3-0 Methylglucose Uptake as a Marker of Nutrient Absorption and Bowel Length in Pediatric Patients
Sigalet, David L
ABSTRACT. Background: Inert carbohydrate probes are commonly used to assess intestinal permeability; we have previously shown that the actively transported moiety 3-0 methylglucose (3-0 MG) is a useful marker of intestinal surface area and nutrient absorption in animal models of short bowel syndrome (SBS). This study examines the correlation of 3-0 MG absorption with nutrient absorption, bowel length, and the tolerance of enteral feeds in pediatric patients. Methods: Fifteen children (1 month to 15 years in age) were studied after intestinal surgery. All had a stoma, 2 were > 1 year of age, the remainder had surgical intervention as a neonate or within the first month of life. Eight had SBS (50% expected bowel length for age). Bowel length was measured intraoperatively. Nutrient absorption was quantified with a 48-hour bowel study, measuring fat, protein, and carbohydrate output directly. 3-0 MG absorption and intestinal permeability were quantified using a solution containing 30 mg/mL 3-0 MG, 20 mg/mL mannitol and 30 mg/mL lactulose (osmolarity 352, given at 1 mL/kg via feeding tube). Subsequent urine production was collected for 8 hours, and probe recovery measured using HPLC. Results: 3-0 MG absorption was significantly correlated with nutrient absorption. The correlation with protein absorption was r^sup 2^ = .59, fat r^sup 2^ = .62 and carbohydrate r^sup 2^ = .56. The correlation between 3-0 MG absorption and bowel length was r^sup 2^ = .58. 3-0 MG absorption was significantly lower in SBS patients vs patients with normal bowel length (15.8 ± 6.7 vs 30.5 ± 10.2%). 3-0 MG absorption also correlated with the ability to tolerate enteral feeds (r^sup 2^ = .38; p
The care of short bowel syndrome (SBS) in pediatric patients is a challenging management problem. Although there are multiple causes of SBS in pediatric patients, their clinical course tends to be similar.1 The small intestine has a considerable capacity to compensate for loss of absorptive surface area, up-regulating nutrient absorption, via the process known as adaptation.2,3 However when >50% of normal bowel length is removed, the patients tend to have ongoing difficulties with malabsorption.1-4 At present, nutrition management of these infants is largely empiric. Clinicians must balance the need for increasing the rate of enteral feeding (which stimulates the adaptive process, and also reduces the secondary cholestasis) against the problems of overfeeding. These can include bacterial overgrowth, osmotic diarrhea, and sepsis.1 Furthermore, bacterial overgrowth and sepsis have been implicated in worsening total parenteral nutrition (TPN) cholestasis.1,5 At present, there is no simple way to determine the nutrient absorptive capacity of the residual bowel in SBS patients. Inert carbohydrate probes are commonly used to measure intestinal permeability.6-8 The 3-0 substituted methyl glucose (3-0 MG) has long been known to reflect active glucose transport in the intestine.9-11 We have examined the use of 3-0 MG absorption as a predictive tool for measuring nutrient absorptive capacity. In animal studies, we have shown that 3-0 MG absorption is correlated with bowel length and absorption of both actively transported (protein) and passively absorbed (fat) macronutrients.12-14 The present study tests the hypothesis that 3-0 MG absorption is correlated with bowel length and macronutrient absorption in human patients. We also tested 2 secondary hypotheses: first that 3-0 MG absorption would discriminate between normal and SBS patients; second, we examined the correlation between 3-0 MG absorption and tolerance of enteral feeds, as a potential predictor of feeding tolerance.
