Journal of Applied Physiology
Vol. 81, No. 6, pp. 2523-2527, December 1996
SYSTEMIC CIRCULATION AND FLUID BALANCE

Enhancement of intestinal water absorption and sodium transport by glycerol in rats

Raul A. Wapnir, Maria C. Sia, and Stanley E. Fisher

Divisions of Perinatal Medicine and Gastroenterology, Department of Pediatrics, North Shore University Hospital-Cornell University Medical College, Manhasset, New York 11030

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Wapnir, Raul A., Maria C. Sia, and Stanley E. Fisher. Enhancement of intestinal water absorption and sodium transport by glycerol in rats. J. Appl. Physiol. 81(6): 2523-2527, 1996.Glycerol (Gly) is a hydrophilic, absorbable, and energy-rich solute that could make water absorption more efficient. We investigated the use of Gly in a high-energy beverage containing corn syrup (CS) by using a small intestine perfusion procedure in the rat, an approach shown earlier to provide good preclinical information. The effectiveness of several formulations with Gly and CS was compared with commercial products and to experimental formulas where Gly substituted for glucose (Glc). The CS-Gly combination was more effective than preparations on the market containing sucrose and Glc-fructose syrups (G-P and G-L, respectively) in maintaining a net water absorption balance in the test jejunal segment [CS-Gly = 0.021 ± 0.226, G-L = 1.516 ± 0.467, and G-P = 0.299 ± 0.106 (SE) µl ^· min^{1 ·} cm¹ (P = 0.0113)] and in reducing sodium release into the lumen [CS-Gly = 133.2 ± 16.2, G-L = 226.7 ± 25.2, and G-P = 245.6 ± 23.4 nmol ^· min^{1 ·} cm¹ (P = 0.0022)]. In other preparations, at equal CS concentrations (60 and 80 g/l, respectively), Gly clearly improved net water absorption over a comparable Glc-containing product [CS60-Gly = 0.422 ± 0.136 and CS80-Gly = 0.666 ± 0.378 vs. CS60-Glc = 0.282 ± 0.200 and CS80-Glc = 1.046 ± 0.480 µl ^· min^{1 ·} cm¹ (P = 0.0019)]. On the basis of the data of this rat intestine perfusion model, Gly could be a useful ingredient in energy-rich beverages and might enhance fluid absorption in humans.

rehydration; corn syrup; glucose

INTRODUCTION

REPLACEMENT OF WATER and electrolyte losses due to acute gastrointestinal disease with oral rehydration solutions (ORS) has been extensively applied in developing and in industrialized nations with considerable success (1, 3). ORS are effective for compensating losses occurring during diarrhea, although the desirable goal of reducing the duration of disease has not yet been achieved. The formulations used in ORS generally contain between 45 and 90 mM of sodium.

In parallel with diarrhea, strenuous exercise frequently results in substantial fluid losses, although comparatively much less of electrolytes than in diarrheal disease. Because normal human sweat under conditions not involving extreme exertion has generally a sodium concentration <30 mM, athletic or recreational beverages have sodium concentrations much lower than those used in the treatment of diarrhea (5). In addition, replacement fluids used in sports activities have often included easily assimilable energy sources, namely, glucose, fructose, and sucrose (21). Mono- or disaccharides can introduce a considerable osmotic load that precludes their addition in excessive amounts to maintain osmolality within reasonable limits. Hypertonicity is a general characteristic of soft drinks and juices that contain sugars. "Diet" beverages, on the other hand, are hypotonic but are not energy replacement sources.

Assimilable maltodextrins, derived from cornstarch or other amylaceous products, have been tested as substitutes for low-molecular-weight sugars in ORS and other beverages. Maltodextrins are glucose polymers that have the advantage of allowing for a higher energy density than sugar-containing beverages of the same osmolality. Their absorption is also more protracted than that of monosaccharides because they are gradually degraded to absorbable glucose by salivary and pancreatic amylases and mucosal glucoamylase in the upper gastrointestinal tract (4, 8, 20, 24, 31). An additional advantage of maltodextrins is that they lower the requirements for sodium at the absorptive site, mandated by the 2:1 or 1:1 sodium-to-glucose stoichiometry during the small intestinal brush-border transport process (9). If not enough sodium is present in a beverage, it must be secreted into the intestinal lumen and taken up again by the enterocyte as the glucose is absorbed.

