induced by sham feeding is accompanied by an increased excretion of University College, London.)

Transcription

1 THE METABOLISM OF THE SALIVARY GLANDS. II. The blood sugar metabolism of the Submaxillary Gland. BY G. V. ANREP AND R. K. CANNAN (Beit Memorial Research Fellow). (From the Institute of Physiology, University College, London.) THE only direct attempts to study the blood sugar changes in a secretory organ are, so far as we are aware, those of Chauveau and Kaufmann(1) and of Asher and Karaulow(2). The former worked on the submaxillary gland of the horse and, although their analytical methods are open to criticism,, they were able to show a diminution in the sugar in the blood in its passage through the active gland. A sh er and K ara ulow on the other hand observed an actual increase in the sugar of the venous blood from the submaxillary gland of the dog during secretion, although they found a decrease immediately succeeding the flow of saliva. Indirect evidence, afforded by studies of oxygen consumption and respiratory quotient, for the submaxillary gland (3) and for the kidney(4) point to a carbohydrate metabolism in rest which is markedly increased in activity, whilst C ohnh eim (5) has shown that the secretory activity induced by sham feeding is accompanied by an increased excretion of carbon dioxide but not of nitrogen. The results of Asher and Karaulow are open to criticism in several particulars but the essential fact which they overlooked was the concentration of blood by loss of water to the saliva. Thus in their first experiment in the two minutes necessary for the collection of 1057 c.c. of venous blood for analysis 4 c.c. of saliva were secreted. That is to say c.c. of arterial blood entered the gland in the same period, and its sugar content was x 1-34 = 19-6 mgms. The sugar in c.c. of venous blood was 20-4 mgms. The difference between these two figures is within the error of the method. At such a blood flow as 5 c.c. per minute it would not be expected that there would be an exchange of sugar between the gland and each c.c. of blood sufficiently large to be detected by the method used? Moreover, it seems doubtful whether, in their experiments, the blood was collected from the submaxillary gland alone. The rates of blood flow they obtained were so high as to suggest

2 SALIVARY GLAND METABOLISM. 249 a large admixture of extrinsic blood. Thus, in a dog of medium size a resting blood flow of c.c. per minute was recorded and during the stimulation of the chorda this fell to 22-1 and c.c. in one minute and on another occasion to c.c. in 1 min. 40 secs. Such a flow is quite unique and no less unusual is the diminution in rate of flow which accompanied stimulation of the chorda. In the three experiments recorded the total saliva secreted was in each case only about 8-5 c.c. and in every case the last stimulation failed to produce any appreciable secretion. Undoubtedly the currents used for stimulation were so strong as to kill the nerve. The limited and contradictory nature of the work done on the carbohydrate metabolism of a secretory organ suggested to us a study of the question, and for this purpose the submaxillary gland of the dog was chosen. The improved methods of blood sugar analysis whereby the dextrose in 1 c.c. of blood may be estimated with an error of less than 0 05 mgm. appeared to present the opportunity of a successful study of the subject in a small animal. Any disappearance of sugar from the blood in its passage through the gland may be accounted for by loss in several directions, It may have (a) been retained by the gland in the form of glycogen, (b) passed into the saliva, (c) passed into the lymph, (d) been consumed by the gland. (a) That the gland does contain glycogen was determined in the case of eight pairs of glands. The amount found varied from 0.1 to 0*4 p.c. and the two glands of any one animal agreed closely. The glands were however taken from dogs which had been submitted to a variety of experimental procedure and it is probable that the normal content is not so variable. Pfluiger's method was used. The variations in glycogen content accompanying activity will form the subject of a future communication and will not be further discussed here. Whatever the fate of the sugar may be-whether it is oxidised, undergoes unoxidative disintegration or is stored in the form of glycogen-in all these cases we are entitled to call it "consumption." (b) Carlson and Ryan(6) have found sufficient sugar in the saliva of the cat to respond sometimes to the common sugar tests. These workers obtained the saliva either by spraying the mouth with ether after a preliminary rinsing or under local ansesthesia directly from the duct by an injection of pilocarpine. The results from the former samples must be altogether discounted as sugar may well have been present in the mouth, and in every case the handling to which the animal had been