MATERIALS AND METHODS
Infants and children undergoing intestinal surgery with an end stoma who were determined to have SBS or requiring long-term TPN were eligible for the study. The study protocol was reviewed and approved by the conjoint ethical review board of the Calgary Health Region. Postsurgery, the families of all patients with an end stoma were approached for consent to join the study and were enrolled if TPN requirements were >2 weeks after surgery. Patients were studied in the early refeeding phase, 1 to 2 weeks after introduction of enteral nutrition, so that postresection malabsorption would be maximal and to reduce the variability of partial adaptation. Fifteen patients were considered eligible and underwent study. Normative values for bowel length were from previously published values, which have been modified for use intraoperatively.1’15
Once patients had established refeeding for >1 week, a 48-hour nutrient balance study was done using standardized methods.16 Stoma output was collected, the volume recorded by the nursing staff, and immediately frozen. Intake was closely monitored, and no changes were made for 2 days before or during the study phase. Most patients were receiving formula diets, and an accurate measurement of macronutrient enterai intake was calculated from the manufacturer’s published values and confirmed using the testing protocol as outlined for the stool studies. In older patients taking a regular mixed diet, intake was calculated using standardized methods (Nutritionist 5, First Databank Inc., San Bruno, CA).17
In Vivo Nutrient Absorption Studies
Quantification of the nutrient content of stool was done using previously described techniques.14 In brief, stool samples were thawed and homogenized. A representative aliquot was then freeze dried, and the percent of moisture in the original sample determined. The freeze-dried material was then assayed for total energy content using energy release in an oxygen bomb calorimeter (PARR 1261 Bomb Calorimeter, Moline, IL). Fat content was determined using the modified Folch technique, protein by the Lowry assay, and fiber using the neutral detergent method.18,19 Carbohydrate content was calculated by subtracting the energy content of measured fat and protein and fiber in each sample from total energy as determined by bomb calorimetry. The fiber, ash, and mineral content of the infant diets was
In Vivo Carbohydrate Probe Recovery
Intestinal permeability and 3-0 MG absorption were measured under typical fed conditions. Feedings were held for a half hour before study, and patients then received 1 mL/kg of a solution containing 30 mg/mL 3-0 MG, 20 mg/mL mannitol, and 30 mg/mL lactulose with an osmolality of 352 mOsm/L. Urine was collected for the subsequent 8 hours, and the total volume recorded and samples were frozen. Older patients had a complete voluntary urine collection done; infants had an indwelling urinary catheter placed. Urine specimens were batch analyzed for the content of 3-0 MG, mannitol, and lactulose using our previously described techniques.8,12-14 Briefly, urine was filtered through a 0.4-µ filter and diluted as necessary. Samples were deionized and injected on to a Dionnex MA-1 ion exchange column (Dionnex Corp, Sunnyvale, CA). Sugars were eluted with NaOH at a flow rate of 0.4 mL/min, with concentrations ranging from 400 to 600 µm/L. The peaks were detected using pulse and amperometric detection on a Dionex HPLC and quantitated as peak areas. Calibration was performed daily with authenticated samples. Data are reported as a percentage of the gavaged probe detected in the urine.
Statistical Methods
Results are expressed as means ± SD. Pearson correlation coefficient analysis was performed using Instat statistical software (Instat Corporation, San Francisco, CA). Comparisons of values for SBS and patients with normal bowel lengths were done using Student’s 2-tailed t test for unpaired data, with appropriate correction for variations in SD, with p
RESULTS
Patient characteristics are recorded in Table I. The majority of patients were infants, with 2 older children (age 6 and 15, respectively). The most common cause of bowel loss was necrotizing enterocolitis (6 patients), followed by volvulus (4), intestinal atresia (3), and 1 case each of trauma and motility disorder. Small bowel length, as measured intraoperatively is recorded, and if this was
Table II shows results of the sugar absorption studies and the in vivo absorption of macronutrients (fat, protein, and carbohydrate). 3-0 MG absorption was significantly correlated with small bowel length r2 = .58 (Fig. 1). 3-0 MG absorption was significantly lower in the SBS us normal patients (average 3-0 MG absorption 15.8 ± 6.7% in SBS patients vs 30.5 ± 10.2 in normals; Table II). The SBS patients were clearly different than the normals in all aspects of feeding tolerance and nutrient absorption as measured by total enterai calories, and fat, protein, and carbohydrate absorption (Table II; Figs. 2-4). 3-0 MG absorption was significantly correlated with each of fat, protein, and carbohydrate absorption and the ability to “tolerate” enterai feeding (see Figs. 2-5).
In these patients, there was no significant difference in the intestinal permeability as measure by lac/man ratio (Table II). In some patients maintained with high volumes of breast milk or lactose-containing formula, the high concentration of lactose in the excreted urine prevented accurate determination of lactulose absorption, and this reduced the number of data points available for analysis so no significant trends could be identified.
DISCUSSION
These results confirm that our hypothesis that 3-0 MG absorption is correlated with bowel length and macronutrient absorption in human patients in the patient population studied. Moreover, the results were consistent with our previous findings in animals, and previous human reports, which showed that 3-0 MG absorption was correlated with small bowel length.11-16,20 The present results extend these observations and show that 3-0 MG absorption is correlated with macronutrient absorption and tolerance of enteral feedings.
The significant correlation between 3-0 MG and carbohydrate absorption seen in this study contrasts with our previous findings in experiments with animals.14 We theorize that in the animal studies, the relatively high amounts of dietary fiber (8% to 10%) and variations in its breakdown within the cecum and colon may have interfered with accurate determination of carbohydrate absorption. In the present study, the majority of patients were maintained on a very low-fiber diet. This suggests that specific validation of the 3-0 MG methodology in patients taking a higher-fiber diet would be required before it could be used for older patients.
