- Email: [email protected]
Absorption of sugars from isolated surviving intestine
Absorption
of Sugars from Isolated W. A. Darlington
From
the Research
Institute,
Surviving
Intestine
and J. H. Quastel
Montreal
General
Hospital,
Montreal,
Canada
Received November 6,1952
VerzWs hypothesis (l), that a process of phosphorylation is involved in the rapid absorption of certain of the hexoses from the small intestine, has been discussed by a number of workers (2-6a). The object of the present study has been to investigate furt,her some of the phenomena underlying this phenomenon, with special reference to the relation between selective absorption of hexoses and respiratory activity. Since t.he available methods are ill suited to this study, it became necessary to develop a more satisfactory technique. The new experiment.al procedure used in this work will, therefore, be described in detail. METHODS
AND MATERIALS
The technique,which haa beenin usein this Institute for over 4 years,is similar in principle to the method recently describedby Fisher and Parsons(7). It con-
sistsin perfusingoxygenatedRinger solution through a sectionof freshly ieolated intestine which is bathed in an oxygenated Ringer solution. The apparatusinvolves two independentcirculatory systems,one running through the lumen of the intestine and the other external to the intestine. Measurementsare madeof the rate of perfusionof substances from the inner to the outer solution. A diagrammaticrepreeentationof the perfusionapparatusthat has been used is shown in Fig. 1.
The inner solution passesfrom a cylinder A, through C and a connecting arm into a small reservoir E which is opento the atmosphere.From E the fluid passesthrough F, which connects with the intestinal aegmentH, through Z and D,
back into the cylinder A. The lower opening in cylinder A is cloeed by a rubber stopper through which paaees the glass tubing of a stopcock. This stopcock serves to drain the inner fluid at the end of the experiment.. The volume of the inner solution is approximately 100 ml. The outer solution, which in cylinder M bathesthe outer surfaceof the intestinal segment H, paeses from the cylinder through N and the connecting arm 0 into the small reservoir P which is also open to the atmosphere. From P the fluid 194
ABSORPTION
OF
195
SUGARS
passes down glass tubing through a three-way stopcock into the cylinder M. The three-way stopcock R is used to drain the outer fluid at the end of the experiment, this fluid being collected and measured. The volume of the outer solution is approximately 110 ml. GA5
FIG. 1. Apparatus
for perfusion
5
of isolated surviving
intestine
(scale 1:5).
The continuous movement of fluid through the two circulations is maintained as follows: Gas under pressure (from a cylinder) is allowed to enter at T and U. It escapes from the apparatus at the outlets E and P. The gas passes through the side connections D and 0, and thus into E and P. In passing through the side connections, which are filled with fluid, the gas carries fluid with it into the small reservoirs E and P. In this way the level of fluid in E and P is made higher than that in C and N. Two circulations, therefore, result. One, called the inner, passes through the lumen of the intestinal segment, and the other, the outer, passes through the
196
W. A. DARLINGTON
AND J. H. QUASTEL
cylinder M which bathes the outside of the segment. Any exchange of substances between the inner and outer solutions must occur through the wall of the intestinal segment. For ease in setting up, and washing, the apparatus is made in sections, which are joined by rubber tubing, shown in the diagram by shading. The intestinal segment H is shown stippled in the diagram. When in use the apparatus is kept in an incubator maintained at 38 f 1°C. Samples may be removed during the experiment from both circulations through the stopcocks L and R, or by pipetting from the small reservoirs E and P. It is necessary to replace an equal volume of fluid to the circulation after withdrawal of a sample. EXPERIMENTAL
PROCEDURE
An animal is killed by decapitation and is bled. The abdomen is opened in the midline and all of the small intestine is removed except that portion adjacent to the stomach (duodenum) which adheres firmly to the surrounding tissue. The intestine is placed in a small beaker of the solution to be used in the experiment, all the solutions having previously been brought to 38°C. and gassed with 93% O2 + 7‘% COZ. In order to obtain a standardized procedure, the intestine in the beaker is removed and that part of the intestine which was 39-40 cm. from the stomach in the intact animal is cut out, and the inside washed out with the solution to be used. Care is taken to maintain the orientation of the intestine so that perfusion is carried out through the segment in the normal direction of flow. The apparatus is now assembled in the following way. The lower end of the segment is tied with a piece of thread to the glass tubing I, this having been pushed through the rubber stopper J as far as it will go, and the stopper is placed in the end of cylinder M. The segment of intestine now protrudes through the open end of cylinder M, and the loose end is fastened to that section of the glass tubing F which passes through the rubber stopper G. The stopper G is placed in position, and glass tubing Z is withdrawn through the stopper .Z until the segment is fully extended, but not stretched. Cylinder A, with stopper K inserted, is now joined to Z by means of rubber tubing, and cylinder A is partially filled with inner solution. The stopper B, which holds the section CDE is placed in position, and the two sections of F are joined with rubber tubing. The inner circulation is now completed but is not yet completely filled with solution. The part of the apparatus NOPQRS, which has been assembled previously, is now placed in position with rubber tubing at N and S, and the stopcock R adjusted so that the flow of fluid is from & to S. The outer solution is now added through P, care being taken that no fluid passes down 0 and into N, and cylinder M is thus filled from the bottom with the displaced air escaping through N. The filling of the inner circulation is completed in the same way. The level of fluid must be slightly above the side openings D and 0 in the small reservoirs E and P. The levels of fluid in E and P are kept even, as a higher level of fluid in E wilI cause distention of the segment of intestine, and also result in a higher hydrostatic pressure in the inside of the segment than on the outside. If the level of P
ABSORPTION
OF
SUGARS
197
is much above that in E, collapse of the segment will occur with a consequent blocking of the inner circulation. Gas is led to the system through rubber tubing which is connected over the glass tubing at T and U, and the perfusion is started. The time taken, from the killing of the animal to the commencement of the perfusion, is from 10 to 12 min. Samples are taken by pipetting from E and P because, with this method, there is no temporary stopping of circulation, which often results in excessive motility of the segment. Male guinea pigs, from a commercial source, weighing 550-350 g. have been usually used. In experiments carried out with an isotonic salt solution, as used by Krebs and Henseleit (S), as the circulatory fluid, severe peristaltic contractions occur in the intestinal segment, in some cases sufficiently vigorous to prevent circulation of the inner fluid, and extremely variable results are obtained. However, if nicotinamide is added to the outer solution this difficulty is partly overcome. In the presence of nicotinamide no motility is shown by the segment during perfusion, but vigorous movements usually occur for a short time after the taking of a sample. Liaci (9) has reported that nicotinamide has a depressing effect on guinea pig intestinal muscle. It has been noted, also, that the segment remains quiet even when samples are taken, if calcium is omitted from the circulating solutions. For this reason, calcium is usually omitted from the solutions bathing the intestine. In order to obtain a measure of the amount of sugar which will cross the intestinal wall by simple diffusion, sorboae has been added to the inner circulatory fluid. Verzar (10) has shown that L-sorbose is an inert sugar in the intestine, and that its absorption from the intestine is by diffusion only. In the experiments to be reported, therefore, sorbose has been added to the perfusion fluid containing the sugar under investigation. The rate of sorbose appearance in the outer solution is used as a measure of the rate of physical diffusion of either sugar through the intestinal wall, it being assumed that there will be little difference between the rates of permeability of sorbose and those of sugars of allied constitution. Sugar Estimations. Total reducing substances (glucose plus sorbose) are determined by the ferricyanide reduction method of Hagedorn and Jensen (11). L-Sorbose, or n-fructose, is determined by the resorcinol method of Roe (12), originally designed for fructose but also applicable to sorbose, although separate standard curves are necessary. Glucose does not interfere with these determinations. The amount of glucose is obtained by difference. Phosphate is determined by the method of Snell and Snell (13), using hydroquinone reduction. Intestine. Intestinal segment used: 30 cm. from stomach; Eength of segment: 10 cm. Perfusion Salt Solution. This consists of 0.714yo NaCl, 0.0365yc KCl, 0.0167yc KH*PO,, 0.0311% MgSOd.7Hz0, 0.0416% NaHCOa. Sampling. One-milliliter samples are pipetted from reservoir P for analysis every 10 or 15 min. after commencement of the perfusion, for 90 min., and an equal volume of solution is immediately replaced. When calculating the results, allowance is made for the amount of material removed in this way. The absorption
198
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
rates are found to be constant throughout the experimental period, for the conditions given in Table I. Histological Appearance. Histological sections on a length of guinea pig intestine, which has been perfused for 90 min., when compared with an adjacent section of the same intestine fixed immediately after removal from the animal, show that there is a partial disintegration of the mucosa, a loss of some cells from the villi into the lumen, and some loosening of the connective tissue. There is no gross stripping of the mucosal layer as reported by VereBr and McDougall (1). Under high power, the cytological lesions are moderate.
