Sucrase insufficiency has been observed in children with of functional bowel disorders (FBD) and symptoms of dietary carbohydrate intolerance may be indistinguishable from those of FBD. A two-phase 13C-sucrose/13C-glucose breath test (13C-S/GBT) was used to assess sucrase activity because disaccharidase assays are seldom performed in adults. When 13C-sucrose is hydrolyzed to liberate monosaccharides, oxidation to 13CO2 is a proportional indicator of sucrase activity. Subsequently, 13C-glucose oxidation rate was determined after a secondary substrate ingestion (superdose) to adjust for individual habitus effects (Phase II). 13CO2 enrichment recovery ratio from 13C-sucrose and secondary 13C-glucose loads reflect the individualized sucrase activity [
Functional bowel disorders (FBD) have an estimated prevalence of 10–21% of the general population for which the pathophysiology remains elusive [
Up to 65% of FBD patients report symptoms exacerbated by meals with or without specific dietary intolerances [
Normal sucrose digestion to glucose and fructose and transporter-mediated monomer absorption that follows have been well-described [
The idea that disaccharide maldigestion, such as sucrose, could be causally involved in FBD is difficult to discount due to the striking similarity of symptoms [
This study investigated the possibility of intestinal sucrase insufficiency as a contributing factor in FBD (functional bloating type) by using stable (
This study was a prospective, open-label survey of adult patients with FBD as defined by Rome criteria and control subjects who presented to Baylor Clinic during a three-month period and subsequently underwent testing using biphasic 13C-S/GBT. A validation substudy was also performed to demonstrate that the biphasic breath test measures sucrose digestion. Additionally, the substudy evaluated four nondiabetic adult women with known sucrase-isomaltase genotypes to validate biphasic 13C-S/GBT.
This study was performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board at Baylor College of Medicine under protocol H-31197. The Institutional Review Board at Baylor College of Medicine is organized, operates, and is registered with the United States Office for Human Research Protections according to the regulations codified in the United States Code of Federal Regulations at 45 CFR 46 and 21 CFR 56. The BCM IRB operates under the BCM Federal Wide Assurance number 00000286. Written documentation of informed consent was obtained from all participants and, in addition to consent for prospective study participation, explicitly permits the use of health information obtained from health records including diagnoses, medications, laboratory data (exclusive of the whole genotype), and demographic information including age, gender, race, and ethnicity; such authorization does not have an expiration date.
Twenty-two subjects participated in this pilot study. Subjects were either asymptomatic healthy controls or symptomatic test subjects that met inclusion criteria for clinical diagnosis of FBD-functional bloating by Rome III criteria (FBD group:
Summary of the clinical features of 11 subjects diagnosed with functional bowel disorder as per gastroenterologist (HTS). All patient subjects experienced recurrent feeling of bloating or visible distension for at least 3 days/month during the prior 3 months and did not meet criteria for a diagnosis of functional dyspepsia, irritable bowel syndrome, or other functional GI disorders. All symptomatic subjects were scored as “severely affected” in accordance with the 2011 Rome Foundation working-team report [
Number | Gender | BMI | 75′ breath test |
Predominant symptoms at least 3 days per month, >6 months [ |
Pertinent history | Stool pattern | Symptoms during or immediately after breath test |
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1 | F | 19 | 83.4 | Abdominal pain, bloating, increased flatus; severity: severe | Lactose intolerant (avoids all lactose), poor response to FODMAP diet & VSL number 3 | Soft, irregular frequency, difficult to evacuate, no constipation, no diarrhea | None reported beyond baseline FBD symptoms |