The selection of patients with end small intestinal stomas for this study improved the reliability of the balance studies.16 In several trial patients, attempts at quantitative stool collections in patients stooling normally were unreliable in the ward setting.16 However, the use of the study population selected limits our ability to predict how these testing methodologies would be useful in patients with an intact enteral stream, stooling normally. We would predict that 3-0 MG/lac/mannitol methodology would be reliable in populations with varying segments of colon intact. Previous studies have shown that lac/man absorption is very specific for the small intestine,8 with
In the present study, we had hoped to analyze intestinal permeability using lactose/mannitol absorption ratios, which have been used as general markers of intestinal integrity. However we found that in patients receiving high concentrations of breast milk or lactosecontaining formulas that urinary lactose interfered with the determination of lactulose. At present we are testing protocols to enzymatically separate these components; alternatively, we may switch to a different disaccharide marker.20
The results confirm our secondary hypothesis. 3-0 MG absorption was clearly less in patients with anatomic SBS, and it did correlate with tolerance of enteral feedings during the early refeeding phase. As discussed further below, these findings support the use of 3-0 MG in monitoring patients clinically and may be extended to helping predict the tolerance of enteral feeding in specific patients.
A potential limitation of this methodology can be the difficulty in obtaining a complete urinary collection. In the present study, all infant patients were catheterized, and so this was not a problem. However, in our initial trials for the study, we found that urinary collection bags were not reliable. There are also theoretic concerns regarding the absorptive capacity of the small intestine and how this may vary, depending on the availability of other nutrients at the time of testing.7,10 In previous studies, we have attempted to improve the discriminatory power of 3-0 MG absorption by using varying concentrations of native glucose to “compete” for the glucose cotransporter. However, this did not improve the precision of the testing,13 and at present we feel that the method described, which uses a weight-based dose of test solution, will give the most reliable results over the wide range of patient size seen in the pediatric population.
It is likely that variations in the patient population studied added to the variability in the results noted. We chose to include this patient population for the practical reasons of increasing the power of the study and determining “the robustness” of using 3-0 MG as a marker. We anticipate refining the precision of measurement according to an improved understanding of the range of “normal” 3-0 MG absorption in different padiatrie populations.
These observations provide a useful starting point for further studies. We would suggest that 3-0 MG absorption, along with lactulose/mannitol uptake studies, may provide useful information in the management of pediatric patients with impaired bowel function. The lac/man ratio, as noted, is widely used in adult patients as an indicator of intestinal damage.6-8 A more widespread use of this index in pediatric patients may provide insight into the relationship between intestinal leakiness and gut function. It may also be predictive of which patients are at risk of developing sepsis secondary to abnormal translocation of normal enteric flora. 3-0 MG absorption could be useful in predicting the “tolerance” of enteric feedings. Given the relative simplicity of the test, it could be repeated sequentially and used to guide enteral refeeding. Our hope would be that this would then help provide refeeding parameters, which would prevent development of bacterial overgrowth and secondary sepsis, which is so commonly a problem in pediatric SBS patients.1 Finally, an increased understanding of the relationship between the intestinal permeability and complications of SBS such as TPN cholestasis in the development of liver disease may provide useful insights into the management of this difficult problem.
In conclusion, these results demonstrate that after intestinal surgery the recovery of an orally administered sugar probe test solution can provide useful information on the available length and nutrient absorptive capacity of the residual bowel. These findings support further studies in using this testing method both to monitor patients, guide enteral refeeding, and to test the effect of other therapies and nutrient absorptive capacity. Given the present lack of therapies for treating SBS, the addition of reproducible and reliable outcome measures can only help advance work in this field.
ACKNOWLEDGMENTS
We acknowledge the support of the Alberta Children’s Hospital Research Foundation for funding. The assistance of Jill Marie Shaw, RD, and Sharla Stoffman, RD, was invaluable in monitoring patients. We also wish to express our appreciation for the help from both the families and nurses on N cluster for their diligence in performing “pooper scooper” duty. The technical assistance of Kim Tran in Dr Meddings’s lab and the secretarial support of Gail Wright-Wilson is gratefully acknowledged. Finally, we wish to express our appreciation for the discussions with Dr Grant Gall, which led to the formulation of this line of inquiry.
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David L. Sigalet, MD, PhD*; Gary R. Martin, BSc[dagger]; and Jon B. Meddings, MD[dagger]
From the * Department of Surgery, Alberta Children’s Hospital; and the [dagger]GI Research Group, University of Calgary, Calgary, Alberta, Canada
Received for publication March 24, 2003.
Accepted for publication January 23, 2004.
Correspondence: Dr. David L. Sigalet, Department of Surgery, Alberta Children’s Hospital, 1820 Richmond Road SW, Calgary, AB T2T 5C7, Canada. Electronic mail may be sent to sigalet@ ucalgary.ca.
Copyright American Society for Parenteral and Enteral Nutrition May/Jun 2004
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