Absorption
of
Inner Outer Gas : No. of
TABLE I Monosaccharides from Isolated Surviving Guinea Pig Intestine solution: As shown in col. 2 + Ringer-bicarbonate (without calcium) pH 7.4 solution: 1% nicotinamide + Ringer-bicarbonate (without calcium) pH 7.4 93% oxygen; 7% carbon dioxide
T,--
Sugar present
expts.
--
Kexose,
mg.lhr. Median deviation
Median
I-
Sorbose,
mg./kr.
I-
Median deviation
Median
Gh3Z.e
025% glucose + 0.25% sorbose
7.1
0.250Jo galactose + 0.25% sorbose
7.25
/
0.65
0.5
0.1
0.05
0.45
0.05
Galactose
,
GlUCO?X
0.25yo fructose
-
3.6
EXPERIMENTAL
t
0.3
0.65
-
0.05
RESULTS
The Absorption of Monosaccharides The isolated surviving guinea pig small intestine examined by the above procedure shows the same relative absorption rates with respect to glucose, galactose, and fructose as those obtained by Cori (14) and Verz&r (1) using other methods. The experimental results are given in Table I. The relative values reported by the above-mentioned workers are: galactose 115, glucose 100, fructose 44, sorbose 30. The results in Table I, expressed in a similar manner, are approximately: galactose 100, glucose 100, fructose 50, and sorbose7. Although the rates for the
ABSORPTION
OF
199
SUGARS
first three sugars are in approximate agreement with those of Cori and VerzBr, that for sorbose differs considerably. The much lower rate of absorption of sorbose in the experiments reported here is probably due to the low concentrations of sugars used whereby diffusion is considerably decreased. Fructose Conversion to Glucose The problem of transformation of fructose into glucose in the small intestine has been discussed by Verzitr and McDougall (1). Our technique affords a ready means of investigating this phenomenon. In the experiments on the absorption of fructose, this was the only sugar placed in the solution circulating through the isolated intestine. The outer solution was analyzed for fructose by the resorcinol technique and also for total reducing substances. TABLE II Fructose Conversion to Glucose on Perfusion Through Isolated Surv@ing Guinea Pig Zntestine Duration of expt. = 90 min.; 37°C.; 93% 02 + 7% CO* Constituents
of perfusion
fluid
(“inner
solution”)
0.50~o fructose-Ringer-bicarbonate solution (free from calcium) Ringer-bicarbonate solution (free from calcium)
Glucose found in outer solution (not&n method)
Fructose found in outer solution m.
57
2.8
0.9
0.0
The rate of fructose appearance in the outer solution, as measured by the resorcinol method, was found to be of the same order as the rate of sorbose appearance (Table I). It is inferred that any fructose that appears in the outer solution, in these experiments, passesthrough the intestinal wall by diffusion. The quantity of total reducing substances in the outer solution, estimated with ferricyanide, however, was found to be much higher than could be accounted for by the fructose present. The additional reducing substance was shown to be glucose by the fact that it is oxidized by glucose oxidase (notatin) which (16) is known to react specifically with glucose. Manometric experiments, carried out with Mr. Louis Fridhandler of this Institute, have shown that aliquots of the outer solution, concentrated in volume ten times, give oxygen uptakes in the presence of notatin corresponding to the amount of reducing substance (glucose) found by the ferricyanide technique (seeTable II for experi-
200
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
mental results). The fructose used for perfusion was free from glucose, as indicated by lack of oxygen uptake when it was added to the glucose oxidase (notatin) preparation. It would seem from these experiments that the absorption of fructose results in a transformation of the monosaccharide into glucose [see also (15, 1, 34)], any fructose present in the outer solution arriving there by / diffusion. Phosphate Changes During
Sugar Absorption
In one experiment measuring glucose absorption and in one measuring galactose absorption, phosphate determinations were carried out on both inner and outer solutions at 30min. intervals. Although active absorption occurred with both these sugars, there was no detectable change in the total phosphate, or apparently in the phosphate distribution, in either solution. It was found also that omission or addition of phosphate to the perfusion fluids was without effect on the rate of absorption of the sugars. The results of Magee and Reid (31), indicating an accelerating action of phosphate on glucose absorption, are apparently due to the buffer action of the phosphate (32, 33). Respiratory
Activity
and Sugar Absorption
A relationship between respiratory activity and absorption of sugars has not previously been established, as none of the experimental methods previously used for absorption measurements has been suited to such a study. The present technique offers a direct method for carrying out such an investigation. The results (quoted in Table III) show that the selective absorption of glucose disappears almost completely, when oxygen in the system is replaced by nitrogen. The permeability of the intestine, however, to sorbose remains unchanged. It is evident that the presence of oxygen, or of respiratory activity, is essential for the phenomenon of selective absorption of glucose in the isolated guinea pig intestine. If respiratory processes are important for the active absorption of glucose, it follows that any substances which interfere with respiration should also interfere with the selective absorption of glucose. For this reason, a number of respiratory inhibitors have been investigated for their effects on glucose absorption.
ABSORPTION
OF
201
SUGARS
E$ects of Cyanide and Azde Both cyanide (0.01 M) and aaide (0.01 M) inhibit completely the active absorption of glucose without altering the diffusion of sorbose TABLE Effects
of Inhibitors
Inner solution: Outer solution: Gas:
on Glucose
III
Absorption Intestine
frvm
the Isolated
0.25yv glucose + 0.250Jo sorbose + Ringer-bicarbonate (without calcium) 1% nicotinamide + Ringer-bicarbonate solution calcium) Bee Table. Inhibitor
Expt. No.