3 250 G. V. ANREP AND R. K. CANNAN. subjected was such as to render probable a high degree of hyperglycaemia. Further, they precipitated the mucin by long contact with acetic acid, a procedure by which sufficient reducing sugar to give positive tests might have been split off from the mucin. They attempted no quantitative determinations. Rattery and Bisset(7) have recently stated that sugar appears in the saliva when the blood sugar exceeds 0 3 p.c. -a very high figure not reached in any of the experiments of this research. To determine if there might be an escape of sugar in aliva in normal concentration of blood sugar, a series of tests were carried out on saliva from dogs. In some cases the secretion was effected by pilocarpine or by stimulation of the chorda, in others samples were taken from a dog with a salivary fistula. Fehling's test and Cramer's delicate test were tried after precipitation of th6 mucin by alcohol. Results were always negative. Attempts to estimate the sugar in 20 c.c. of saliva were also without results. It was concluded that no sugar was lost to the saliva under the conditions of our experiments. (c) Barcroft(12) has studied the lymph flow of the submaxillary gland and found that whilst it is inappreciable in rest it increases with the flow of saliva. He used the hemoglobin concentration of the blood as a means of measuring the exudation from the gland and attributed the difference to the flow of lymph. He found that in good conditions of the resting gland there was no concentration of the blood due to escape of fluid, i.e. lymph. Bainbridge(ii) made some direct determinations and, although his experiments are open to criticism, he also found hardly any lymph flow in a resting gland. The amount of sugar in the lymph was not determined in our experiments but systematic determinations of the haemoglobin ratio of the corresponding arterial and venous blood samples were made and the lymph flow calculated. To control our results it was assumed that the sugar in the lymph was equal to that in the plasma, and the amount thus calculated to have been lost in the lymph was subtracted from the total disappearance of sugar. This assumption is to a certain extent justified by determinations of sugar in lymph from the thoracic duct. The concentration was found to be of the same order as that of the arterial plasma. This does not, however, mean that the lymph coming from an active organ would retain its concentration of sugar as the thoracic lymph is not comparable to that of the gland. Further, the gland lymph is the fluid from which the cells actually obtain their supplies of material and one would expect that the lymph coming from a working organ would be deprived of its sugar to a greater extent than the blood circulating through the organ.

4 SALIVARY GLAND METABOLISM. 251 The routine determinations made were (1) sugar of the venous blood, (2) sugar of the arterial blood, (3) haemoglobin ratio, (4) venous blood flow, (5) flow of saliva. The prevailing methods of blood sugar analysis are, broadly, of two kinds-titrimetric and colorimetric. Comparison of a standard method of each class usually shows a slightly lower series of values by the former than by the latter and it is probable that these lower results more closely represent the amount of actual dextrose in the blood(s). The method of MacLean(9), of which we had had experience, being simple, rapid and economical was well suited to these experiments. Duplicate estimations were made and showed the close agreement the author claims for his method. Glycolysis. In the first three experiments each estimation was carried as far as the protein-free filtrate immediately upon the collection of the blood in order to prevent any glycolysis. We afterwards employed neutral formalin as recommended by Denis and Aldrich(lo) as an anti-glycolytic agent, preserving all blood samples for analysis after the conclusion of the experiment. Although formaldehyde is a weak reducing agent it was found that no reduction of MacLean's solution by formalin was given, probably because of the low alkalinity of the reagent. Further, not only was the sugar concentration of the blood maintained over 24 hours in the presence of formalin but the concentration in the plasma was unchanged. It was therefore possible to make analyses of both blood and plasma on specimens preserved in this way. One drop of neutral (40 p.c.) formalin was added to each sample of about 4 c.c. of blood. In Table I is given an example of some of these experiments on the effect of formalin. TABLE I. Blood sugar in mgms. per 100 c.c. blood. Blood+formalin Blood Blood alone Blood+formalin +sugar +sugar Blood Plasma Blood Plasma Blood Plasma Blood I. Estimated at once After 5 hours II. Estimated at once After 3 hours After 18 hours III. Estimated at once After 24 hours Anti-coagulant. The blood was received into dry tubes containing a very little powdered potassium oxalate. The pipette in which the venous blood was collected was likewise thinly dusted with the same powder.