Many individuals who need to replace fluid losses during exercise can do well with water alone, but strenuous protracted effort benefits from simultaneous energy replacement (5). Plain water reaching the duodenum and jejunum will elicit temporary sodium secretion/efflux (E) to achieved isotonicity with the extracellular fluid, as is clearly shown in the same experimental setup as used in the present study (27). This E-influx (I) movement constitutes a "futile cycle," which could be avoided by supplying absorbable metabolizable solutes at a physiological osmolality in the replacement fluid. One candidate solute is glycerol. Glycerol fulfills many desirable features because it does not require sodium for absorption. Glycerol can be considered nontoxic when ingested at concentrations at or below isotonicity (12). It is mildly sweet and palatable. In the rat, during experiments measuring copper absorption, it has been shown that glycerol, when used as an osmotic agent, enhanced both water and copper intestinal uptake (29). In humans, glycerol can induce hyperhydration (10, 13, 17) and can serve as a gluconeogenic substrate (2, 14). The present study was directed to experimentally test the hypothesis that the addition of glycerol could increase the rates of water absorption and limit, or reverse, the outflow of sodium into the intestinal lumen in formulations with high carbohydrate content. The experiments were not designed to estimate carbohydrate or glycerol absorption.

MATERIALS AND METHODS

The physiological effects of glycerol on water and sodium absorption were tested in two series of experiments. In the first series, a formula containing corn syrup and glycerol (CS-Gly), plus a small concentration of sodium and potassium, was contrasted against two forms of a commercial product extensively used as recreational and thirst-quenching beverages for sports activities. The commercial products are identified here as G-P and G-L, depending on their being sold as a powder for extemporaneous preparation or as a ready-to-drink liquid, respectively. The G-P powder was dissolved as indicated on the container. The CS-Gly preparation was made with regular corn syrup (not treated with invertase; 42-43° Baumé; Roquette America, Keokuk, IA). Glycerol, sodium citrate, and potassium chloride were purchased from Sigma Chemical (St. Louis, MO). The composition of the solutions is given in Table 1. The second series of experiments was directed at examining the effectiveness of glycerol as a water absorption stimulant in a formula in which the main energy source, corn syrup, remained constant, whereas the most readily absorbed energy sources were either glucose or glycerol. The composition of these preparations is given in Table 2.

Table 1. Composition of tested products G-P G-L CS-Gly Total carbohydrate, g/la 62.5b,e 58.3c,e 78.0d,e Free glucose, g/l 22.3e 26.2e 25.0e Sodium (tri) citrate, g/l ND ND 2.35   Sodium, mM [19.9]e [19.9]e [24.0]e Potassium chloride, g/l 0.37   Potassium, mM [2.7]e [3.2]e [5.0]e Glycerol, g/l 9.2 [100] Osmolality, mosmol/kg 313 385 350 Energy density, kcal/l 250f 208f 352g (233)h G-P, G-L: commercial preparations using sucrose and glucose-fructose syrups, respectively; CS, corn syrup; Gly, glycerol. a Expressed as glucose equivalents; b prepared as indicated on label (carbohydrate described as sucrose and dextrose); c as sucrose syrup and glucose-fructose syrup, as indicated on label; d introduced as regular corn syrup; e by actual analysis; f as per manufacturer's label description; g estimated from formula; h calculated from actual analysis; ND, present, as per label; concentration not determined. Bracketed nos., concentration (in mM).