Gas: 93yv nitrogen;
solution (without
7oJ, carbon dioxide ;
8::
Gas: 93% oxygen; 7% carbon dioxide
0.01
Guinea Pig
lpz+&z&
-
1 2
3 4 5 6 7 8 9 10 11 12 13 14 15
Surviving
M NaCNO
0.01 M NaNa 0.001 M sodium fluoroacetate 0.01 M sodium fluoroacetate 0.02 M sodium malonate 0.02 M sodium malonate” 0.005 M chloretone” 0.01 M chloretone” 0.0025 M quinine sulfate” 0.0025 M quinine sulfatern 0.001 M 2,4-dinitrophenol 0.0001 M 2,4-dinitrophenol
7.4 7.4 7.5 7.4 7.4 7.4 7.4 7.4 7.4 7.5 7.5 7.5 7.5
1
::i
~ 8::
7.0 0.6 0.2 5.0 3.1 5.7 3.9 6.5 1.4 1.3 1.4 1.6 1.7
0.6 1.0 1.0 0.8 0.8 0.3 0.5 1.4 1.0 0.9b 0.P 0.5 0.6
0 Inhibitor present in outer fluid as well as inner. b Fructose in mg./hr.
(Table III). Since oxygen is present in these experiments, it follows that absorption of the sugar depends on the respiratory activity of the tissue (which is poisoned by aside or cyanide) rather than upon the presence of oxygen.
202
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
Effects of Malonate and Fluoroacetate Malonate, well known as a competitive inhibitor of succinic dehydrogenase (19) and for its effect on the tricarboxylic acid cycle, inhibits the active absorption of glucose by isolated guinea pig intestine only feebly at 0.02 M. The effect is heightened when malonate is present in both the outer and inner solution to which the intestine is exposed (Table III). Sodium fluoroacetate, known to inhibit respiration by its interference with the tricarboxylic cycle (17, 18), inhibits active absorption of glucose at 0.001 M, the effect being considerably increased at 0.01 M (Table III). E$ect of Narcotics It has been shown by Quastel and Wheatley (21) that narcotics have the common property of inhibiting at low concentrations respiratory activities of tissues, particularly those aspects concerned with the oxidation of glucose and pyruvic acid. Recent evidence (22) indicates that narcotics at low concentration inhibit the oxidative synthesis of adenosine triphosphate. Chloretone (0.01 M) decreases glucose absorption in the guinea pig intestine in &fro (Table III). Such an inhibition is consistent with the conclusion that glucose absorption is a process greatly dependent on the respiratory activity of the intestine. Since oxidation of succinate is apparently not diminished by the presence of the narcotic (20), an attempt was made to reverse the inhibition caused by 0.01 M chloretone by adding to the inner solution 0.04 M sodium succinate. The inhibition, however, was not affected. E$ect of Quinine Quinine sulfate has been reported as either having no effect on glucose absorption (4), or as producing a substantial stimulation (23). Present experiments (Table III) show that the presence of quinine sulfate greatly depresses the active absorpt,ion of glucose by the isolated surviving guinea pig intestine. The diffusion of fructose, however, is unchanged. Effect of 2 ,&Dinitrophenol The results of the experiments, quoted above, on the active absorption of glucose by isolated intestine, either anaerobically or with inhibitors of respiratory processes, lead to the conclusion that such absorption depends on the integrity of the intestinal respiratory system.
ABSORPTION
OF
SUGARS
203
It is known (24) that 2,4-dinitrophenol at concentrations ranging from 0.00005 M to 0.0002 M “uncouples” phosphorylations from respiratory processes. In view of the phosphorylation theory for active absorption of sugar, it was of interest to discover the effects of such “uncoupling” agents on glucose absorption by guinea pig intestine. Results given in Table III show that when 2,4-dinitrophenol is added to the perfusion fluid of the isolated guinea pig intestine, absorption of glucose is considerably decreased. It is significant that the inhibition of absorption occurs at low concentrations (e.g. 0.0001 M) that are ineffective in bringing about respiratory inhibition. This result is contrary to that of Bruckner (30) whose experimental conditions differ markedly from ours. It is logical to conclude that 2,4-dinitrophenol exerts its inhibition by its well-known effect on the relation between phosphorylation and oxidation. This interpretation of the action of low concentrations of 2,4dinitrophenol indicates that phosphorylation reactions are involved in the selective absorption of glucose by intestine. The inhibitory effects of respiratory poisons on the absorption of sugar are probably due, therefore, to the inhibition of phosphorylations, which are dependent for their optimal rates on a constant supply of high-energy phosphate produced as a result of respiratory activities. Absorption
of o-Gkosamine
n-Glucosamine is only absorbed at a very slow rate, in the isolated surviving guinea pig intestine (Table IV). There is, moreover, no apparent inhibition of conversion of fructose to glucose when either n-glucosamine or N-acetylglucosamine is present (25). Absorption
of Hexose Diphosphate
Experiments with isolated guinea pig intestine show that the rate of appearance of reducing substances in the outer solution is slower when hexose diphosphate is perfused, than when glucose or fructose is perfused. Whether this is due to a slow rate of diffusion of this molecule into the cells, or to a slow rate of dephosphorylation of the molecule, it is impossible at present to say. The results agree, qualitatively, with those of Mathieu (27). Absorption Reducing l-phosphate
of Glucose 1 -Phosphate
substances appear as fast, during the perfusion of glucose through the isolated intestine, as during the perfusion of
204
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
glucose (Table IV). This apparently rapid absorption of glucose l-phosphate is in contrast to the slow absorption of glucose 6-phosphate and fructose 6-phosphate observed by Mathieu (27). It is interesting to note in this connection that Sutherland and Cori (28) have claimed that the -liver cell is permeable to glucose l-phosphate. TABLE The Absorption
IV
of Sugar Derivatives from the Isolated Surviving Guinea Pig
Intestine (The concentrations of sugar derivatives used were equimolar with 0.25yo glucose) Inner solution: as in col. 2 + Ringer-bicarbonate solution (without calcium) Outer solution: 1% nicotinamide + Ringer-bicarbonate solution (without calcium) Gas : see Table Expt.
No.
Substances
present
PH
/
I
/
Glucose
1 Sorbose
/ mg./kr. 1 mg./kr
Gas: 93yc oxygen; 7% carbon dioxide n-Glucosamine -I- sorbose Hexose diphosphate” Hexose diphosphate Glucose l-phosphate + sorbose” Glucose l-phosphate + sorboseD n-Glucosamine + fructose N-Acetyl-n-glucosamine + fructose Gas: 93% nitrogen; 8 9
Glucose l-phosphate Hexose diphosphate
7.5 7.3 7.3 7.4 7.4 7.5 7.5
1.1 1.5 2.4 6.6 6.2 4.5 3.5
0.2 0.0 0.0 0.7 0.4 0.5” 0.5”
7% carbon dioxide
+ sorbose
(1 KHtPO, omitted from Ringer solution. b Fructose in mg./hr.
on the Absorption of Glucose l-Phosphate and Hexose Diphosphate Results given in Table IV show that, under anaerobic conditions, apparently no absorption occurs with either glucose l-phosphate, or hexose diphosphate. It seemslikely from this finding that both these hexose phosphates
E$ect
undergo
of Anaerobiosis
dephosphorylation
in the intestine,
the resulting
sugar requiring
energy, from respiratory processes,before active absorption takes place.
