Intestinal absorption of sucrose in man: interrelation of hydrolysis and monosaccharide product absorption.
G M Gray, F J Ingelfinger
J Clin Invest. 1966;45(3):388-398. https://doi.org/10.1172/JCI105354.
Research Article
Find the latest version: https://jci.me/105354/pdf Journal of Clinical Inxestigation Vol. 45, No. 3, 1966
Intestinal Absorption of Sucrose in Man: Interrelation of Hydrolysis and Monosaccharide Product Absorption * GARY M. GRAY t AND FRANZ J. INGELFINGER (From the Evans Memorial Department of Clinical Research, University Hospital, and the Department of Medicine, Boston University School of Medicine, Boston University Medical Center, Boston, Mass.) Disaccharides are hydrolyzed by their specific amounts of the hydrolysis products accumulate enzymes present in the intestinal mucosa (1-3). intraluminally during the process of sucrose ab- Although some current textbooks still state that sorption in man and that these monosaccharides these enzymes are secreted into the intestinal lu- appear to move back from their mucosal site of hy- men (4-7), the concentrations of monosaccharide drolysis to the lumen (13). products free in intestinal contents during disac- The present work is concerned with the rela- charide absorption in vitro (8-10) have been found tion of hydrolysis of sucrose to the absorption of insufficient to support the concept of intraluminal its monosaccharide components, glucose, an ac- hydrolysis. In addition, the low disaccharidase tively absorbed monosaccharide (14, 15, 17), and activity of intestinal contents during the absorp- fructose, which is passively absorbed (14, 15). tion process in vivo (11-13) strongly suggests that the disaccharide either enters the cell before Methods being hydrolyzed or else is hydrolyzed at the cell Thirty-two normal young subjects were studied on 105 surface by mucosa-bound enzyme. The released occasions by use of a double-lumen tube placed at various monosaccharide products presumedly are then levels of the intestine (13, 18). Polyethlene glycol 4000 transported across the intestinal cell (8, 14, 15). (PEG) was used as the nonabsorbable water-soluble Little information is available that relates disac- marker and was determined by a modification of Hyd&i's method (19). Sucrose solution or an equivalent mix- charide hydrolysis to absorption of the component ture of glucose and fructose made isotonic with NaCl monosaccharides. Wilson and Vincent (14) com- (290 ± 10 mOsm per L) was infused at 15 ml per minute mented on the accumulation of monosaccharides in through the proximal orifice of the tube. In some stud- the mucosal medium during the process of disac- ies, galactose was also infused. Intestinal samples were charide absorption in hamster gut sac preparations, collected by siphonage from the distal orifice, which was located 15 or 30 cm from the site of infusion. The ini- and Dahlqvist and Thomson have reported that tial 30 minutes of an infusion period allowed steady state large amounts of free fructose accumulate intra- conditions to be approached so that, thereafter, concen- luminally during sucrose absorption in the intact tration of PEG and the test sugar or sugars in succes- rat (16). Despite these findings in animals, sively collected samples showed little variation (13). Dahlqvist and Borgstr6m found little free intra- When successive experiments were performed in the same intestinal segment, the equilibration period also luminal monosaccharides during the process of served to prevent the contamination of one test by the disaccharide absorption in man (12). It was re- residuals of a preceding test. Absorption rates deter- cently demonstrated, however, that appreciable mined by infusing a specific solution changed little (mean ± 10%) when experiments were repeated during the *Submitted for publication June 10, 1965; accepted course of a 10-hour period; thus there was no evidence of December 2, 