5 252 G. V. ANREP AND R. K. CANNAN. The hemoglobin ratio of the corresponding arterial and venous blood samples was determined by comparing a 05 p.c. solution of blood in the form of carboxy-haemoglobin in a Dubosq colorimeter. The acid haematin method was tried but was less accurate in our hands. Dissection. Dogs were used in all experiments. The animal was ansesthetised with C.E. mixture, without previous injection of morphia, in order to avoid the usual secretion of saliva caused by the latter and. so to keep the glands in as nearly a resting condition as possible. With the same object both lingual nerves were cut above the points at which the chordse tympani leave them. The vago-sympathetic trunk on the same side as the gland chosen for the experiment was severed. Then only was morphia or chloralose injected, as little volatile anaesthetic as possible being given. Morphia and chloralose were thought to be more suitable than any of the volatile aneesthetics as the depth of anaesthesia is more regular, and for that reason the blood sugar is less likely to show fluctuations. In the first four experiments before injection of morphia, the suprarenal veins were clipped in order to avoid stimulation of the sympathetic nerve endings in the submaxillary gland or fluctuations of blood sugar by a possible increased discharge of adrenalin into the blood. This procedure was abandoned in the subsequent experiments since it had been found that there was no dependen'ce of the sugar consumption of the gland on the blood sugar level. A cannula was tied into the submaxillary duct, another into one femoral artery for the collection of the blood and a third into the second femoral for recording blood-pressure. The dissection of the submaxillary veins and the collection of the blood were performed as recommended by Barcroft 3). The blood-pressure was recorded at each collection. The blood flow was measured by timing the flow with a stop watch over 2 c.c. in a graduated pipette connected to the cannula which had been introduced into the vein. Two readings were taken, and if the time differed by more than about 5 p.c. the two specimens of blood were analysed separately, otherwise they were mixed to form a sample of about 4 c.c. and the mean time of flow taken. Sufficient blood was allowed to escape before collection to ensure that the sample taken corresponded to the condition under observation. The flow of saliva in the time taken to collect 2 c.c. of blood was measured in a similar pipette connected to the cannula in the duct. Again two readings were taken, and if not in fair agreement, the corresponding blood samples were kept separate. About 8 c.c. of arterial blood were taken each time to allow sufficient for sugar determinations in blood and plasma. It was early discovered that the blood sugar was capable:

6 SALIVARY GLAND METABOLISM. 253 of quite rapid fluctuations and therefore care was taken to collect the arterial blood in the middle of the period of collection of the corresponding venous blood. The resting gland. The first observation made in the course of the experiments was that in every case there was a disappearance of sugar from the blood in its passage through the gland, and this disappearance calculated per unit time was generally of the same order in any one animal. Now in the resting condition, since there is no disappearance of the fluid from the blood either in the form of saliva or lymph, the whole difference between the arterial and the venous blood sugar may be taken to represent the amount of sugar consumed by the gland. All the figures given for consumption are calculated on the basis of consumption per gram of the gland per hour. The actual determinations of typical experiments are given in the tables at the end of the paper. Here will be discussed only the main results. (Exps. IX-XV will be dealt with more fully in a later paper.) If for each experiment the mean resting consumption per gram of the gland per hour be taken the following figures are given: I II III IV V VI VII VIII IX X xi XII XIII XIV XV 2-4 1* Mean 2-07 mgms. It is interesting to compare this figure with that given by B arcroft for the oxygen consumption of the resting gland. Barcroft(12) gives figures for the resting consumption of oxygen by the submaxillary gland whose mean is about 1 2 c.c. per gram per hour. Assuming the substance oxidised to be glucose this would imply a consumption of 1-8 mgms. of glucose in the same units-a figure similar to that obtained above. Blood sugar consumption and the level of blood sugar. It was almost invariably found that the blood sugar concentration gradually diminished as an experiment proceeded. These variations in blood sugar were in no way reflected in changes in consumption. Sometimes, particularly when the animal was not in a satisfactory condition, the consumption gradually rose though the blood sugar went down-thus in Exp. II the blood sugar fell through mgms. p.c., while the corresponding consumptions were 10, 12, 2-0 mgms. per gram per hour. In other cases, as in Exp. VI, the blood sugar fell from 152 to 67, while the consumption fluctuated irregularly between 3.3 and 09. It must be acknowledged that we did not in our experiments succeed in obtaining such close figures as did B arcroft for oxygen consumption. We can, PH. LVI. 1 7