ABSORF'TION OF SUGARS
205
DISCUSSION The technique used in this work for the study of absorption in the isolated surviving intestine has the advantage of simplicity and ease of construction. The fact that active absorption, as apart from passive diffusion, of sugars may take place is doubtless due to the fact that the intestine during the time of experiment (up to 90 min.) receives ample oxygenation and is, apparently, in a healthy physiological state. It was to be expected that absorption rates observed in the isolated surviving intestine preparation would be lower than those obtained in intact animals because of the additional barrier imposed by the intestinal wall and serosa. This condition, however, does not necessarily alter the relative absorption rates of different sugars, and the method is therefore suited to an investigation of the mechanisms, or factors, that influence intestinal absorption. Although fructose is known to occupy an intermediate position between the selectively absorbed sugars (glucose or galactose) and the inert sugars (sorbose and the pentoses), Wainio (29), after consideration of the available evidence, has concluded that the major fraction of the fructose which enters the organism does so by diffusion. Although it has been shown (1, 15) that the small intestine can convert fructose into glucose in vitro, the part that this transformation plays in the active absorption of fructose has not been established. The experiments reported here indicate that such a transformation accounts for all of the active absorption of fructose in vitro. The rate of diffusion of fructose is no greater, apparently, than that of sorbose. The observation that the presence of phosphate is not necessary in the perfusion fluids for selective absorption to take place does not necessarily oppose the phosphorylative mechanism of absorption. It is conceivable that a phosphorylation-dephosphorylation cycle with a rapid turnover of phosphate would require only catalytic amounts of phosphate which, no doubt, are already present in the tissue. It is demonstrated that selective absorption by isolated intestine depends on an intact respiratory system. This result is consistent with the phosphorylation hypothesis, and it is to be expected that respiratory poisons such as cyanide or azide, or a narcotic, such as chloretone, will inhibit oxidative synthesis of adenosine triphosphate in the tissue and hence the rate of phosphorylation of sugars. The inhibitory action of 2,4-dinitrophenol at low concentrations pro-
206
W. A. DARLINGTON
AND J. H. QUASTEL
vides more direct evidence of a phosphorylation being essential for the active absorption of glucose in the isolated surviving intestine. ACKNOWLEDGMENTS We are grateful to the National Cancer Institute of Canada and to the Sugar Research Foundation for grants-in-aid. One of us (W.A.D.) is indebted to the National Research Council (Canada) for a research studentship. We wish to thank Mrs. R. Fridhandler for technical assistance throughout this work. Grateful acknowledgment is due to Prof. C. P. Leblond of McGill University for his kindness in preparing and interpreting the histological sections of intestine used in this work. SUMMARY
1. A new apparatus and technique have been described for the perfusion of isolated surviving guinea pig small intestine, and the technique has been applied to the study of sugar absorption. 2. The isolated guinea pig intestine selectively absorbs glucose, fructose, and galactose. Sorbose enters the intestine by diffusion. The relative absorption rates for the selectively absorbed sugars are similar to those that have been observed in intact animals. 3. The absorption of fructose by the intestine results in a transformation into glucose. Any absorption of fructose as such is due to a diffusion process in the isolated guinea pig intestine, the rate of fructose diffusion being no greater than that of sorbose. 4. No concomitant transfer of phosphate during the absorption of glucose or galactose has been demonstrated, and the presence of phosphate in the perfusion fluids is not necessary for active glucose absorption. 5. An active respiratory system is essential for glucose absorption from the isolated guinea pig intestine, as shown by the inhibitory effects of anaerobiosis, cyanide, azide, fluoroacetate, malonate, and chloretone. The diffusion of sorbose or fructose is not altered by the presence of these respiratory inhibitors. Quinine sulfate is also inhibitory to active absorption of glucose. 6. 2,4-Dinitrophenol exercises, at low concentration, a highly inhibitory effect on active absorption of glucose. This observation is held to constitute direct evidence for the phosphorylation hypothesis of the mechanism
of glucose absorption
by isolated
intestine.
7. Glucose absorption from a glucose l-phosphate solution is as rapid as from a glucose solution in the surviving guinea pig intestine.
ABSORPTION
OF SUGARS
207
8. Anaerobiosis inhibits glucose absorption from glucose l-phosphate or hexose diphosphate solution, indicating that energy-requiring reactions are required for the phenomenon. REFERENCES 1. VERZ~R, F., AND MCDOUGALL, E. J., Absorption from the Intestine. Longmans, Green and Co., London, 1936. 2. WESTENBRINK, H. G. K., Ned. Tijdschr. Geneesk. 93, (III), 2217 (1949). 3. BECK, L. V., J. Biol. Chem. 143,403(1942). 4. OHNELL, R., AND HOBER, R., J. Cellular Comp. Physiol. 13, 161 (1939). 5. KALCKAR, H., Nature 130, 872 (1935). 6. SHAPIRO, B., Biochem. J., (London) 41, 151 (1947). 6a. NAITB, Y., J. Biochem. (Japan) 36, 131 (1944). 7. FISHER, R. B., AND PARSONS, D. S., J. Physiol. (London) 110, 36 (1949). 8. K~EBS, H. A., AND HENSELEIT, K., Z. physiol. Chem. 210, 33 (1932). 9. LIACI, L., Arch. furmacol. sper. 69, 2&l (1940). 10. VERZAR, F., Biochem. Z. 276, 17 (1935). 11. HAQEDORN, H. C., AND JENSEN, B. N., Biochem. Z. 136, 46 (1923); ibid. 137, 92 (1923). 12. ROE, J. H., J. BioZ. Chem. 107, 15 (1934). 13. SNELL, F, D., AND SNELL, C. T., Calorimetric Methods of Analysis. Chapman and Hall, London, 1936. 14. CORI, C. F., J. BioZ. Chem. 66, 691 (1925). 15. BOLLMAN, J. L., AND MANN, F. C.? Am. J. PhysioZ. 98, 683 (1931). 16. KEILIN, D., AND HARTREE, E. F., Biochem. J. (London) 42,221 (1948). 17. BARTLETT, G. R., AND BARRON, E. S. G., J. BioE. Chem. 170, 67 (1947). 18. ELLIOTT, W. B., AND KALNITSKY, G., J. BioZ. Chem. 186, 487 (1950). 19. QUASTEL, J. H., AND WOOLDRIDGE, W. R., Biochem. J. (London) 22, 689 (1928). 20. QUASTEL, J. H., AND WHEATLEY, A. H. M., Proc. Roy. Sot. (London) $112, 60 (1932). 21. QUASTEL, J. H., AND WHEATLEY, A. H. M., Biochem. J. (London) 28, 1521 (1934). 22. QUASTEL, J. H., Current Researches Anesthesia & Analgesia 31, 151 (1952). 23. ROY, A., AND SEN, B., Ann. Biochem. and ExptZ. Med. (India) 3, 1 (1943). 24. LOOMIS, W. F., AND LIPMANN, F., J. Biol. Chem. 173, 807 (1948). 2.5. CS~KY, T., Z. physiol. Chem. 2T7, 47 (1942). 26. HARPUR, R. P., AND QUASTEL, J. H., Nature 164, 693 (1949). 27. MATHIEU, F., Biochem. Z. 276,49 (1935). 28. SUTHERLAND, E. W., AND CORI, C. F., J. BioZ. Chem. 172, 737 (1948). 29. WAINIO, W. W., Sugar Research Foundation (N. Y.) Sci. Rept. Ser. No. 12, 6 (1948). 30. BRUCKNER, J., Helv. Physiol. et PharmacoZ. Acta 9, 259 (1951). 31. MAQEE, H. E., AND REID, E., J. Physiol. (London) 73, 181 (1931). 32. LASZT, L., Biochem. Z. 276, 40 (1935). 33. PONZ, F., AND LARRALDE, J., Nature 168, 912 (1951). 34. GODA, T., Biochem. Z. 294, 259 (1937).