1965. "fatigue" of the intestinal segment under study, and ab- Supported in part by U. S. Public Health Service re- sorption of different carbohydrate solutions could be search grant AM 03560-04 and 05 and by training grant compared. The test solutions were infused in random T1 AM 5025-07 and 08 from the National Institute of order, and specimens obtained from the distal orifice were Arthritis and Metabolic Diseases. collected by use of the precautions previously outlined in Presented in part at the Annual Meeting of the Amer- order to insure stability of the carbohydrates (13). ican Society for Clinical Investigation, Atlantic City, Assay of carbohydrates in intestinal samples. After N. J., May 3, 1964. Ba(OH)a-ZnSO4 deproteinization (20), sucrose,' glu- t Address requests for reprints to Dr. Gary M. Gray, U. S. Army Tropical Research Medical Laboratory, 1 a-D-Glucopyranosyl-P-D-fructofuranoside, analytical APO, New York, N. Y. 09851. grade, Merck Co., Rahway, N. J. 388 INTESTINAL ABSORPTION OF SUCROSE IN MAN 389 cose,2 and total hexose (glucose plus fructose) 3 were Sucrose absorption can also be expressed in terms of determined by the specific enzymatic methods previously the individual monosaccharides: described (13). Glucose was substracted from total Glucose product absorption hexose to determine fructose. = [Si V-S,-V-(Pi/P,)J - [G..V'(Pi/P,)], [3] In some of the samples containing only fructose and glucose, fructose was analyzed by Dische and Devi's and ketohexose method (21); values obtained were within fructose product absorption 3% of those found by use of the enzymatic method. = [S1-V-Se-V-(P1/P.) ]-[F.*V (P1/P.)]. [4] Galactose 4 was assayed by a modification of the galac- tose oxidase assay described by Avigad, Amaral, Asensio, The use of Equations 3 and 4 is necessary if absorption and Horecker (22). The reagent consisted of 125 U of sucrose is to be compared with that of an equivalent galactose oxidase,5 3 mg peroxidase,6 0.6 ml of 1% glucose-fructose mixture. o-dianisidine in 95% ethanol, 100,000 U catalase,5 and 65 ml of 0.5 M Tris buffer (23) at pH 7.0. It was neces- Results sary to prepare the reagent daily since the chromogen rapidly became colored. To 1 ml of deproteinized sample Comparison of absorption of sucrose versus glu- 2.5 ml of the reagent was added. The reaction mixture cose-fructose mixture. To investigate the relation was incubated at 370 C for 20 minutes and the reaction between the hydrolysis of the disaccharide and stopped with 0.1 ml of 1 N H2SOs Extinction was de- transport of its monosaccharides, we undertook termined in a spectrophotometer at 395 m/A and a 1-cm light path used. A linear relationship of optical density paired experiments comparing absorption from su- to concentration occurred when 50 to 125 ,ug of galactose crose (73 mM) with that from an equivalent mix- was in the cuvette. Amounts of galactose less than 50 ture containing glucose plus fructose (73 mM each /Ag produced little absorbance. Sucrose, glucose, and monosaccharide). Experiments at a given intesti- fructose in 25 times the concentration of galactose read nal level were performed over successive 60-minute the same as the reagent blank and did not interfere with the reaction. Recovery of galactose added to intestinal intervals and the solutions infused in random contents was 100 ± 4%. order. A 30-cm distance between infusion orifice Calculations. Sucrose disappearance over the intesti- and collection orifice was used. As shown in Ta- nal segment may be considered as equal to the sucrose ble I, glucose and fructose absorption rates were hydrolyzed, since appreciable amounts of sucrose do not not significantly different whether the disaccharide disappear by absorption into blood of the intact disac- charide; this was discussed in a previous report (13). was infused (mean: glucose, 35 mmoles per hour; Considerable quantities of the monosaccharide hydroly- fructose, 