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Effect of Gel-Forming Gums on the Intestinal Unstirred Layer and Sugar Transport in Vitro

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Effect of Gel-Forming Gums on the Intestinal Unstirred Layer and Sugar Transport in Vitro Gut: first published as 10.1136/gut.22.5.398 on 1 May 1981. Downloaded from Gut, 1981, 22, 398-403 Effect of gel-forming gums on the intestinal unstirred layer and sugar transport in vitro I T JOHNSON AND JENNIFER M GEE ARC Food Research Institute, Colney Lane, Norwich SUMMARY The effect of two gel-forming polysaccharide gums, guar gum and Na-carboxymethyl- cellulose (CMC), on glucose transport in vitro was investigated using everted sacs of rat jejunum. The gums were added to the mucosal bathing media to give apparent viscosities in the range 1-110 Pascal seconds x 10-3, mPa.s(cP). Serosal glucose transport fell steeply by about 60% as the vis- cosities of the mucosal media rose to 20mPa.s, and levelled off thereafter. A similar effect was observed in sacs preincubated with guar gum (15 minutes) and exposed to glucose in a subsequent guar-free incubation. Glucose transport with and without the addition of guar gum was found to be sensitive to mucosal stirring, so that, when shaken at 130 oscillations per minute, sacs exposed to guar gum (0.25 %, viscosity c.a. 16 mPa.s (cP) transported glucose at a similar rate to sacs incubated without guar at 80 oscillations per minute. By measuring the time course for the establishment of osmotic induced potentials, it was shown that incubation with guar or CMC led to an increase in the apparent thickness of the unstirred fluid layer overlying the mucosa (guar-free thickness =317±15[*, guar treated thickness =468+25tx). It is suggested that the presence of a polysaccharide gum in the fluid film surrounding the villi increases its viscosity, and thus gives rise to a thickening of the rate-limiting unstirred layer. If such an effect occurs in vivo, this could contribute to the http://gut.bmj.com/ diminished post-prandial glycaemia observed in human subjects fed guar gum. In recent years, a promising advance in the dietary the small intestine may slow absorption at the management of diabetes has come about with the mucosal surface, but little evidence exists for or observation that post-prandial glycaemia in man is against this proposition.7 82 In the present study we strongly influenced by dietary fibre.1 Studies with have investigated the effect of viscous gums on on September 30, 2021 by guest. Protected copyright. human subjects have demonstrated that glucose glucose transport in vitro, thus avoiding the influence absorption is slowed by the simultaneous ingestion of intestinal motility and other systemic effects. We of viscous dietary gums such as guar gum, pectin, or demonstrate that low levels of guar gum and sodium methylcellulose, and this effect has been shown to carboxymethylcellulose cause a pronounced reduc- increase with the viscosity of the ingested gum.23 tion in glucose transport, and we show that this is Conversely, the reduction in hyperglycaemia asso- probably brought about by an increase in the ciated with the ingestion of guar gum, the most effectiveness of the mucosal diffusion barrier. effective material, is nullified by mild hydrolysis, which renders it non-viscous.3 Methods The mechanism underlying the effect of viscous forms of dietary fibre is still a matter of controversy. PHYSIOLOGICAL PREPARATIONS Recently, Holt et aL.4 have demonstrated that Male Wistar strain rats (150-250 g) were allowed relatively large doses of guar gum and pectin food and water ad libitum, before being killed by markedly reduce the rate of gastric emptying in man, stunning and decapitation. Groups of everted but their conclusion that this in itself is sufficient to jejunal sacs were prepared from each animal, filled account for the observed reduction of glucose with glucose-free Krebs bicarbonate buffer, and absorption has been disputed."6 Earlier workers assigned randomly or by means of a latin square have suggested that the presence of viscous fibre in design to control and treatment incubation flasks. Glucose transport experiments were carried out in Received for publication 25 November 1980 25 ml conical flasks containing 10 ml of medium, 398 Gut: first published as 10.1136/gut.22.5.398 on 1 May 1981. Downloaded from Effect ofgel-forming gums on the intestinal unstirred layer and sugar transport in vitro 399 incubated at 37°C in a shaking water bath at 80 (seconds), D=diffusion constant for solute (cm-2/s). oscillations per minute, unless otherwise stated. In In this study, cannulated sacs were rapidly some experiments a second incubation stage was transferred from a tube containing the normal introduced. Sacs preincubated in glucose free media incubation medium to one containing medium plus were suspended in a jacketed organ bath, containing 50 mmol mannitol. The transmural potential differ- medium, gently stirred by a