of Sugars from Isolated W. A. Darlington
From
the Research
Institute,
Surviving
Intestine
and J. H. Quastel
Montreal
General
Hospital,
Montreal,
Canada
Received November 6,1952
VerzWs hypothesis (l), that a process of phosphorylation is involved in the rapid absorption of certain of the hexoses from the small intestine, has been discussed by a number of workers (2-6a). The object of the present study has been to investigate furt,her some of the phenomena underlying this phenomenon, with special reference to the relation between selective absorption of hexoses and respiratory activity. Since t.he available methods are ill suited to this study, it became necessary to develop a more satisfactory technique. The new experiment.al procedure used in this work will, therefore, be described in detail. METHODS
AND MATERIALS
The technique,which haa beenin usein this Institute for over 4 years,is similar in principle to the method recently describedby Fisher and Parsons(7). It con-
sistsin perfusingoxygenatedRinger solution through a sectionof freshly ieolated intestine which is bathed in an oxygenated Ringer solution. The apparatusinvolves two independentcirculatory systems,one running through the lumen of the intestine and the other external to the intestine. Measurementsare madeof the rate of perfusionof substances from the inner to the outer solution. A diagrammaticrepreeentationof the perfusionapparatusthat has been used is shown in Fig. 1.
The inner solution passesfrom a cylinder A, through C and a connecting arm into a small reservoir E which is opento the atmosphere.From E the fluid passesthrough F, which connects with the intestinal aegmentH, through Z and D,
back into the cylinder A. The lower opening in cylinder A is cloeed by a rubber stopper through which paaees the glass tubing of a stopcock. This stopcock serves to drain the inner fluid at the end of the experiment.. The volume of the inner solution is approximately 100 ml. The outer solution, which in cylinder M bathesthe outer surfaceof the intestinal segment H, paeses from the cylinder through N and the connecting arm 0 into the small reservoir P which is also open to the atmosphere. From P the fluid 194
ABSORPTION
OF
195
SUGARS
passes down glass tubing through a three-way stopcock into the cylinder M. The three-way stopcock R is used to drain the outer fluid at the end of the experiment, this fluid being collected and measured. The volume of the outer solution is approximately 110 ml. GA5
FIG. 1. Apparatus
for perfusion
5
of isolated surviving
intestine
(scale 1:5).
The continuous movement of fluid through the two circulations is maintained as follows: Gas under pressure (from a cylinder) is allowed to enter at T and U. It escapes from the apparatus at the outlets E and P. The gas passes through the side connections D and 0, and thus into E and P. In passing through the side connections, which are filled with fluid, the gas carries fluid with it into the small reservoirs E and P. In this way the level of fluid in E and P is made higher than that in C and N. Two circulations, therefore, result. One, called the inner, passes through the lumen of the intestinal segment, and the other, the outer, passes through the
196
W. A. DARLINGTON
AND J. H. QUASTEL
cylinder M which bathes the outside of the segment. Any exchange of substances between the inner and outer solutions must occur through the wall of the intestinal segment. For ease in setting up, and washing, the apparatus is made in sections, which are joined by rubber tubing, shown in the diagram by shading. The intestinal segment H is shown stippled in the diagram. When in use the apparatus is kept in an incubator maintained at 38 f 1°C. Samples may be removed during the experiment from both circulations through the stopcocks L and R, or by pipetting from the small reservoirs E and P. It is necessary to replace an equal volume of fluid to the circulation after withdrawal of a sample. EXPERIMENTAL
PROCEDURE
An animal is killed by decapitation and is bled. The abdomen is opened in the midline and all of the small intestine is removed except that portion adjacent to the stomach (duodenum) which adheres firmly to the surrounding tissue. The intestine is placed in a small beaker of the solution to be used in the experiment, all the solutions having previously been brought to 38°C. and gassed with 93% O2 + 7‘% COZ. In order to obtain a standardized procedure, the intestine in the beaker is removed and that part of the intestine which was 39-40 cm. from the stomach in the intact animal is cut out, and the inside washed out with the solution to be used. Care is taken to maintain the orientation of the intestine so that perfusion is carried out through the segment in the normal direction of flow. The apparatus is now assembled in the following way. The lower end of the segment is tied with a piece of thread to the glass tubing I, this having been pushed through the rubber stopper J as far as it will go, and the stopper is placed in the end of cylinder M. The segment of intestine now protrudes through the open end of cylinder M, and the loose end is fastened to that section of the glass tubing F which passes through the rubber stopper G. The stopper G is placed in position, and glass tubing Z is withdrawn through the stopper .Z until the segment is fully extended, but not stretched. Cylinder A, with stopper K inserted, is now joined to Z by means of rubber tubing, and cylinder A is partially filled with inner solution. The stopper B, which holds the section CDE is placed in position, and the two sections of F are joined with rubber tubing. The inner circulation is now completed but is not yet completely filled with solution. The part of the apparatus NOPQRS, which has been assembled previously, is now placed in position with rubber tubing at N and S, and the stopcock R adjusted so that the flow of fluid is from & to S. The outer solution is now added through P, care being taken that no fluid passes down 0 and into N, and cylinder M is thus filled from the bottom with the displaced air escaping through N. The filling of the inner circulation is completed in the same way. The level of fluid must be slightly above the side openings D and 0 in the small reservoirs E and P. The levels of fluid in E and P are kept even, as a higher level of fluid in E wilI cause distention of the segment of intestine, and also result in a higher hydrostatic pressure in the inside of the segment than on the outside. If the level of P
ABSORPTION
OF
SUGARS
197
is much above that in E, collapse of the segment will occur with a consequent blocking of the inner circulation. Gas is led to the system through rubber tubing which is connected over the glass tubing at T and U, and the perfusion is started. The time taken, from the killing of the animal to the commencement of the perfusion, is from 10 to 12 min. Samples are taken by pipetting from E and P because, with this method, there is no temporary stopping of circulation, which often results in excessive motility of the segment. Male guinea pigs, from a commercial source, weighing 550-350 g. have been usually used. In experiments carried out with an isotonic salt solution, as used by Krebs and Henseleit (S), as the circulatory fluid, severe peristaltic contractions occur in the intestinal segment, in some cases sufficiently vigorous to prevent circulation of the inner fluid, and extremely variable results are obtained. However, if nicotinamide is added to the outer solution this difficulty is partly overcome. In the presence of nicotinamide no motility is shown by the segment during perfusion, but vigorous movements usually occur for a short time after the taking of a sample. Liaci (9) has reported that nicotinamide has a depressing effect on guinea pig intestinal muscle. It has been noted, also, that the segment remains quiet even when samples are taken, if calcium is omitted from the circulating solutions. For this reason, calcium is usually omitted from the solutions bathing the intestine. In order to obtain a measure of the amount of sugar which will cross the intestinal wall by simple diffusion, sorboae has been added to the inner circulatory fluid. Verzar (10) has shown that L-sorbose is an inert sugar in the intestine, and that its absorption from the intestine is by diffusion only. In the experiments to be reported, therefore, sorbose has been added to the perfusion fluid containing the sugar under investigation. The rate of sorbose appearance in the outer solution is used as a measure of the rate of physical diffusion of either sugar through the intestinal wall, it being assumed that there will be little difference between the rates of permeability of sorbose and those of sugars of allied constitution. Sugar Estimations. Total reducing substances (glucose plus sorbose) are determined by the ferricyanide reduction method of Hagedorn and Jensen (11). L-Sorbose, or n-fructose, is determined by the resorcinol method of Roe (12), originally designed for fructose but also applicable to sorbose, although separate standard curves are necessary. Glucose does not interfere with these determinations. The amount of glucose is obtained by difference. Phosphate is determined by the method of Snell and Snell (13), using hydroquinone reduction. Intestine. Intestinal segment used: 30 cm. from stomach; Eength of segment: 10 cm. Perfusion Salt Solution. This consists of 0.714yo NaCl, 0.0365yc KCl, 0.0167yc KH*PO,, 0.0311% MgSOd.7Hz0, 0.0416% NaHCOa. Sampling. One-milliliter samples are pipetted from reservoir P for analysis every 10 or 15 min. after commencement of the perfusion, for 90 min., and an equal volume of solution is immediately replaced. When calculating the results, allowance is made for the amount of material removed in this way. The absorption
198
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
rates are found to be constant throughout the experimental period, for the conditions given in Table I. Histological Appearance. Histological sections on a length of guinea pig intestine, which has been perfused for 90 min., when compared with an adjacent section of the same intestine fixed immediately after removal from the animal, show that there is a partial disintegration of the mucosa, a loss of some cells from the villi into the lumen, and some loosening of the connective tissue. There is no gross stripping of the mucosal layer as reported by VereBr and McDougall (1). Under high power, the cytological lesions are moderate.