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2 | F | 25 | 57.0 | Abdominal pain, bloating, increased flatus; severity: severe | Good response to FODMAP diet | Irregular frequency, difficult to evacuate, no constipation, no diarrhea | None reported beyond baseline FBD symptoms |
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3 | M | 28 | 83.8 | Abdominal pain, bloating, increased flatus; severity: severe | Some pain relief with eating, but not predictable | Irregular frequency, fluctuating consistency, no constipation, no diarrhea | Increased A/P and bloating near test completion |
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4 | F | 42 | 75.1 | Abdominal pain, bloating, nausea, increased flatus;severity: severe | Marginal response to FODMAP diet | Irregular frequency, fluctuating consistency, no constipation, no diarrhea | Increased A/P and bloating near test completion |
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5 | F | 31 | 72.5 | Abdominal pain, bloating; severity: severe | Benign other history | Irregular frequency, fluctuating consistency, no constipation, no diarrhea | None reported beyond baseline FBD symptoms |
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6 | M | 35 | 78.3 | Abdominal pain, bloating; severity: severe | History of diverticulitis | Irregular frequency, fluctuating consistency, no diarrhea | None reported beyond baseline FBD symptoms |
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7 | F | 26 | 58.0 | Abdominal pain, bloating; severity: severe | Benign other history | Irregular frequency, fluctuating consistency, no diarrhea | None reported beyond baseline FBD symptoms |
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8 | F | 33 | 138.8 | Abdominal pain, bloating, increased flatus; severity: severe | Lactose intolerant (avoids all lactose) | Irregular frequency, fluctuating consistency, no constipation, no diarrhea | None beyond baseline FBD symptoms |
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9 | F | 25 | 77.3 | Abdominal pain, bloating; severity: severe | History of rectal carcinoid, resolved | Irregular frequency, fluctuating consistency, no constipation, no diarrhea | Increased A/P and bloating near test completion |
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10 | F | 25 | 75.4 | Abdominal pain, bloating; severity: severe | Benign other history | Irregular frequency, no constipation, no diarrhea | None beyond baseline FBD symptoms |
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11 | M | 20 | 45.6 | Abdominal pain, bloating, increased flatus (significant); severity: severe | Good response to low carbohydrate diet, strong family history of similar issues | Increased frequency, frequent loose stool (not frank diarrhea), no constipation | None beyond baseline FBD symptoms |
Affected patients reporting any alarming issues, such as gastrointestinal bleeding, were not referred for study participation. Other exclusion criteria included, pregnancy, current lactation, allergy to food coloring, other causes of abdominal pain or altered bowel habits (e.g., celiac disease, pancreatitis, malignancy, or inflammatory bowel disease), history of diabetes mellitus, gastroparesis, recent febrile illness (5 days prior to the study), ingestion of an investigational drug or placement of an investigational device 30 days prior to the study start date, and absence of the mental capacity necessary to comprehend the protocol. Symptom severity to assess FBD was determined by patient reports, behavior of symptoms, and impact on health-related quality of life [
The asymptomatic control group (
To validate the breath test, four additional nondiabetic adult women were recruited as subjects for a substudy based upon their sucrase-isomaltase (SI) genotype (mutation status) and detailed self-reported dietary sucrose tolerance. As described below, 13CO2 breath test enrichment was compared with 13C-plasma enrichment since the only source of the tracer could be sucrose digestion with resultant monosaccharide absorption. One subject was symptomatic and homozygous for congenital sucrase-isomaltase (SI) deficiency, two subjects were heterozygous for congenital sucrase-isomaltase deficiency in the sucrase gene domain and moderately symptomatic, and one subject was sucrose tolerant (completely asymptomatic) without any of the known mutations in the sucrase-isomaltase gene (3q25.2-q26.2).