7 254 G. V. ANREP AND R. K. CANNAN. however, be satisfied that the fluctuations in our values are related neither to changes in blood sugar level nor to the velocity of the blood flow through the gland. We must ascribe these irregular results to the greater error of the blood sugar determination as compared with that for oxygen. To be on the safe side one must consider, in each experiment,,the range of these fluctuations. Effect of atropine on the sugar consumption. If we compare the mean resting consumption already quoted with the mean consumption of the atropinised gland we find a close agreement. Resting consumption Exp. III IV VI VIII xi XIV Mean Before atropine After atropine X2 The differences fall well within the fluctuations of the resting consumptions for each particular experiment and justify the conclusion that atropine does not affect the consumption of the resting gland. Rate of bloodflow and sugar consumption. The question of the relation of blood flow and sugar consumption will be more fully dealt with in a later communication. Here it will be sufficient to note that in Exp. II there was a rising consumption and a steady blood flow, in Exp. III a falling consumption and a rising blood flow, in Exp. IV a rising consumption and an irregularly oscillating blood flow, whilst in Exp. VI (cp. Appendix) there was a steady blood flow and a fluctuating consumption. These examples would seem to show that small variations in blood flow do not affect the consumption and that the latter may vary within certain limits when the blood flow remains steady. Effect of pilocarpine on sugar consumption. The consumption of the gland secreting under pilocarpine was much increased, and this increase was broadly related to the activity of the gland as measured by the flow of saliva. Many examples occurred of the observation of Asher and Karaulow that the sugar concentration of the venous blood was above that of the arterial but this relation was always reversed when the correction for concentration of the blood was applied. The method of calculating was as follows: The figures for the first pilocarpine period of Exp. VII are: Venous blood sugar 1-65 mgms. per 1 c.c. Venous blood flow 1 c.c. in 0 33 min. Saliva flow 0-26 c.c. in 0*33 min. Hence 1 c.c. venous blood corresponded to 1-26 c.c. arterial blood which contained 1-26 x 1-57 mgms. sugar = 1-98 mgms. Hence the consumption was 0-33 mgm. in 0 33 min. Weight of gland 5 grms. Hence consumption was 12 mgms. per gram per hour.

8 SALIVARY GLAND METABOLISM. 255 Results were controlled for lymph flow in the way already explained, the calculation being as follows: Plasma sugar 1-95 mgms. per 1 c.c. Ratio of hemoglobin concentration, venous: arterial 1F32: 1. Hence total exudation per 1 c.c. venous blood was 0-32 c.c. saliva,,,,,, 026 c.c. Therefore lymph,,,,,, 0-06 c.c. And lymph sugar was 006 x 1-95 =0-117 mgm c.c. of arterial blood correspanded to 1.00 c.c. venous and contained 2-07 sugar. Therefore consumption in 033 min. was (1.65, 0-117), i.e. 0303, i.e mgms. per gram per hour. The above example was chosen as showing the greatest divergence that has been obtained between the results calculated by the two methods. The figures given in the Appendix are calculated without consideration of the lymph flow. This is justified on the grounds that it is not at present known whether the lymph leaving a working gland carries away sugar or whether it leaves as a sugar free fluid. In the former case the consumption recorded would be too great by the amount of such sugar escape, in the latter case the recorded concentration of the blood would be less than the actual by the amount of the lymph flow and for that reason the determined consumption would be somewhat too low. Table II demonstrates the differences in results which were obtained (a) by ignoring lymph flow, (b) assuming the lymph removes sugar in concentration equal to that of arterial blood. The difference is not of an order to affect the conclusions drawn from the experiments. TABLE II-Exp. VII. Comparison of results calculated (1) without allowing for lymph flow, (2) assuming the lymph to have the same concentration of sugar as the arterial blood. Consumption mgms. sugar Hemoglobin Lymph c.c. Ignoring Including Period ratio per min. lymph lymph sugar * pilocarpine * * * ' pilocarpine * In every case the active gland consumed more sugar than when in the resting condition. Moreover, there was a broad dependence of the 17-2