Absorption
of
Inner Outer Gas : No. of
TABLE I Monosaccharides from Isolated Surviving Guinea Pig Intestine solution: As shown in col. 2 + Ringer-bicarbonate (without calcium) pH 7.4 solution: 1% nicotinamide + Ringer-bicarbonate (without calcium) pH 7.4 93% oxygen; 7% carbon dioxide
T,--
Sugar present
expts.
--
Kexose,
mg.lhr. Median deviation
Median
I-
Sorbose,
mg./kr.
I-
Median deviation
Median
Gh3Z.e
025% glucose + 0.25% sorbose
7.1
0.250Jo galactose + 0.25% sorbose
7.25
/
0.65
0.5
0.1
0.05
0.45
0.05
Galactose
,
GlUCO?X
0.25yo fructose
-
3.6
EXPERIMENTAL
t
0.3
0.65
-
0.05
RESULTS
The Absorption of Monosaccharides The isolated surviving guinea pig small intestine examined by the above procedure shows the same relative absorption rates with respect to glucose, galactose, and fructose as those obtained by Cori (14) and Verz&r (1) using other methods. The experimental results are given in Table I. The relative values reported by the above-mentioned workers are: galactose 115, glucose 100, fructose 44, sorbose 30. The results in Table I, expressed in a similar manner, are approximately: galactose 100, glucose 100, fructose 50, and sorbose7. Although the rates for the
ABSORPTION
OF
199
SUGARS
first three sugars are in approximate agreement with those of Cori and VerzBr, that for sorbose differs considerably. The much lower rate of absorption of sorbose in the experiments reported here is probably due to the low concentrations of sugars used whereby diffusion is considerably decreased. Fructose Conversion to Glucose The problem of transformation of fructose into glucose in the small intestine has been discussed by Verzitr and McDougall (1). Our technique affords a ready means of investigating this phenomenon. In the experiments on the absorption of fructose, this was the only sugar placed in the solution circulating through the isolated intestine. The outer solution was analyzed for fructose by the resorcinol technique and also for total reducing substances. TABLE II Fructose Conversion to Glucose on Perfusion Through Isolated Surv@ing Guinea Pig Zntestine Duration of expt. = 90 min.; 37°C.; 93% 02 + 7% CO* Constituents
of perfusion
fluid
(“inner
solution”)
0.50~o fructose-Ringer-bicarbonate solution (free from calcium) Ringer-bicarbonate solution (free from calcium)
Glucose found in outer solution (not&n method)
Fructose found in outer solution m.
57
2.8
0.9
0.0
The rate of fructose appearance in the outer solution, as measured by the resorcinol method, was found to be of the same order as the rate of sorbose appearance (Table I). It is inferred that any fructose that appears in the outer solution, in these experiments, passesthrough the intestinal wall by diffusion. The quantity of total reducing substances in the outer solution, estimated with ferricyanide, however, was found to be much higher than could be accounted for by the fructose present. The additional reducing substance was shown to be glucose by the fact that it is oxidized by glucose oxidase (notatin) which (16) is known to react specifically with glucose. Manometric experiments, carried out with Mr. Louis Fridhandler of this Institute, have shown that aliquots of the outer solution, concentrated in volume ten times, give oxygen uptakes in the presence of notatin corresponding to the amount of reducing substance (glucose) found by the ferricyanide technique (seeTable II for experi-
200
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
mental results). The fructose used for perfusion was free from glucose, as indicated by lack of oxygen uptake when it was added to the glucose oxidase (notatin) preparation. It would seem from these experiments that the absorption of fructose results in a transformation of the monosaccharide into glucose [see also (15, 1, 34)], any fructose present in the outer solution arriving there by / diffusion. Phosphate Changes During
Sugar Absorption
In one experiment measuring glucose absorption and in one measuring galactose absorption, phosphate determinations were carried out on both inner and outer solutions at 30min. intervals. Although active absorption occurred with both these sugars, there was no detectable change in the total phosphate, or apparently in the phosphate distribution, in either solution. It was found also that omission or addition of phosphate to the perfusion fluids was without effect on the rate of absorption of the sugars. The results of Magee and Reid (31), indicating an accelerating action of phosphate on glucose absorption, are apparently due to the buffer action of the phosphate (32, 33). Respiratory
Activity
and Sugar Absorption
A relationship between respiratory activity and absorption of sugars has not previously been established, as none of the experimental methods previously used for absorption measurements has been suited to such a study. The present technique offers a direct method for carrying out such an investigation. The results (quoted in Table III) show that the selective absorption of glucose disappears almost completely, when oxygen in the system is replaced by nitrogen. The permeability of the intestine, however, to sorbose remains unchanged. It is evident that the presence of oxygen, or of respiratory activity, is essential for the phenomenon of selective absorption of glucose in the isolated guinea pig intestine. If respiratory processes are important for the active absorption of glucose, it follows that any substances which interfere with respiration should also interfere with the selective absorption of glucose. For this reason, a number of respiratory inhibitors have been investigated for their effects on glucose absorption.
ABSORPTION
OF
201
SUGARS
E$ects of Cyanide and Azde Both cyanide (0.01 M) and aaide (0.01 M) inhibit completely the active absorption of glucose without altering the diffusion of sorbose TABLE Effects
of Inhibitors
Inner solution: Outer solution: Gas:
on Glucose
III
Absorption Intestine
frvm
the Isolated
0.25yv glucose + 0.250Jo sorbose + Ringer-bicarbonate (without calcium) 1% nicotinamide + Ringer-bicarbonate solution calcium) Bee Table. Inhibitor
Expt. No.