Commercial Mylar® breath collection bags (1.3 L and 0.25 L) fitted with one-way valves (
13C-tracer isotopes (
At study initiation, subjects were asked to hold their breath for a 10-second count and then inflate the reference breath sample for isotopic comparison later with timed breath samples. 13C-sucrose substrate solution with 15% excipient was then ingested (at time 0). At 60 minutes post-13C-sucrose ingestion, a breath sample was collected, followed by another breath sample at 75 minutes. Immediately after
Measurement of 13CO2 sample enrichment was performed for each 60-minute and 75-minute breath collection using 13CO2 mass dispersive infrared spectrometer (
Because of body habitus and age-related variations in carbon dioxide production [
Considering the tremendous resources that would be required to conventionally validate the 13C-sucrose breath test using duodenal/jejunal biopsies obtained from adult patients coincidentally undergoing gastroduodenal endoscopy, an alternative validation substudy approach was undertaken in four genetically distinct subjects. The subjects were identified based upon their sucrase-isomaltase (SI) genotype and tolerance to dietary sucrose and, in two cases, mucosal biopsy results were available which demonstrated diminished sucrase activity by Dahlqvist-assay. The substudy was performed by comparison of 13CO2 appearance in the breath with the timed appearance of 13C-monosaccharides in the plasma, the primary products of sucrose digestion.
Successful sucrose digestion results in absorption of 13C-fructose and 13C-glucose, for which 13C-fructose is mostly taken up by the liver on first-pass portal circulation while most resultant 13C-glucose passes to the systemic circulation for immediate use by vital organs [
Subject-A was a healthy, asymptomatic 45.9-year-old woman that routinely ingested dietary sucrose without adverse reactions; her SI genotype (WT/WT) revealed no genetic aberrations (ABI PRISM SNaPshot Assay Life Technologies, performed by LabCorp test 511570 SI, RTP, North Carolina). Subject-D was a profoundly symptomatic 46.7-year-old woman that avoided dietary sucrose and required sacrosidase supplementation with most meals to avoid typical symptoms of maldigestion; her SI genotype revealed homozygous aberrations [c.3218G>A (p.G1073D)]. A recent mucosal biopsy revealed no sucrase activity (only one sample obtained). Subject-B was a moderately symptomatic 23.3-year-old woman that restricted dietary sucrose to avoid typical symptoms. She was the biologic daughter of Subject-D and her sucrase-isomaltase genotype revealed heterozygous aberrations [c.3218G>A; (p.G1073D)/WT]. She has not required sacrosidase supplementation and she manages her symptoms by dietary sucrose restriction. Subject-C was a moderately symptomatic 43.9-year-old woman with a different SI mutation [c.5234T>G; (p.P1745C)] that also restricted dietary sucrose to avoid typical symptoms and periodically required sacrosidase supplementation. A sucrase assay of mucosal biopsy performed 13 years earlier demonstrated diminished sucrase activity (25
The plasma samples were processed in a closed reaction system where aliquots of plasma (1.00 mL) were incubated (37°C) overnight with a 20 mL culture of baker’s yeast (Red Star, Cedar Rapids, IA). Each test tube was fitted to a standard 250 mL Mylar breath collection bag and each system was primed with 20.0 mL oxygen (100%). After 24 hours of elapsed time, the system was purged with 100.0 mL of carrier gas containing 3% unlabeled (12C) carbon dioxide. Samples were analyzed for 13CO2 enrichment by comparison with a reference standard using mass dispersive spectrophotometry, adjusted for individual plasma volume (DOB
Deidentified clinical data were abstracted from the Baylor Clinic EPIC health care software (
We were unable to directly validate the adult control group using comparative enzyme assays on biopsy sample; therefore, a diagnostic cut-off point for the breath test was determined by calculating the lower 95% confidence interval of 13CO2 enrichment data obtained for the asymptomatic control group (CGO-S cut-off ~ 85%). For the purpose of estimating a reference diagnostic cut-off point from normal subjects, a