rising column ofbubbles. ence was monitored with KCl/Agar bridges led via For transmural potential difference measurements, calomel half-cells to a Vibron Electrometer (model sacs were ligatured over glass cannulae (ca. 2.5 cm 33C) with attached chart recorder (Servoscribe IS) in length) and suspended in 15 ml glass centrifuge runing at 120 mm/min. All incubation media tubes containing incubation media, which were contained glucose (28 mmol). Under these condi- gassed continuously with 95% 02: 5% CO2, and tions the osmotic induced potential had been maintained at 37°C in a water bath. previously shown to be linearly related to mannitol concentration up to at least 150 mmol. INCUBATION MEDIA All incubation media were prepared from Krebs OXYGEN CONSUMPTION MEASUREMENTS bicarbonate buffer, pregassed with 95 % 02:5 % CO2, The metabolic activities of tissue samples pre- having a final pH of 7.4. Dispersions of guar gum incubated with and without guar gum were compared (Sigma Ltd) and high viscosity sodium carboxy- by means of Warburg manometry. Everted sacs from methylcellulose (BDH Ltd) were prepared daily by adjacent lengths of jejunum were incubated for 15 adding the dry powder to vigorously stirred buffer minutes in the presence and absence of guar gum. (Ultra-Turrax, TP18/2N). Both were prepared at the Everted rings of tissue were then prepared from each highest concentration required, diluted as necessary, sac, and their oxygen consumptions were measured and maintained at 37°C in gently stirred, closed in paired Warburg manometers.14 Q02 values were flasks for at least two hours before use, to ensure subsequently calculated for the dried tissue samples. the attainment of stable viscosity. All incubation media contained glucose (28 mmol) unless otherwise EXPERIMENTAL DESIGN stated, and were gassed continuously with 95 % 02: To study the effect of guar gum or CMC on serosal 5% CO2. glucose transport, four sacs were prepared from each The viscosities of the guar and sodium carboxy- animal and allotted at random to flasks containing http://gut.bmj.com/ methylcellulose (CMC) dispersions were determined either gum-free medium, or one of three concen- on a rotary viscometer (Contraves Rheomat 15) at a trations of gum, such that the apparent viscosities of continuous shear rate of 50/s wherever possible. the media covered the range 1-110 mPa.s (cP). To observe the effect of a single concentration of MEASUREMENT OF MUCOSAL DIFFUSION guar gum on glucose transport over a range of BARRIER concentrations, 10 sacs were prepared from each The resistance to diffusion of the mucosal barrier animal, adjacent pairs being assigned to test and on September 30, 2021 by guest. Protected copyright. was measured using a modification of the technique control flasks by means of a latin square design. developed by Diamond9 for the measurement of Test flasks contained buffer, guar gum (0.25 %, unstirred layers in gall bladder, and later applied to viscosity ca. 16 mPa.s (cP)) and glucose at a concen- intestine by other workers.10 11 The method depends tration of 5, 10, 20, 35, or 50 mmol. Control flasks on the accurate measurement of the half-time for were identical except that guar gum was omitted the establishment of an osmotic induced potential, from the medium. which results from the application ofan osmotic load After incubation the sacs were rinsed in glucose. to the mucosal surface.'2 When a suitable intestinal free buffer, the serosal solutions were collected in preparation is suddenly transferred to a medium pre-weighed glass vials, and the tissue was dried containing a solute at an appropriate concentration, overnight at 80°C. Serosal solutions were weighed the osmotic induced potential is established only as and assayed for glucose by the GOD/PERID rapidly as the solute can diffuse up to the mucosal spectrophotometric method (Boehringer Mannheim). surface. The effective thickness of the unstirred layer The total serosal glucose transport for each sac was across which the solute must diffuse can be calculated calculated and expressed in terms of tissue dry using the following equation.'39 weight. In experiments designed to study the effect of d= (D*t)\ preincubation in guar containing media, four sacs 0.38 were prepared and alloted in random pairs to where d=unstirred layer thickness (microns), glucose-free media with or without guar gum ti=half-time for the establishment of new potential (0.25 %, viscosity ca. 16 mPa.s (cP)), and incubated Gut: first published as 10.1136/gut.22.5.398 on 1 May 1981. Downloaded from 400 Johnson and Gee Table 1 Serosal glucose transport in presence ofguar 'Sac 2' was incubated for 15 minutes in gum-free gum and CMC medium before measurement, and 'sac 3' was pre- incubated for 15 minutes in a medium containing Viscosity Glucose transport (mPo-s) (timol/gl3o min) either guar gum (0 5 % w/v, viscosity ca. 100 mPa.s or in Guar gum concentration* (cP)), CMC (0.7 % w/v, viscosity ca.
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