Participants
Patients were candidates for inclusion in this study if they had been diagnosed with functional dyspepsia who were aged 8–17 years (inclusive), had a demonstrated a lack of clinic response to acid-reduction therapy, and were undergoing endoscopy to evaluate FD. Patients were excluded from the study if any of the following criteria were present: 1) previous abdominal surgery; 2) any chronic non-gastrointestinal illness requiring regular medical care (e.g., diabetes mellitus, juvenile rheumatoid arthritis, cystic fibrosis, cancer); 3) any history of an adverse reaction to lactulose or mannitol; 4) any use of aspirin within one week prior to the study; 5) any use of antacids or laxatives within 1 week prior to the study; 6) any use of steroids, antihistamines or antihistamine-like drugs within 4 weeks prior to the study; 7) any use of antibiotics within 4 weeks prior to the study; 8) pregnancy; or, 9) non-English speaking. Fifty-five patients were approached and 22 patients agreed to participate in the study, yielding a 40% recruitment rate. Three patients who participated in the study demonstrated pathology on biopsy and were excluded from data analysis, leaving 19 participants in the patient group (13 females, 6 males; mean age = 13 ± 2.5 years). Of these, one patient also was found to have eosinophilic esophagitis on biopsy, but was retained in the study as this was not an exclusion criterion.
For the control group, all respondents aged 8–17 years (inclusive) were candidates for inclusion in the study. In addition to the exclusion criteria noted above, control participants were not admitted to the study if there was any history of gastrointestinal symptoms (including abdominal pain or discomfort, nausea, vomiting, bloating, diarrhea, or constipation) within the previous six months. Twenty-four potential control participants responded to the study advertisement. Twenty-one of the respondents met eligibility criteria. Of these, two refused, leaving 19 controls enrolled in the study (10 females, 9 males; mean age = 13 ± 2.6).
Measures
Intestinal permeability
Intestinal permeability is measured by evaluating absorption of macromolecules which are not typically absorbed across intestinal cells, but may pass through the paracellular space when tight junction disruption is present[5]. Lactulose is a non-digestible sugar and is absorbed through the paracellular space following damage to the tight junction; it is then rapidly metabolized by colonic bacteria upon entry into the colon.As such, lactulose often is utilized to test intestinal permeability as part of a Sugar Absorption Test (SAT)[5]. Mannitol is a monosaccharide that is readily absorbed through the transcellular pathway and is used to control for mucosal (e.g. intestinal surface area) and non-mucosal (e.g. gastric empting, intestinal transit, and impaired renal function) factors which may affect lactulose absorption and metabolism. Sucrose is added to make a hyperosmolar solution which has been shown to also improve sensitivity of the test. In the SAT procedure, urine is collected for 5 hours following administration of an oral lactulose/mannitol/sucrose solution. The concentration of lactulose and mannitol in the urine is determined and results are expressed as the ratio of lactulose to mannitol. An increased ratio indicates increased paracellular permeability due to tight junction dysfunction.
Urinary excretion of lactulose and mannitol was determined in the Toxicology Laboratory, CMH, KC, MO 64108 using an enzymatic method (available from INstruchemie, AC Delfzijl, The Netherlands) adapted to the V-Twin automated chemistry analyzer. Recovery of the sugar probes were expressed both quantitatively and as the lactulose/mannitol ratio. A urine creatinine was determined in order to express quantitative results in mmol/mol creatinine. Urine creatinine was performed in the Toxicology Laboratory, Children’s Mercy Hospital using the V-Twin automated chemistry analyzer.
Gastrointestinal eosinophils and mast cells
Eosinophil density was determined from the routine histology slides (hematoxylin and eosin stain) by counting eosinophils beginning in what appeared to be the most involved area after scanning the entire specimen. Five consecutive fields (400× magnification) were evaluated. Peak count was defined as the highest count of the five fields, while mean count was defined as the average of the five fields.
Mast cell density was evaluated utilizing immunohistochemical techniques. Serial 3-μm paraffin sections were air dried and heat fixed on slides. The sections were deparaffinized with xylene and iodine and rehydrated in a graded series of alcohol. Sections were stained on an automated Dako Autostainer 3400 using Dako’s LSAB + kit with streptoavidin conjugated to horseradish peroxidase. The antibody utilized was tryptase monoclonal mouse antihuman mast cell tryptase, clone AA1, Dako. Mast cell density was determined by counting mast cells beginning in what appeared to be the most involved area after scanning the entire specimen. Five consecutive fields (400× magnification) were evaluated. Peak count was defined as the highest count of the five fields, while mean count defined as the average of the five fields.
Anxiety and depression
Symptoms of anxiety and depression were assessed using the Behavioral Assessment System for Children (BASC)[9]. The BASC is a paper-and-pencil questionnaire assessing psychosocial functioning in youth with different versions available for children (ages 8–11 years), adolescents (ages 12–18 years), and parents (different versions for parents of children 6–11 years and 12–18 years). The BASC has demonstrated criterion-related and construct validity, has good internal consistency for most individual subscales, and is widely used in both clinical and research settings[9]. Standardized T scores for anxiety and depression were used for the present study.
Procedures
This pilot study was a single-site, case-controlled, single-blind observational design. Participants for the patient group were recruited in a single hospital-based clinic specializing in the evaluation and treatment of children with chronic or recurrent abdominal pain. Participants for the control group were recruited by advertisement at the same hospital. Informed parental permission and participant assent were obtained prior to the initiation of any study procedures.
The study involved one visit to the hospital-based gastroenterology clinic. After providing informed parental permission and participant assent, all participants completed the Behavior Assessment System for Children (BASC) and then underwent a differential sugar absorption test (SAT) using lactulose/mannitol/sucrose solution to evaluate intestinal permeability. Participants emptied their bladders immediately prior to the start of the SAT and then ingested 2 mL/kg (maximum 100 ml) of the sugar solution. The SAT solution contained 5 grams lactulose, 2 grams mannitol and 40 grams sucrose dissolved in demineralized water for a total volume of 100 mL. Participants did not eat or drink for 2 hours following ingestion of the sugar solution. After 2 hours, participants could consume any fructose-free foods and were asked to consume at least 1 cup of water hourly. Urine was collected for 5 hours following administration of the lactulose/mannitol solution. Finally, for participants in the patient group only, an endoscopy with biopsy was performed on a separate day as part of standard care and a minimum of 4 mucosal biopsies were obtained from the duodenum for later evaluation.
All study personnel performing procedures and assessing outcomes were blinded to group assignment. The study protocol was approved by the Children’s Mercy Kansas City Institutional Review Board.
Sample size
A priori analysis provided a sample size estimate of 18 per group which would allow 80% power to detect a difference for mean permeability between 0.015 and 0.045 assuming a standard deviation of 0.03.
Statistical analysis
Independent-sample t- tests (2-tailed) were used to evaluate mean differences in the lactulose/mannitol ratio in the patient group compared to the control group. In the FD patients, Pearson correlation coefficients were calculated and used to evaluate associations between the lactulose/mannitol ratio and mast cell and eosinophil densities, as well as the BASC anxiety and depression subscale scores, respectively.
SPSS 18.0 for Windows computer program, SPSS Inc. Chicago, IL was used to complete the analyses. All analyses used a set significance limit of α = 0.05.