The four genetically SI-characterized subjects undertook 13C-S/GBT with simultaneous breath and blood sampling to validate breath test outcomes. High enrichment values, derived from effective sucrase activity, resulted in the appearance of high 13CO2 tracer from the plasma and vice versa. Results are shown in Figure
The four genetically SI-characterized (chromosome 3-NC_000003.12) subjects undertook 13C-S/GBT with simultaneous breath and blood sampling for plasma fermentation to release 13CO2 to validate breath sample enrichment outcomes; Phase I 13C-sucrose oxidation data and plasma fermentation data are shown. (a) 45.9 y/o healthy, asymptomatic woman without suspected SI gene mutation and normal CGO for 13C-sucrose (>80%); (Chr. 3q25.2-26.2) WT/WT. (b) 23.3 y/o symptomatic woman with heterozygous SI gene mutation and abnormal mean CGO for 13C-sucrose (<80%); Chr. 3q25.2-26.2 [c.3218G>A (p.G1073D)/WT]. (c) 53.9 y/o symptomatic woman with heterozygous SI gene mutation and abnormal mean CGO for 13C-sucrose (<80%); Chr. 3q25.2-26.2 [c.5234T>G (p.P1745C)/WT]. (d) 46.7-year-old symptomatic woman with homozygous SI gene mutation and abnormal (very low) mean CGO for 13C-sucrose (c/o < 80%); Chr. 3q25.2-26.2 [c.3218G>A (p.G1073D)/c.3218G>A (p.G1073D)]. B: 13CO2 breath sample enrichment (delta per mil); P: plasma sample 13CO2 enrichment (delta per mil); C: theoretical target 13CO2 plasma enrichment derived from
Collectively, the results obtained from the blood assay (fermentation products of digestion) closely correlated (
To validate breath sample enrichment outcomes, the relationship between 13CO2-breath sample oxidation data and 13CO2-plasma fermentation data was determined for the four genetically SI-characterized subjects (chromosome 3-NC_000003.12). Subjects undertook 13C-S/GBT with simultaneous blood sampling for plasma glucose fermentation to release 13CO2. Phase I 13C-sucrose oxidation data and plasma fermentation data are shown.
Eleven patients with FBD and 11 paired control subjects were enrolled and underwent the 13C-S/GBT (Figure
Between-group comparisons of biphasic 13C-sucrose/13C-glucose breath test data obtained from symptomatic patients with FBD and asymptomatic adult control subjects. Patients with FBD demonstrated decreased coefficient of glucose oxidation values for 13C-sucrose at 60-, 75-, and mean 60–75-minute time points when compared with asymptomatic controls.
Three subjects experienced typical, mild abdominal bloating and abdominal discomfort during the test. In sharp contrast, only one control subject (9%) demonstrated diminished sucrose digestive capacity at the 60-minute, 75-minute, and 60–75-minute time points but did not report any adverse symptoms
13CO2 Enrichment Coefficient of Glucose Oxidation for 13C-Sucrose (CGO-S) data values obtained at 60-minute, 75-minute, and the mean 60–75-minute values are shown in Supplemental Table
Free sucrose is a relatively modern component to the human diet. Sucrose is pervasive in commercially prepared foods, is frequently added to drinks and recipes, and is, therefore, difficult to avoid in the habitual diet. The specific association between sucrose intolerance and FBD-like symptoms has been previously observed [
Approximately two-thirds of the glucose and one-quarter of the fructose (or half of the total stable isotopic-tracer load) were expected to appear in the systemic circulation and to be available as a proportional indicator of normal digestion since very little free sucrose is passively absorbed by the intestines. After fasting, most absorbed fructose is converted to trioses in the liver over time, and trioses are subsequently released into the systemic circulation as lactate for oxidation in extrahepatic tissues or stored as glycogen. Plasma content of 13C-labeled monosaccharides closely paralleled breath sample enrichment as the appearance of 13CO2 in either breath or plasma and are both dependent upon mucosal sucrase activity. As such, 13CO2 breath sample enrichment is a valid measure of sucrase activity.