Table 2. Composition of rehydration formulas with and without glycerol CS60-OSE CS80-OSE CS60-Gly CS80-Gly Corn syrup (42-43 Baumé), g/l 60 80 60 80 Sodium citrate, g/l 2.35 [8] 2.35 [8] 2.35 [8] 2.35 [8] Potassium chloride, g/l 0.37 [5] 0.37 [5] 0.37 [5] 0.37 [5] Glucose, g/l 18 [100] 18 [100] Glycerol, g/l 9.2 [100] 9.2 [100] Osmolality, mosmol/kg 256-268 308-314 254-262 324-329 CS60-OSE, CS80-OSE: perfusions containing CS at concentrations of 60 and 80 g/l, respectively, and electrolyte with glucose; CS60-Gly, CS80-Gly: perfusion with CS at concentrations of 60 and 80 g/l, respectively, and electrolyte with glycerol. Bracketed nos., concentration (in mM).

The testing procedure consisted of a jejunal perfusion of rats under anesthesia, as described in earlier publications (27-30). In brief, 60- to 80-g male rats (Sprague-Dawley, Zivic-Miller, Zelienople, PA) that had been acclimated to the institutional housing facility for no less than 48 h were fasted overnight, anesthetized with urethan (1.3 g/kg ip), and laparotomized, and an 18- to 28-cm-long jejunal segment was cannulated at both ends with polyethylene tubing, with the proximal part inserted through the duodenum. At surgery, an estimated jejunal section was selected, while disturbance of the vascular bed was minimized. Actual length was determined at the end of perfusion, as indicated below. The location of the test segment was chosen to focus on the main absorptive site of water and sodium in the upper small intestine. Phenol red (20 mg/l) and ~1 µCi/l (37 MBq/l) of ³H₂O were added to all solutions, including the commercial products, as net water absorption and unidirectional water flow markers, respectively. The perfusates were pumped at a rate of 0.17-0.20 ml/min and were maintained in a warm-water bath to allow their entering the animal at body temperature. Only one solution was perfused through the small intestine of each rat. To minimize biological variation, either all three or four solutions evaluated in each series of experiments were tested the same day. Up to 12 rats were perfused each time. The perfusion procedure consisted of a 60-min equilibration period followed by eight 15-min collections of the effluents through the distal cannula. Each 15-min sample was separately analyzed. Between-sample variation was 15%. Only one value of absorption rates per animal was tabulated. The samples were immediately refrigerated before analysis within 24 h. Once the perfusion ended, the rats were killed by exsanguination from the abdominal aorta. The intestinal segment between the cannulas was extended with a 3-g weight, and its length was measured. The procedures were carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" [DHEW Publication No. (NIH) 86-21, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892]. The protocol was approved by the institutional Animal Care and Utilization Committee.

Effluents and original solutions were spectrophotometrically analyzed (model 21D, Milton Roy, Rochester, NY) for phenol red concentration to determine net water absorption (22); ³H₂O was counted in a Beckman LS-3800 beta-scintillation counter; sodium was determined by atomic-absorption spectrophotometry (SpectrAA 10, Varian Instruments, Sunnyvale, CA), and osmolality was measured by vapor pressure changes (model 5500, Wescor, Logan, UT). Free glucose content was determined by an enzymatic procedure (Sigma 510). Total carbohydrate was analyzed by the anthrone method (18). The algorithms used in the computation of intestinal absorption rates and unidirectional water fluxes have been previously published (7). All results are presented as means ± SE. The significance of differences among solutions was determined by the Kruskal-Wallis test and Bonferroni adjustment, when indicated, with the use of a computer program (32).

RESULTS

There were significant differences in the performance of the G-P, G-L, and CS-Gly solutions regarding net water (P = 0.0113) and sodium absorption (P = 0.0022). The commercial preparations G-P and G-L produced a net secretory effect in the perfused segment of the small intestine. In contrast, the CS-Gly solution was approximately in equilibrium regarding net water movement from lumen to circulation (Fig. 1A) and differed significantly from G-L (P = 0.0064). G-P and G-L results were indistinguishable (P > 0.05). Net sodium movement across the mucosa was in the direction of the lumen in all three preparations (Fig. 1B). However, the rates of sodium secretion into the lumen were considerably less for the CS-Gly preparation than for G-P (P = 0.0027) and G-L (P = 0.0043). The differences in net water absorption could be linked to the unidirectional fluid movement. Thus, although neither the lumen-to-mucosa I nor the mucosa-to-lumen E rates differed among the three groups of rats, the poor result of the G-L in terms of net water absorption was consistent with its presenting the lowest I and highest E rates (Table 3). In consequence, the influx-to-efflux ratio (I/E) was significantly lower (P = 0.0043) in the G-L group than in the CS-Gly.