Gas: 93yv nitrogen;
solution (without
7oJ, carbon dioxide ;
8::
Gas: 93% oxygen; 7% carbon dioxide
0.01
Guinea Pig
lpz+&z&
-
1 2
3 4 5 6 7 8 9 10 11 12 13 14 15
Surviving
M NaCNO
0.01 M NaNa 0.001 M sodium fluoroacetate 0.01 M sodium fluoroacetate 0.02 M sodium malonate 0.02 M sodium malonate” 0.005 M chloretone” 0.01 M chloretone” 0.0025 M quinine sulfate” 0.0025 M quinine sulfatern 0.001 M 2,4-dinitrophenol 0.0001 M 2,4-dinitrophenol
7.4 7.4 7.5 7.4 7.4 7.4 7.4 7.4 7.4 7.5 7.5 7.5 7.5
1
::i
~ 8::
7.0 0.6 0.2 5.0 3.1 5.7 3.9 6.5 1.4 1.3 1.4 1.6 1.7
0.6 1.0 1.0 0.8 0.8 0.3 0.5 1.4 1.0 0.9b 0.P 0.5 0.6
0 Inhibitor present in outer fluid as well as inner. b Fructose in mg./hr.
(Table III). Since oxygen is present in these experiments, it follows that absorption of the sugar depends on the respiratory activity of the tissue (which is poisoned by aside or cyanide) rather than upon the presence of oxygen.
202
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
Effects of Malonate and Fluoroacetate Malonate, well known as a competitive inhibitor of succinic dehydrogenase (19) and for its effect on the tricarboxylic acid cycle, inhibits the active absorption of glucose by isolated guinea pig intestine only feebly at 0.02 M. The effect is heightened when malonate is present in both the outer and inner solution to which the intestine is exposed (Table III). Sodium fluoroacetate, known to inhibit respiration by its interference with the tricarboxylic cycle (17, 18), inhibits active absorption of glucose at 0.001 M, the effect being considerably increased at 0.01 M (Table III). E$ect of Narcotics It has been shown by Quastel and Wheatley (21) that narcotics have the common property of inhibiting at low concentrations respiratory activities of tissues, particularly those aspects concerned with the oxidation of glucose and pyruvic acid. Recent evidence (22) indicates that narcotics at low concentration inhibit the oxidative synthesis of adenosine triphosphate. Chloretone (0.01 M) decreases glucose absorption in the guinea pig intestine in &fro (Table III). Such an inhibition is consistent with the conclusion that glucose absorption is a process greatly dependent on the respiratory activity of the intestine. Since oxidation of succinate is apparently not diminished by the presence of the narcotic (20), an attempt was made to reverse the inhibition caused by 0.01 M chloretone by adding to the inner solution 0.04 M sodium succinate. The inhibition, however, was not affected. E$ect of Quinine Quinine sulfate has been reported as either having no effect on glucose absorption (4), or as producing a substantial stimulation (23). Present experiments (Table III) show that the presence of quinine sulfate greatly depresses the active absorpt,ion of glucose by the isolated surviving guinea pig intestine. The diffusion of fructose, however, is unchanged. Effect of 2 ,&Dinitrophenol The results of the experiments, quoted above, on the active absorption of glucose by isolated intestine, either anaerobically or with inhibitors of respiratory processes, lead to the conclusion that such absorption depends on the integrity of the intestinal respiratory system.
ABSORPTION
OF
SUGARS
203
It is known (24) that 2,4-dinitrophenol at concentrations ranging from 0.00005 M to 0.0002 M “uncouples” phosphorylations from respiratory processes. In view of the phosphorylation theory for active absorption of sugar, it was of interest to discover the effects of such “uncoupling” agents on glucose absorption by guinea pig intestine. Results given in Table III show that when 2,4-dinitrophenol is added to the perfusion fluid of the isolated guinea pig intestine, absorption of glucose is considerably decreased. It is significant that the inhibition of absorption occurs at low concentrations (e.g. 0.0001 M) that are ineffective in bringing about respiratory inhibition. This result is contrary to that of Bruckner (30) whose experimental conditions differ markedly from ours. It is logical to conclude that 2,4-dinitrophenol exerts its inhibition by its well-known effect on the relation between phosphorylation and oxidation. This interpretation of the action of low concentrations of 2,4dinitrophenol indicates that phosphorylation reactions are involved in the selective absorption of glucose by intestine. The inhibitory effects of respiratory poisons on the absorption of sugar are probably due, therefore, to the inhibition of phosphorylations, which are dependent for their optimal rates on a constant supply of high-energy phosphate produced as a result of respiratory activities. Absorption
of o-Gkosamine
n-Glucosamine is only absorbed at a very slow rate, in the isolated surviving guinea pig intestine (Table IV). There is, moreover, no apparent inhibition of conversion of fructose to glucose when either n-glucosamine or N-acetylglucosamine is present (25). Absorption
of Hexose Diphosphate
Experiments with isolated guinea pig intestine show that the rate of appearance of reducing substances in the outer solution is slower when hexose diphosphate is perfused, than when glucose or fructose is perfused. Whether this is due to a slow rate of diffusion of this molecule into the cells, or to a slow rate of dephosphorylation of the molecule, it is impossible at present to say. The results agree, qualitatively, with those of Mathieu (27). Absorption Reducing l-phosphate
of Glucose 1 -Phosphate
substances appear as fast, during the perfusion of glucose through the isolated intestine, as during the perfusion of
204
W.
A.
DARLINGTON
AND
J.
H.
QUASTEL
glucose (Table IV). This apparently rapid absorption of glucose l-phosphate is in contrast to the slow absorption of glucose 6-phosphate and fructose 6-phosphate observed by Mathieu (27). It is interesting to note in this connection that Sutherland and Cori (28) have claimed that the -liver cell is permeable to glucose l-phosphate. TABLE The Absorption
IV
of Sugar Derivatives from the Isolated Surviving Guinea Pig
Intestine (The concentrations of sugar derivatives used were equimolar with 0.25yo glucose) Inner solution: as in col. 2 + Ringer-bicarbonate solution (without calcium) Outer solution: 1% nicotinamide + Ringer-bicarbonate solution (without calcium) Gas : see Table Expt.
No.
Substances
present
PH
/
I
/
Glucose
1 Sorbose
/ mg./kr. 1 mg./kr
Gas: 93yc oxygen; 7% carbon dioxide n-Glucosamine -I- sorbose Hexose diphosphate” Hexose diphosphate Glucose l-phosphate + sorbose” Glucose l-phosphate + sorboseD n-Glucosamine + fructose N-Acetyl-n-glucosamine + fructose Gas: 93% nitrogen; 8 9
Glucose l-phosphate Hexose diphosphate
7.5 7.3 7.3 7.4 7.4 7.5 7.5
1.1 1.5 2.4 6.6 6.2 4.5 3.5
0.2 0.0 0.0 0.7 0.4 0.5” 0.5”
7% carbon dioxide
+ sorbose
(1 KHtPO, omitted from Ringer solution. b Fructose in mg./hr.
on the Absorption of Glucose l-Phosphate and Hexose Diphosphate Results given in Table IV show that, under anaerobic conditions, apparently no absorption occurs with either glucose l-phosphate, or hexose diphosphate. It seemslikely from this finding that both these hexose phosphates
E$ect
undergo
of Anaerobiosis
dephosphorylation
in the intestine,
the resulting
sugar requiring
energy, from respiratory processes,before active absorption takes place.