We note that reference values for mucosal disaccharidase assay activities are also determined by mathematical analysis of the distribution of the means of thousands of samples (
A monophasic 13C-sucrose breath test was previously applied to a rat model with chemotherapy-induced gastrointestinal mucositis and varying degrees of sucrose activity [
The findings of this study also point to the need for clinicians to carefully obtain a dietary history during the evaluation, admittedly a time-consuming task that in this day and age could be initially approached using a handheld device application if a dietician is not readily available [
As predicted, the sucrose load used in this study was well tolerated but does not eliminate the possibility of fructose intolerance that could be confirmed by additional 13C-fructose testing [
The concern over excessive amounts of maldigested sucrose contributing to the FBD condition has been the subject of some debate [
Since the 1980s, various sucrose-load breath tests have been used as a means to estimate intestinal absorptive function and sucrase activity in humans, rodents, and swine [
The modus of the biphasic 13C-S/GBT exploits the principle that the constituents of sucrose (glucose and fructose) should be promptly oxidized after ingestion and undergo glycosidic hydrolysis, portal absorption, and systemic oxidation such that the timed appearance and magnitude of 13CO2 recovery from breath sampling provide a good assessment of the digestive process. Modified Scofield equations have been used in the past to adjust 13C-urea breath test outcomes in children [
The application of a secondary, superdosed 13C-glucose breath test also addressed the issue of variable habitus, leaving only mucosal sucrase activity as the isolated physiological difference. In theory, 13C-glucose undergoes the same metabolic fate as the hydrolyzed sucrose-derived glucose and fructose monomers but requires no further digestion before oxidation [
Delayed digestion is expected to contribute to distal fermentation and symptoms related to the production of small molecule effectors, such as N-acyl-3-hydroxypalmitoyl-glycine (commendamide) [
The diagnostic cut-off value used in this study (<0.85) was derived indirectly by determining 95% confidence intervals bracketing the breath test outcomes of the symptom-free control group. However, this diagnostic cut-off point should be considered tenuous at this time, and the issue should be revisited by a larger, direct comparative study of nonmutants with endoscopic mucosal sucrase assays and/or blood enrichment assays.
Sucrase deficiency appears to be a factor in the FBD condition. 13C-S/GBT was successfully used to assess sucrose digestion and the results reported herein support the need for a larger study. Sucrose maldigestion may also be an indicator of a larger issue, starch maldigestion. This is considered since isomaltase is concurrently expressed with sucrase (same gene product 3q25.2-q26.2) and because starch is the major dietary staple that is difficult to limit. A careful history identifying a link between GI symptoms and starchy food intake, in addition to sucrose intake, should be helpful in predicting the contribution of dietary intolerances to recurrent GI symptomatology [
Breath test
13Carbon-sucrose/13C-glucose breath test
Coefficient of Glucose Oxidation for Sucrose
Delta
Functional bowel disorder
Gastrointestinal
Irritable bowel syndrome
Not significantly different
Plasma volume in liters
Sucrase-isomaltase
Wild type.
Albert M. Balesh and Harold T. Shelby are coauthors.
Antone R. Opekun’s laboratory, through Baylor College of Medicine, has a standing service-for-fee contract for research breath testing with QOL Medical, LLC, makers of a sacrosidase product. Authors Albert M. Balesh and Harold T. Shelby declare that they have no competing interests.
Antone R. Opekun, as principal investigator, conceived the study idea, supervised execution of the studies, interpreted literature, data analyses, paper writing, and revisions. Albert M. Balesh performed clinical testing, reviewed and interpreted cited literature, and drafted aspects of the preliminary paper under the close supervision of the principal investigator. Harold T. Shelby provided interpretation of cited literature and recruited study subjects.
The authors thank Ms. Susan A. Kennedy who provided editorial review and Karol M. Hernandez for stable isotope substrate preparation and spectrophotometer analysis of samples. They also thank students Christina Lecea-Plaisance, B. S., and Katie Fracoli, PA-SIII, for assistance with some breath sample collections associated with this project. QOL Medical, LLC graciously supported the genetic testing. This work was supported, in part by, NIH Digestive Disease Center Grant P30 DK56338 which funds the Texas Medical Center Digestive Disease Center and by a generous contribution from D.R. and G.P. Laws to the BCM G.I. Research Laboratory Fund.