21 mmoles per hour) or the monosac- sis products of sucrose accumulate intraluminally during charide mixture was infused (mean: glucose, 39 the absorption process, and therefore amounts of the mmoles per hour; fructose, 25 mmoles per hour). monosaccharide found at the collecting orifice must be Also, a similar relation prevailed in 13 paired subtracted from the sucrose hydrolyzed (sucrose that disappeared) to determine absorption (13). studies when 29 mM sugars were infused. This is summarized in Table I. Sucrose hydrolysis=2[S1 V-Se-V-(P1/P.)], and [1] sucrose absorption = 2 [S.V -S.eV- (P1/P.) ] Figure 1 relates glucose absorption rates in in- - [(Ge + F) V (P1/Pe)J, [2] dividual paired experiments and demonstrates that where hydrolysis and absorption are expressed as mil- there is close correlation to linearity (r = 0.87, limoles monosaccharide per hour, and symbols refer to p < 0.001) as well as to the theoretical line repre- millimolar concentrations as follows: St = sucrose in in- senting identical absorption from either infusion fusion; S. = sucrose in collected samples; P1 = PEG in solution. A significant correlation, but of some- infusion; P. = PEG in collected samples; G. = glucose what lower order, was also found for fructose ab- in collected samples; F. =fructose in collected samples. V = volume (liters infused in 1 hour). sorption (r = 0.62, p < 0.01) (Figure 2). Effect of galactose on sucrose hydrolysis and 2 D-Glucose, analytical grade, Merck Co., Rahway, N. J. 3.D-Fructose, C. P. Pfanstiehl Co., Waukegan, Ill. absorption. In our experiments the rate of su- 4 D-Galactose, C. P. Pfanstiehl Co., Waukegan, Ill. crose hydrolysis was greatly different in jejunum Although all other carbohydrates used were chromato- and ileum (Table I) (13). Despite this, similar graphically pure (less than 1% impurity), D-galactose amounts of monosaccharide products accumulated contained 5% D-glucose (as measured by glucose oxidase at all levels of intestine (Figure 3), suggesting the and hexokinase reagents) and a trace of an oligosac- charide. possibility that the hydrolytic process was inhibited 6 Worthington Biochemical Corp., Freehold, N. J. in both jejunum and ileum once the intraluminal INutritional Biochemicals Corp., Cleveland, Ohio. concentration of monosaccharides reached a criti- 390 GARY M. GRAY AND FRANZ J. INGELFINGER
TABLE I Comparison of absorption rates of sucrose with equimolar mixture of glucose plus fructose*
Glucose 73 mM + fructose§ Sucrose 73 mM§ 73 mM Intestinal Sucrose Glucose Fructose Glucose Fructose Subjectt level: hydrolysis absorption absorption absorption absorption cm mmokes disac- mmoks/hr mmoles/hr charide/hr P.C. 100 23 22 7 24 18 G.Ha.a 95 32 30 26 33 22 G.Ha.b 135 54 52 38 49 33 D.Z. 120 33 27 13 52 36 G.Ha.IIa 112 37 34 22 50 32 G.Ha.IIb 240 41 25 11 32 20 W.C.a 160 48 46 34 46 30 W.C.b 232 22 20 9 26 18 O.H.a 115 48 47 38 48 33 O.H.a 130 51 49 41 53 17 O.H.b 240 20 19 11 31 16 D.Z.a 165 38 32 12 32 21 D.Z.b 210 30 26 7 22 14 D.Go.a 145 38 36 19 43 28 D.Go.b 210 34 31 22 38 26 H.L.a 150 50 45 26 41 27 H.L.b 200 32 29 15 28 19 H.L.c 255 31 27 12 26 23 L.R. 140 52 51 27 48 34 A.Q.a 140 63 62 46 61 38 A.Q.b 230 31 25 9 27 16 Mean i 2 SE 39 4t 6 35 4 6 21 4 6 39 i 5 25 4 4 * Thirty-cm tube distance from infusion to collection orifice. t Roman numerals after subjects' initials refer to a second study session 4 or more weeks after the initial experiment, and letters indicate first (a), second (b), or third (c) day of a study session. T Centimeters from incisor teeth to collecting orifice: 110 to 170 cm = jejunum; 170 to 260 cm = ileum. § Glucose and fructose absorption not significantly different whether disaccharide or monosaccharide mixture in- fused (p > 0.3). 90- cal level. Preliminary experiments comparing the