ABSORF'TION OF SUGARS
205
DISCUSSION The technique used in this work for the study of absorption in the isolated surviving intestine has the advantage of simplicity and ease of construction. The fact that active absorption, as apart from passive diffusion, of sugars may take place is doubtless due to the fact that the intestine during the time of experiment (up to 90 min.) receives ample oxygenation and is, apparently, in a healthy physiological state. It was to be expected that absorption rates observed in the isolated surviving intestine preparation would be lower than those obtained in intact animals because of the additional barrier imposed by the intestinal wall and serosa. This condition, however, does not necessarily alter the relative absorption rates of different sugars, and the method is therefore suited to an investigation of the mechanisms, or factors, that influence intestinal absorption. Although fructose is known to occupy an intermediate position between the selectively absorbed sugars (glucose or galactose) and the inert sugars (sorbose and the pentoses), Wainio (29), after consideration of the available evidence, has concluded that the major fraction of the fructose which enters the organism does so by diffusion. Although it has been shown (1, 15) that the small intestine can convert fructose into glucose in vitro, the part that this transformation plays in the active absorption of fructose has not been established. The experiments reported here indicate that such a transformation accounts for all of the active absorption of fructose in vitro. The rate of diffusion of fructose is no greater, apparently, than that of sorbose. The observation that the presence of phosphate is not necessary in the perfusion fluids for selective absorption to take place does not necessarily oppose the phosphorylative mechanism of absorption. It is conceivable that a phosphorylation-dephosphorylation cycle with a rapid turnover of phosphate would require only catalytic amounts of phosphate which, no doubt, are already present in the tissue. It is demonstrated that selective absorption by isolated intestine depends on an intact respiratory system. This result is consistent with the phosphorylation hypothesis, and it is to be expected that respiratory poisons such as cyanide or azide, or a narcotic, such as chloretone, will inhibit oxidative synthesis of adenosine triphosphate in the tissue and hence the rate of phosphorylation of sugars. The inhibitory action of 2,4-dinitrophenol at low concentrations pro-
206
W. A. DARLINGTON
AND J. H. QUASTEL
vides more direct evidence of a phosphorylation being essential for the active absorption of glucose in the isolated surviving intestine. ACKNOWLEDGMENTS We are grateful to the National Cancer Institute of Canada and to the Sugar Research Foundation for grants-in-aid. One of us (W.A.D.) is indebted to the National Research Council (Canada) for a research studentship. We wish to thank Mrs. R. Fridhandler for technical assistance throughout this work. Grateful acknowledgment is due to Prof. C. P. Leblond of McGill University for his kindness in preparing and interpreting the histological sections of intestine used in this work. SUMMARY
1. A new apparatus and technique have been described for the perfusion of isolated surviving guinea pig small intestine, and the technique has been applied to the study of sugar absorption. 2. The isolated guinea pig intestine selectively absorbs glucose, fructose, and galactose. Sorbose enters the intestine by diffusion. The relative absorption rates for the selectively absorbed sugars are similar to those that have been observed in intact animals. 3. The absorption of fructose by the intestine results in a transformation into glucose. Any absorption of fructose as such is due to a diffusion process in the isolated guinea pig intestine, the rate of fructose diffusion being no greater than that of sorbose. 4. No concomitant transfer of phosphate during the absorption of glucose or galactose has been demonstrated, and the presence of phosphate in the perfusion fluids is not necessary for active glucose absorption. 5. An active respiratory system is essential for glucose absorption from the isolated guinea pig intestine, as shown by the inhibitory effects of anaerobiosis, cyanide, azide, fluoroacetate, malonate, and chloretone. The diffusion of sorbose or fructose is not altered by the presence of these respiratory inhibitors. Quinine sulfate is also inhibitory to active absorption of glucose. 6. 2,4-Dinitrophenol exercises, at low concentration, a highly inhibitory effect on active absorption of glucose. This observation is held to constitute direct evidence for the phosphorylation hypothesis of the mechanism
of glucose absorption
by isolated
intestine.
7. Glucose absorption from a glucose l-phosphate solution is as rapid as from a glucose solution in the surviving guinea pig intestine.
ABSORPTION
OF SUGARS
207
8. Anaerobiosis inhibits glucose absorption from glucose l-phosphate or hexose diphosphate solution, indicating that energy-requiring reactions are required for the phenomenon. REFERENCES 1. VERZ~R, F., AND MCDOUGALL, E. J., Absorption from the Intestine. Longmans, Green and Co., London, 1936. 2. WESTENBRINK, H. G. K., Ned. Tijdschr. Geneesk. 93, (III), 2217 (1949). 3. BECK, L. V., J. Biol. Chem. 143,403(1942). 4. OHNELL, R., AND HOBER, R., J. Cellular Comp. Physiol. 13, 161 (1939). 5. KALCKAR, H., Nature 130, 872 (1935). 6. SHAPIRO, B., Biochem. J., (London) 41, 151 (1947). 6a. NAITB, Y., J. Biochem. (Japan) 36, 131 (1944). 7. FISHER, R. B., AND PARSONS, D. S., J. Physiol. (London) 110, 36 (1949). 8. K~EBS, H. A., AND HENSELEIT, K., Z. physiol. Chem. 210, 33 (1932). 9. LIACI, L., Arch. furmacol. sper. 69, 2&l (1940). 10. VERZAR, F., Biochem. Z. 276, 17 (1935). 11. HAQEDORN, H. C., AND JENSEN, B. N., Biochem. Z. 136, 46 (1923); ibid. 137, 92 (1923). 12. ROE, J. H., J. BioZ. Chem. 107, 15 (1934). 13. SNELL, F, D., AND SNELL, C. T., Calorimetric Methods of Analysis. Chapman and Hall, London, 1936. 14. CORI, C. F., J. BioZ. Chem. 66, 691 (1925). 15. BOLLMAN, J. L., AND MANN, F. C.? Am. J. PhysioZ. 98, 683 (1931). 16. KEILIN, D., AND HARTREE, E. F., Biochem. J. (London) 42,221 (1948). 17. BARTLETT, G. R., AND BARRON, E. S. G., J. BioE. Chem. 170, 67 (1947). 18. ELLIOTT, W. B., AND KALNITSKY, G., J. BioZ. Chem. 186, 487 (1950). 19. QUASTEL, J. H., AND WOOLDRIDGE, W. R., Biochem. J. (London) 22, 689 (1928). 20. QUASTEL, J. H., AND WHEATLEY, A. H. M., Proc. Roy. Sot. (London) $112, 60 (1932). 21. QUASTEL, J. H., AND WHEATLEY, A. H. M., Biochem. J. (London) 28, 1521 (1934). 22. QUASTEL, J. H., Current Researches Anesthesia & Analgesia 31, 151 (1952). 23. ROY, A., AND SEN, B., Ann. Biochem. and ExptZ. Med. (India) 3, 1 (1943). 24. LOOMIS, W. F., AND LIPMANN, F., J. Biol. Chem. 173, 807 (1948). 2.5. CS~KY, T., Z. physiol. Chem. 2T7, 47 (1942). 26. HARPUR, R. P., AND QUASTEL, J. H., Nature 164, 693 (1949). 27. MATHIEU, F., Biochem. Z. 276,49 (1935). 28. SUTHERLAND, E. W., AND CORI, C. F., J. BioZ. Chem. 172, 737 (1948). 29. WAINIO, W. W., Sugar Research Foundation (N. Y.) Sci. Rept. Ser. No. 12, 6 (1948). 30. BRUCKNER, J., Helv. Physiol. et PharmacoZ. Acta 9, 259 (1951). 31. MAQEE, H. E., AND REID, E., J. Physiol. (London) 73, 181 (1931). 32. LASZT, L., Biochem. Z. 276, 40 (1935). 33. PONZ, F., AND LARRALDE, J., Nature 168, 912 (1951). 34. GODA, T., Biochem. Z. 294, 259 (1937).