel. II. Ueber die Wirkung von Morphium, Amylnitrit, Cocain, Hoppe-Seyler's Zeitschrift fur Physiologische Chemie, 1891, xv, 546- 561. Arthus, M. : Applications de la dialyse a la solution de quelques questions de chime physiologique, Zeitschrift fur Biologie, 1896, xxxiv, 432-446. Bang, I.: Der Blutzucker, Wiesbaden, 1913. BoE, G.: Untersuchungen iiber Alimentare Hyperglykamie, Biochemische Zeitschrift, 1913, Iviii, 106-118. Bernard, Claude: Lemons sur le diabete et la glycogenese animale, Paris, 1877. Bock, C, and F. A. Hoffmann: Ueber eine neue Entstehungsweise von Melliturie, Du Bois-Reymond's Archiv fiir Anatomy und Physiologic, 1871, 550-560. BoEHM, R., and F. A. Hoffmann: Beitrage zur Kenntnis des Kohlen- hydratstoffwechsels, Archiv fiir Experimentelle Pathologie und Pharmakologie, 1878, viii, 271-308. Burton-Opitz, R.: Ueber die Veranderung der Viscositat des Blutes unter dem Einfluss verschiedener Ernahrung und experimenteller Eingriffe, Archiv fiir die gesammte Physiologie, 1900, Ixxxii, 447-473. Cannon, W. B.^: The movements of the intestine studied by the Rontgen rays, American Journal of Physiology, 1901, vi, 250-277. Cannon, W. B.-: The emergency function of the adrenal medulla in pain and the major emotions, American Journal of Physiology, 1914, x.xxiii, 357- 372. Cannon, W. B., and A. T. Shohl, and W. S. Wright: Emotional glyco- suria, American Journal of Physiology, 191 1, xxix, 280-287. Dehn, Wm. M., and F. A. Hartman: The picrate colorimetric method for the estimation of carbohydrates. Journal of the American Chemical Society, 1914, xxxvi, 403-409. EcKHARD, C: Zur Deutung der Entstehung der vom vierten Ventrikel aus erzeugbaren Hydrurien, Zeitschrift fiir Biologie, 1903, xliv, 407-440. Content of Sugar in the Blood Under Laboratory Conditions 309 Fisher, Gertrude, and Mary B. Wishart: Animal calorimetry. IV. Observations on the absorption of dextrose and the effect it has upon the composition of the blood, Journal of Biological Chemistry, 1912, xiii, 49-61. Frerichs, Fr. Th.: Ueber den Diabetes, Berlin, 1884. Harlay: Cited by Tiegel. Hawk, P. B.: On the influence of ether anesthesia, American Journal of Physiology, 1903, x, p. xxxvii. Hill, Leonard: Philosophical Transactions of the Royal Society, 1900, 193 B. HiRSCH, E., and H. Reinbach: Die Fesselungshyperglykamie und Fessel- ungsglj'kosurie des Kaninchens, Zeitschrift fiir physiologische Chemie, 1913, Ixxxvii, 122-141. Jacobsen, a. Th. B.': Untersuchungen iiber den Einfiuss des Chloral- hydrats auf experimen telle Hyperglykamieformen, Biochemische Zeitschrift, 1913, li, 443-462. Jacobsen, A. Th. B.^: Untersuchungen iiber den Einfiuss verschiedener Nahrungsmittel auf den Blutzucker bei normalen, zuckerkranken und graviden Personen, Biochemische Zeitschrift, 1913, Ivi, 471-494. Lee, F. S.,: Some chemical features of the diaphragm and other skeletal muscles, American Journal of Physiology, 19 14, xxxiii, p. xxiv. Lee, F. S., and C. C. Harrold: The action of phlorhizin on muscle, American Journal of Physiology, 1900, iv, p. ix-x. Lesser, E. J.: Ueber eine Fehlerquelle bei Blutzuckerbestimmungen in Frosch und Schildkrotenblut, Biochemische Zeitschrift, 1913, liv, 252-255. Lewis, R. C, and S. R. Benedict: A method for the estimation of sugar in small quantities of blood, Proceedings of the Society for Experimental Biology and Medicine, 1914, xi, 57-58. LoEB, A.: Beziehungen zwischen Zuckergehalt der Erythrocyten und Glykolyse, Biochemische Zeitschrift, 1913, xlix, 412-425. LoEWY, A., and S. Rosenberg: Ueber die normale Hohe des Blutzuckerge- halts bei Kaninchen und Hunden, Biochemische Zeitschrift, 1913, Ivi, 114-116. LusK, G.: The influence of cold baths on the glycogen content in man, American Journal of Physiology, 1911, xxvii, 427. LuzzATTO, R.: Ueber die Natur und die Ursachen der Morphinglykosurie, Archiv fiir Experimentelle Pathologie und Pharmakologie, 1905, Hi, 95-115. Macleod, J. J. 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VITA I WAS born on August i8th, 1877, in Kinsman, Ohio, and graduated from the Kinsman High School with the class of 1897. In 1902 I received the degree of Bachelor of Science from Ohio Wesleyan University. Immediately upon graduation I entered the United States Coast and Geodetic Survey. While in this service the greater part of my time was spent in hydrographic work in Chesapeake Bay. I began my duties as instructor in sciences in the Western High School, Bay City, Michigan, in September, 1903, and remained there until April, 1910, when I accepted a fellowship in the Department of Physiology of the University of Chicago. With the beginning of the following summer quarter my rank was raised to that of assistant. While in the University of Chicago a part of my time was taken up with instruction in laboratory courses and a part was given to study and research. In the spring of 191 1 I was elected to Sigma Xi and at the close of the summer quarter received the degree of Master of Science. My thesis, which represented an experimental investigation of the influence of intravenous injections of an extract of the pancreas on experimental pancreatic diabetes, was pubUshed in the American Journal of Physiology, volume twenty-nine, 191 2. In the autumn of 191 1 I entered upon my duties as assistant professor of physiology in the University of Kansas. At the close of one year I resigned this position to accept an instructorship in physi- ology in Columbia University. I have now been advanced to an associateship in Columbia for 19 14-15. (Signed) ERNEST LYMAN SCOTT May 10, 1914. COLUMBIA UNIVERSITY LIBRARIES 'wya«"W^T7y This book is due on the date indicated below, or at the *ji^^«^/n; expiration of a definite period after the date of borrowing, '^SwSg" as provided by the library rules or by special arrangement '^ '^>^^^^^^^ with the Librarian in charge. w DAtE BORROWED DATE DUE DATE BORROWED DATE DUE C28 (665) 50M '%¥i&iw'-^'^-'^ WM -1 Scott QP99.3«B5 Sco8 1914 ■ ».v.;,' -A' V-.. ■sTjj^T-i'srii; The content of sugar in the blood. '^'■■**!«C>V THE • PLIMPTON • PRESS • NORWOOD • MASS • U • S • A