80- effect of added glucose and fructose on hydrolysis sucrose use Om rates of by of the 30-cm infusion-to- w zV 70- LL collection distance did not show definite inhibition, and the added glucose made it impossible to de- X 60 termine glucose product absorption from the su- I-° crose. Further, these 30-cm segment studies .0 showed very efficient hydrolysis (greater than mD(IW= 40-
0 OU) 0S 80%o) when sucrose (29 mM) alone was infused, OD A 30 and it was possible that the intestine's maximal () 0 20- capacity was not really being determined. If this D 0 J 0 were the case, inhibition by the added monosac- 10_
TABLE II lb io 3o 40 50 6o io go go GLUCOSE ABSORPTION FROM SUCROSE. Comparison of sucrose to glucose-fructose in mM (mMoles/hr) 29 studies* FIG. 1. CORRELATION OF GLUCOSE ABSORPTION FROM SU- Sucrose Glucose +fructose CROSE (73 MM) WITH ABSORPTION FROM AN EQUIVALENT No. of Glucose Fructose Glucose Fructose MONOSACCHARIDE MIXTURE CONTAINING GLUCOSE (73 MM) studies absorption absorption absorption absorption AND FRUCTOSE (73 MM) (r =.87, p < 0.001). Each plot mmoles/hr mmoles/hr represents results of a paired experiment in a single sub- 13 19-42 1143 22:142 11:4:2 ject, as related to the theoretical line representing equal absorption from each solution. * A 30-cm segment was used. Values expressed as mean -I- 2 SE. INTESTINAL ABSORPTION OF SUCROSE IN MAN 391
910- .62 IOFUSKNs SWUROSE 73 8446 M M a00 cO = .sFRUlCTOSE 36- ° *.GLUGOSE LL 0 7110 34. @ 0 0 Z 32- E 6 0 Is W 30- 00 p w 5io-oo0 28- IJJ 0 26- 4 0o a Cc ° 0~~~~~ IaJ 24- 0o 0 ° 0 0 00 0 22- To 0 io- 0 0
TABLE HI Effect of galactose on sucrose hydrolysis and absorption*
Sucrose 40 mM Sucrose 40 mM + galactose 40 mM Glucose Fructose Fructose Galactose Intestinal absorp- absorp- Glucose ab- absorp- absorp- Subjectt levelt Hydrolysis tion tion Hydrolysis: sorptiont tion tion cm mmois mmoics/hr imoaes % A mmoks/hr %A mmin es/hr disac- disac- charide/hr charide/hr D.Go.IIa 105 25 20 21 18 -28 16 -20 14 16 D.Go.IIb 210 12 10 3.7 11 - 8 7.6 -24 6.0 9.5 V.W. 130 26 23 13 22 -is 20 -13 8.0 8.5 J.C.a 145 26 23 17 22 -15 19 -17 17 15 J.C.b 220 9.0 8.5 5.8 7.9 -12 6.5 -24 5.6 10 A.B.a 160 13 12 3.4 10 -23 6.4 -47 4.3 7.0 A.B.b 250 27 21 5 17 -37 13 -38 I 10 Mean 20 17 11 15 -20 13 -20 95 11
*- iviteen-cm;z-u n tubeA- custanceA n._ from4ron ifusiongnun r_ato collectioniection orifice.vonnce See Table I for explanation. Both ssucrose hydrolysis and glucose absorption were significantly lower when galactose was in the infusion (paired analysis, t test, p <0.02). Fructose absorption did not change significantly (p > 0.2). 5 Specimen lost; mean fructose values therefore based on six studies only. 392 GARY M. GRAY AND FRANZ J. INGELFINGER reported (29-31). To determine whether there is any kinetic relation of concentration of disac-
A,82.0 .0) - C4 C4 - C~~0 00 charide to its rates of hydrolysis and absorption, C4 we perfused solutions containing 10, 20, 40, 80, N~~~~ CO and 160 mM sucrose through 15-cm study seg- '02 0) ments. These solutions were given in random or-
(.0 der to a subject over a single day. The relations mNtm N obtained are shown in Table IV and representative I1 data plotted in Figure 4. There appeared to be a .0) > limiting velocity for both hydrolysis and absorp- S tion in all experiments. Figure 5 shows the Line- ...N m00N 0400- N N4fo "° weaver-Burk plot (32) for sucrose hydrolysis; R there is no significant deviation from linearity 2 ,u X 0) .0) -4: C4 4m4 -4C 4WC400o r4 (analysis of variance, p < 0.001). An almost oa .00 identical curve can also be shown for glucose prod- - *20 uct absorption. The kinetics of these two proc- u0)0.0 esses are compared at different intestinal sites in CO Table V. Fructose product absorption conformed *0 0 00 0 000 C .e' e 7.0 poorly to Michaelis-Menten kinetics and is not i) b-.. N- represented 93 graphically. 0 S.2 2- :0 0 _ Co00 00 CdO asU N )W InX 04 -orOn X Discussion 0). 