Table 3. Comparison of unidirectional water fluxes of G-P, G-L, and CS-Gly n I E I/E G-P 9 4.313 ± 0.451  4.686 ± 0.525  0.935 ± 0.021  G-L 11 4.097 ± 0.150  5.614 ± 0.536  0.776 ± 0.055* CS-Gly 11 4.619 ± 0.243  4.598 ± 0.324  1.031 ± 0.051 Values are means ± SE; n = no. of rats. Data expressed as µl · min1 · cm1. I, influx; E, efflux. * P = 0.0043 vs. CS-Gly.

When glycerol was the low-molecular-weight solute in combination with corn syrup at 60 and 80 g/l (CS60-Gly and CS80-Gly, respectively), net water absorption performance was better (P = 0.0019) than when glucose was included (CS60-OSE and CS80-OSE, respectively) (Fig. 2A). The data did not allow for the ascertainment of the differences among individual solutions. As in the preceding series, there were no differences in the water I (P = 0.7055) or E rates (P = 0.0963) (Table 4). However, because the mucosa-to-serosa reverse flow (E) tended to be higher in the glucose-containing preparations, the I/E of the two glycerol-containing preparations taken together was significantly higher (P = 0.0032) than that of the corresponding glucose-containing solutions, suggesting that glycerol facilitates the transfer of fluid from the intestinal lumen to the circulation. In contrast, there were no differences in sodium transport, regardless of the presence of either glucose or glycerol in the perfusing media (Fig. 2B).

Table 4. Unidirectional water fluxes of CS60-OSE, CS80-OSE, CS60-Gly, and CS80-Gly n I E I/E CS60-OSE 5 5.236 ± 0.425  5.518 ± 0.487  0.953 ± 0.040  CS80-OSE 5 5.376 ± 0.237  6.422 ± 0.628  0.861 ± 0.067  CS60-Gly 5 5.402 ± 0.540  4.979 ± 0.568  1.094 ± 0.030* CS80-Gly 5 5.445 ± 0.760  4.780 ± 0.761  1.169 ± 0.082* Values are means ± SE; n = no. of rats. Data are expressed as µl · min1 · cm1. * P = 0.0032 vs. CS60-OSE and CS80-OSE combined.

DISCUSSION

Glycerol crosses the mucosal brush border dragging an atmosphere of water surrounding each molecule. Because its molecular weight is about one-half that of glucose or fructose, this offers an obvious advantage. These characteristics may explain the comparative greater effectiveness of preparations with glycerol vis-a-vis comparable solutions with monosaccharides. Thus addition of glycerol to a beverage has the potential to enhance or accelerate the rehydration process as ingested fluid reaches the duodenum and jejunum. If the hydrating drink is iso- or hypotonic, no dilution stage is required before absorption, and no energetic penalty associated with electrolyte fluxes is exacted. Glycerol also compares favorably with fructose, another widely explored alternative to glucose (cf. data for G-L). Fructose-rich syrups are an inexpensive source of beverage sugars. However, fructose absorption has limitations in humans (16, 19) and the same osmotic constraints as glucose. In addition, the diffusion of glycerol across a chemical gradient does not depend on sodium cotransport as glucose does.

Providing energy replacement is one of the major goals of sport activities beverages: simple natural sugars or fructose-rich hydrolyzed corn syrup are frequently the major ingredients. They supply rapidly assimilable carbohydrate at low cost and high palatability. A logical way to increase the energy value of the beverage is to provide as a source of energy partially hydrolyzed soluble starch that carries only a modest osmotic load. Many such products readily available in the food industry are quite palatable or can be discretionarily flavored. Commercial corn syrup, for instance, contains a wide array of glucose short-chain oligomers and long-chain polymers that allow not only the rapid absorption of mono- and oligosaccharides but the progressive hydrolysis of larger polymers, attacked by amylases of salivary, lingual, pancreatic, and mucosal origin (8, 23). It has recently been shown that a 50-unit glucose polymer enhances water absorption more than shorter-chain-length products in humans (25).