9 256 G. V. ANREP AND R. K. CANNAN. sugar consumption upon the rate of salivary secretion. A few examples will suffice (see also the illustrative experiments given in the Appendix). Exp. I. Gland 5 grms. Rest Pilocarpine Rest A Saliva c.c. per grm. of gland *96 per hour Consumption mgms. sugar per grm. gland per hour 1-6 Exp. III. Gland 2-9 grms. Rest Pilocarpine Rest Saliva Consumption X8 1X8 1X3 Exp. VII. Rest Pilocarpine Rest Pilo. Rest Saliva V Consumption *2 0.5 If, in any one experiment, the difference be taken between the mean resting consumption per gland and the consumption per gland for a given active condition a figure may be obtained for the sugar used to produce 1 c.c. of saliva. Thus, in Exp. II the mean resting consumption was 0-09 mgm. per gland per min. when the gland was secreting 0-13 c.c. saliva in a minute. That is to say, 0413 c.c. saliva was produced at the expense of = 0-23 mgm. sugar, so that the glucose used to produce 1 c.c. of saliva would be 1-8 mgms. The means of the values. thus calculated in each experiment are: I II III IV V VI vii viii i' Mean1i5 mgms. These show the wide variation, that would be expected in any attempt to give a quantitative expression on a loose differential basis to several unknown factors-nature of the saliva secreted, glycogen changes and time relations of secretion and consumption. Indeed, such a quantitative expression may only be used with the greatest reserve, and is here deduced merely for comparison with results from studies of oxygen consumption. Barcroft gives 0-6 c.c. of oxygen as a very approximate figure for the oxygen used in the production of 1 c.c. of saliva under the action of adrenalin. This expressed in terms of glucose oxidised gives. 0-8 mgm. glucose-a figure about half of ours.

10 SALIVARY GLAND METABOLISM. 257 CONCLUSIONS. 1. The resting submaxillary gland consumes blood sugar. 2. The mean rate of blood sugar consumption varied in 15 experiments, from 0O8 to 2*9 per gram of gland per hour, being fairly constant for any one experiment. 3. The average figure for all the experiments was 2-1 per gram per hour. 4. Atropine does not change the blood sugar consumption of the resting gland. 5. Pilocarpine increases the blood sugar consumption. The increase being within broad limits proportional to the rate of salivary secretion. 6. The increase in sugar consumption per 1 c.c. of saliva secreted varied in eight experiments from 07 to 2-7 mgms., being also fairly constant in any single experiment. 7. The average figure of blood sugar consumption per 1 c.c. of saliva secreted was 1-5 mgms. per gram of gland per hour. The expenses of this research were defrayed out of a grant from the Medical Research Council. REFERENCES. (1) Chauveau and Kaufmann. C.R. 104, p (2) Asher and Karaulow. Biochem. Ztsch. pp. 25, (3) Barcroft. Journ. Physiol. 27, p (4) Barcroft. Respiratory Function of the Blood (5) Cohnheim. Ztsch. physiol Chem. 45, p (6) Carlson and Ryan. Amer. J. Physiol. 21, p (7) Rattery and Bisset. Presse Med. 28, p (8) Host and Hallehol. Journ. Biol. Chem. 42, p (9) Maclean. Biochem. Journ. 13, p (10) Denis and Aldrich. Journ. Biol. Chem. 44, p (11) Bainbridge. Journ. Physiol. 16, p (12) Barcroft and Priper. Journ. Physiol. 44, p

11 258 G. V. ANREP AND R. K. CANNAN. APPENDIX. Mgms. sugar per 100 c.c. blood c.c. per minute Time Venous Arterial Ven. blood Saliva Exp. I. Wt. of gland 5-0 grms :55' : mgms. pilocarpine injected subcutaneously 2: : F : Exp. VI. Wt. of gland 3-1 grms. 0: : o90-0 : : : : : : 30 2*0 mgms. pilocarpine 1: : : o : mgmis. atropine 1: : Exp. VII. Wt. of gland 5 0 grms. 0: : 8 0'3 grm. morphia 0: : : mgms. pilocarpine 1: : *07 1: '0 1: : : mgms. pilocarpine 2 : *6 2 : mgm. atropine 2: Exp. VIII. Wt. of gland 4.3 grms. 0: : : : mgm. pilocarpine 0 : : : : mgms. pilocarpine 1 : : : mgms. pilocarpine 2 : *05 2: mgm. atropine : Consumption per grm. per hour 2 3 mgms. dextrose 2*4,, 6*8,, 6*7 1*6, 1 6 to *7 V$ 1*7, 2*0, 2-2,, 09,, 135,, 4-8,, 2*4,, 3*6,, 2 3,, * *8 0' * _ *8 0* Just ceased _ 1*7 1-7