000ok '4C'.00 C' -00O+C'40s 06bO o .0)4 CO UI i~When the absorption of sucrose was considered en in terms of its component monosaccharides,7 glu- E~ N 00 _%OC')es4 cose was absorbed considerably more rapidly than 0 fructose, and sucrose hydrolysis exceeded glucose 0)X C'j%0_r ItOO_i Ooi IN + e-i o +o' ll absorption only slightly (Table I). Figure 6 dia- .ll. 4.) cO N grams what appears to occur during sucrose ab- .0 0 sorption in man, the mean results of infusing a 73 U2 esd 2~ v 2 0NO 00 0 0 00 0 0o 0t S mM solution being shown. Of 66 mmoles of su- 0 000.0 b .2 crose infused in 1 hour, 39 mmoles was hydrolyzed C0 It V) R)e e U t% CuAie to the monosaccharide products. Of the 39 0sC Ct C4 -0 - 04 mmoles of glucose released, 35 was absorbed, 08 whereas 4 was present or accumulated in the in- 7 'oo14t-t0'000 Estimation of the sucrose absorption process in these 00.0 0),v * terms depends upon the assumption that each molecule e 0)O < of sucrose is split by sucrase to one molecule of glucose 0 O co NO W)u0m m and one of fructose. As well as having hydrolytic ac- O 0 0 0 0 0 14 0 0 )t')Cd 00. 000%lo0000000000'v t,.te. , tivity, all sucrases that have been purified thus far also 00 r have been found to catalyze the formation of small
0 C'I. 00 C00- oN0 'C' 00 0 ^ amounts of oligosaccharides when another sugar, rather 04-) g than water, is used as a receptor (33-35). With mam- a malian sucrase, this transglycosylation process may cause E a net release of smaller amounts of free glucose than fructose (36). Table VI demonstrates that such sucrase Cs4 B activity as there is in intestinal contents results in re- lease of essentially equal amounts of glucose and fruc- .4- C. --N_-N_ - - _ ^ - tose. of collected intestinal sam- -- O. .4-._C8 Furthermore, analysis 2 ples by paper chromatography revealed no sugars other >, than sucrose, glucose, and fructose. Thus sucrose is split * to essentially equal amounts of glucose and fructose un- der the conditions of our study. INTESTINAL ABSORPTION OF SUCROSE IN MAN 393
RK 40 MA ]a 30- 30-
20- 20-
10- 10-
b '-o' do io o' b 'ik 1,Ao5 10 90i 50. 1 7'0 1 9'0 111 1 0 15
0 -.* SUCROSE HYDROLYSIS DG ia mw a: -- GLUJCOSE PRODUCT ABSORPTION cn o FRUCTOSE PRODUCT ABSORPTION 0
0a] w DG llb N'I 0: o 210- -r o11 0- 10 30 io70 90 1 130 150 0 2 E AH 60- SP 50- 50-
40 40-
30- 30-
20- 20-
10- 10-
I 30o0g707 9 I 130 110 10 50 T0 90 110 130 150
MEAN INTRALUMINAL SUCROSE (mMolar)
FIG. 4. RELATION OF MEAN INTRALUMINAL SUCROSE CONCENTRATION TO HYDROLY- SIS OF SUCROSE AND ABSORPTION RATES OF ITS GLUCOSE AND FRUCTOSE PRODUCTS. A 15-cm test segment was used. traluminal contents. In contrast, of the 39 mmoles the current belief that the disaccharides enter the ,of fructose released, only10 21 was absorbed, and 18 intestinal cell before being hydrolyzed (1, 9, 10, accumulated intraluminally. Thus, nearly half 12, 16, 38-41). Eichholz and Crane have recently -of the fructose released from hydrolysis of sucrose localized sucrase to the plasma-membrane of the was still present intraluminally at the point of the microvillus (42), and Crane has set up a hypo- collection orifice. This reflects the slow absorp- thetical model for intestinal transport of sugars tion rate of fructose as compared to glucose (14, in which hydrolysis of disaccharides occurs within 17, 31, 37). These values for glucose and fruc- the outer portion of brush border plasma-mem- tose accumulation are minimal values, since ab- brane whereas the released monosaccharides are sorption of released monosaccharides presumably transported across a .permeability barrier located occurs over the whole length of the segment under deeper in the cell, in the inner portion of the same -study. membrane (43). Transport across this barrier The magnitude of this intraluminal accumula- presumedly requires utilization of a carrier mech- tion of monosaccharides is interesting in view of anism (15, 44, 45), since carbohydrates have a 394 GARY M. GRAY AND FRANZ J. INGELFINGER