Under physiological conditions, sodium is not the rate-limiting factor in the cotransport of glucose and water by osmotic drag; however, in an assessment of events occurring in an isolated intestinal segment, an outflow of sodium into the lumen is consistently observed whenever the sodium concentration presented to the absorptive segment falls <60-65 mM. This occurs both in the human (6) and in the rodent (27). The similarity of the physiological process in both species is remarkable and validates the technique used in this study. Also, previous work has shown that the most effective solutions in isolated perfused rat intestinal segments in situ (28) were similarly better in clinical trials (15). Greater water and sodium absorption in rats equated with reduced fluid and sodium stool losses in children with diarrhea, respectively. The healthy organism may tolerate well the temporary sodium efflux because the electrolyte is recirculated immediately as a participant of the glucose-sodium uptake process. A malnourished or dehydrated individual may be otherwise stressed or discomforted by the outflow of water into the lumen under the same conditions, hence the dichotomy existing in the optimization of ORS, either as a therapy for diarrhea or as thirst-quenching beverages.

Under the experimental conditions chosen for these studies, the CS-Gly using regular corn syrup proved to be superior to a commercial beverage in which the carbohydrate source was sucrose and glucose-fructose syrup (Fig. 1A). Although sodium concentration was comparable and well below the break-even point discussed above, the G-P and G-L preparations resulted in a greater efflux of sodium into the lumen than with the experimental CS-Gly preparation (Fig. 1B). These results support the contention that the addition of glycerol is probably responsible for accelerating water removal from the perfused intestinal segment and further diminishing the natural sodium outflow due to the low sodium concentration in the hydration solution. Although the water influx rates of the CS-Gly preparation were 10-20% greater than the G-P and G-L solutions, an examination of the E and the I/E ratio supports the view that a greater water E is the cause of a poorer rate of net water transport by the G-L product. This finding is consistent with the slower absorbability of fructose and, hence, the creation of a temporary osmotic drag into the lumen (16, 19).

The second approach in the assessment of the hydration stimulatory properties of glycerol was on the basis of a comparison of transport rates of preparations with two concentrations of corn syrup and either 100 mM of glucose or glycerol as low-molecular-weight energy sources. The salt content was comparable to that of the experimental solutions tested in the first part of the study. The solutions containing 60 g/l of corn syrup were hypotonic, whereas those with 80 g/l of corn syrup were slightly hypertonic (Table 2). For each of the two concentrations of corn syrup, the addition of glycerol resulted in significantly greater rates of net water absorption than when glucose was the simple sugar added (Fig. 2A). In contrast to the effects observed on net water absorption, neither the level of corn syrup used (and hence the osmolality) nor the presence of glycerol affected the transport of sodium, which was frankly secretory in the four variants tested.

The results of the present study are consistent with the success of human experiments of glycerol-induced hyperhydration characterized by lower urine output (10). Glycerol is metabolically important during lipolysis occurring in fasting because it can contribute to >20% of glucose production. The gluconeogenic contribution is similar to that of lactate (2, 14). On a weight basis, glycerol has a somewhat higher caloric density than glucose and is considered to be safe for oral consumption in the amounts used in the experiments presented here (10, 13, 14, 17). When used in isotonic or slightly hypotonic formulations, as done in this study, osmolality adjustment and solute dilution by endogenous water (5) is obviated. When glycerol is added as a potential enhancer of water absorption, in the presence of an energy-dense source such as corn syrup, the formulation embodies two advantageous properties: fluid absorption enhancement and high energy content, a combination that deserves future field assessment.

ACKNOWLEDGEMENTS

We thank Dr. Martin L. Lesser and Nina Kohn for statistical assistance and Mark A. Wingertzahn for technical help.

FOOTNOTES

Address for reprint requests: R. A. Wapnir, Dept. of Pediatrics, North Shore Univ. Hospital, Manhasset, NY 11030.

Received 22 November 1995; accepted in final form 16 August 1996.

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