TABLE VI Comparison of amounts of monosaccharide released from sucrase activity of intestinal contents sn vitro* Glucose/ Sample Glucose Fructose Fructose no. released released ratio mmoles/hr/L in- testinal contents V Xlo'100 A 3 3 1.0 X B 5 6 0.84 C 5 6 0.84 D 9 9 1.0 E 8 9 0.89 50- F 7 8 0.88 G 6 7 0.86 H 19 19 1.0 I 9 8 1.1 0 20 40 60 80 100 * Incubation at 370 C for 1 hour in sucrose substrate. IX go charide absorption process because both entry of FIG. 5. LINEWEAVER-BURK PLOT FOR SUCROSE HYDROLY- sis. Values are calculated from Table IV and expressed sucrose into the cell and subsequent exit of re- as mean ± 2 SE. Units for S (substrate concentration) leased glucose and fructose from the cell back into and V (rate of hydrolysis) are millimoles per liter and the lumen would require carrier-mediated trans- millimoles per hour, respectively. The correlation with port across the permeability barrier. Thus, our linearity is significant (analysis of variance, p < 0.001). results, although compatible with the intracellular hydrolysis hypothesis, are perhaps more consistent hydrated molecular diameter larger than the cal- with the hypothesis that sucrose is hydrolyzed by culated pore size of the permeability barrier and brush border enzyme while the disaccharide is hence cannot cross it by simple diffusion (46, 47). still within the lumen, especially if the intestinal Our findings of large amounts of monosaccharide permeability barrier were shown to be located at products within the lumen are consistent with the limiting plasma-membrane of the cell. Crane's hypothesis, since the disaccharide could The finding of equal absorption rates for su- be split in the outer brush border and some of the crose and the monosaccharide mixture at two dif- released monosaccharides could then move back into the lumen by diffusion without crossing a per- meability barrier. However, the exact location of G35mlM this barrier in the intestinal cell is uncertain, and it may well be located at the outermost portion of the cell, i.e., at the limiting plasma-membrane of the microvillus. If this were the case, intracellu- lar hydrolysis would result in an inefficient disac-
TABLE V sucrose saturation studies* 4mM Kinetic data from 39mlIPA GLUOSE
Sucrose Glucose prod- SUCROSE 66mM hydrolysis uct absorption FRUCTOSE 18mM No. of Intestinal studies site Vmax Km Vmax Km Mmoles/ mmoles/ mmoles/ mmoles/ hr/ L hr/ L SUCROSE27mM 15-cm 15-cm segment segment FIG. 6. SUCROSE ABSORPTION IN HUMAN INTESTINAL 8 Jejunum 83 142 83 154 PERFUSION EXPERIMENTS. A diagrammatic representation 6 Ileum 36 74 25 50 of what occurs over a 30-cm segment of intestine when sucrose is infused. * Derived from data in Table IV. Vmax = maximal velocity; Km (73 mM) Values refer to millimoles Michaelis constant. per hour. See text for elaboration. INTESTINAL ABSORPTION OF SUCROSE IN MAN 395 ferent infusion concentrations suggests that the nately, it does not distinguish intracellular hy- hydrolytic step is not rate limiting in the over-all drolysis from hydrolysis at the cell surface. If the process of sucrose absorption. This is in accord disaccharide were split at the cell surface, there with the in vitro guinea pig experiments of Frid- might be a high concentration of monosaccharide in handler and Quastel (8) as well as with the work the fluid immediately adjacent to the mucosal sur- of Dahlqvist and Thomson, who fed sucrose and face with a gradient of decreasing concentrations an equivalent glucose-fructose mixture to rats toward the center of the lumen. Analysis of the (16). It is tempting to conclude that the indi- collected luminal fluid reveals only the mean in- vidual transport rates for glucose and fructose traluminal concentration. define the sucrose absorption process. However, The fact that the presence of galactose in the little is known about the importance of local physi- infusing solution appeared to cause an inhibition cal conditions in the absorption process, and it is of sucrose hydrolysis (Table IV) must be inter- conceivable that rate of contact between sugar preted with caution. Galactose competes with glu- molecules and mucosal surface may occur more cose for the active transport process (24-27), and slowly than either the hydrolytic or transport hence any glucose displaced from this process by processes. Whether physical conditions are rate galactose might accumulate and inhibit sucrase, limiting in the over-all process of absorption can- thereby retarding sucrose hydrolysis. It seems not be determined from our experiments. unlikely that the trace impurities of glucose or In these comparative studies, a measure of the oligosaccharide in the galactose would have such dependence of absorption upon monosaccharide a marked effect on sucrose hydrolysis. Appar- concentrations within the lumen is provided by de- ently, then, sucrose hydrolysis in man may be in- termining the ratio of absorbed to intraluminal hibited in the presence of galactose by 1) galac- monosaccharide (Table VII). It is notable that tose itself, 2) glucose released from sucrose and sucrose has a marked advantage over the mono- displaced from the active transport process by the saccharide mixture since this ratio is three to six galactose (product inhibition), or 3) feedback times greater for glucose and two times greater inhibition of sucrase by a saturated active trans- for fructose than that found for the monosac- port mechanism that must transport the added charide mixture, indicating that absorption rates galactose as well as glucose. The last hypothesis of the released glucose and fructose products do not would require an intimate interrelation between depend on their concentrations within the luminal disaccharide hydrolysis and the active transport fluid. Instead, it is probable that a local high process for glucose. Such a relation has indeed concentration, comparable to the luminal concen- been suggested by Crane (43), and more recently tration of an equivalent monosaccharide mixture, by Semenza, Tosi, Vallotton-Delachaux, and Mill- is provided from splitting of sucrose at the brush haupt (48), who found that Nat is not only im- border membrane and maintained for the trans- portant for active glucose transport (49), but also port mechanism at the outermost portion of the activates sucrase. intestinal cell. This finding is analogous to the When sucrose in 10 to 160 mM concentrations intracellular-medium ratios found by Miller and was infused in 15-cm segments, every study ap- Crane in gut sac preparations (9). Unfortu- peared to show hydrolysis and absorption rates that approached a maximum (Table IV, Figure 4), but the relation of these rates to mean intra- TABLE VII luminal sucrose concentration varied from subject Ratio of absorbed to intraluminal monosaccharides* to subject. Sucrose hydrolysis and glucose prod- uct absorption did appear to conform reasonably Infusion: Infusion: glucose well to Michaelis-Menten kinetics when mean Infusion sucrose +fructose concen- values of the studies were plotted. Figure 5 dem- tration Glucose Fructose Glucose Fructose onstrates this for sucrose hydrolysis. A nearly 29 mM 19 1.2 5.5 0.73 identical relation prevailed for glucose product 73 mM 8.8 1.2 1.4 0.61 absorption, suggesting a close interrelation between * Derived from mean values of paired studies (Tables I and II). hydrolysis of sucrose and absorption of the re- 396 GARY M. GRAY AND FRANZ J. INGELFINGER leased glucose. When jejunal and ileal studies 3) Comparison of absorption from solutions hav- were evaluated separately, values for the maxi- ing different concentrations of sucrose demon- mal velocity and the Michaelis constant were strated the kinetic relationships to be variable higher for jejunum than ileum (Table V). This from subject to subject, but Lineweaver-Burk probably indicates that jejunum has a greater ca- plots revealed saturation kinetics that were nearly pacity for absorption of sucrose at high concen- identical for sucrose hydrolysis and glucose prod- trations of sugar, whereas the ileum performs quite uct absorption, suggesting an interdependence of efficiently at the low concentrations that still re- these two processes. main after the major portion has been absorbed 4) The addition of galactose to the sucrose solu- from jejunum. tion infused caused an appreciable decrease in su- The restricted surface area used in intestinal per- crose hydrolysis and glucose product absorption, fusion experiments may result in saturation phe- but had no effect on fructose product absorption. nomena irrespective of the presence or absence of Since galactose is known to compete with glucose specific binding sites for hydrolysis or transport, for the active transport process, this suggests that and demonstration of a maximal rate is not, of intestinal sucrase is inhibited either by the glu- itself, evidence for enzymatic hydrolysis or car- cose product or by a feedback inhibition by a satu- rier-mediated transport. However, the apparent rated active transport mechanism. Michaelis constant for sucrose hydrolysis in these studies in man is remarkably close to that re- Acknowledgments cently found by Dahlqvist and Thomson for in- The authors are grateful to Dr. Robert K. Crane for tact rabbit intestine in vitro (50). It is important his interest and suggestions and to Mrs. Priscilla Taggart to note that the kinetic relationships shown for for technical assistance. the hydrolytic process should not be assumed to equal those for sucrase itself, since the geometric References relations important for contact between sugar and 1. Miller, D., and R. K. Crane. The digestive function enzyme are undoubtedly quite different for intact of the epithelium of the small intestine. II. Local- intestinal tissue as compared to a homogenate or ization of disaccharide hydrolysis in the isolated purified tissue extract. Thus it has been shown brush border portion of intestinal epithelial cells. that Michaelis constants for intact intestine are a Biochim. biophys. Acta (Amst.) 1961, 52, 293. 2. Dahlqvist, A. Specificity of the human intestinal full order of magnitude higher than those for disaccharidases and implications for hereditary homogenates (50). disaccharide intolerance. J. clin. Invest. 1962, 41, 463. Summary 3. Auricchio, S., A. Rubino, R. Tosi, G. Semenza, M. Landolt, H. Kistler, and A. Prader. Disacchari- 1) Sucrose absorption was studied in man and dase activities in human intestinal mucosa. En- expressed in terms of sucrose hydrolysis and ab- zymol. biol. clin. (Basel) 1963, 3, 193. sorption of its component monosaccharides, glu- 4. White, A., P. Handler, E. L. Smith, and D. W. cose and fructose. Stetten. Principles of Biochemistry. New York, 2) Sucrose hydrolysis rates exceeded the mono- Blakiston, 1959, p. 381. 5. Thomas, J. E. Secretion and absorption in the in- saccharide product absorption rates, and the glu- testine in The Physiologic Basis of Medical Prac- cose component was absorbed considerably more tice, 7th ed., C. H. Best and N. B. Taylor, Eds. rapidly than the fructose component. Moreover, Baltimore, Williams & Wilkins, 1961, p. 669. paired experiments demonstrated the same ab- 6. Cantarow, A., and B. Schepartz. Biochemistry, 3rd sorption rates of these monosaccharides from su- ed. Philadelphia, W. B. Saunders, 1962, p. 270. crose as from an equivalent glucose-fructose mix- 7. Hoffman, W. S. The biochemistry of clinical medi- ture. Hence, hydrolysis does not appear to be cine, 3rd ed. Chicago, Year Book, 1964, p. 65. 8. Fridhandler, L., and J. H. Quastel. Absorption of rate limiting in the process of sucrose absorption, sugars from isolated surviving intestine. Arch. and the individual absorption rates for glucose and Biochem. 1955, 56, 412. fructose may define the rate of the disaccharide 9. 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