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,2
* Gastroenterology Unit, Women’s and Children’s Hospital, North Adelaide, South Australia;
Department of Physiology, University of Adelaide, Adelaide, South Australia;
** Department of Paediatrics, University of Adelaide, Adelaide, South Australia
2To whom correspondence should be addressed. E-mail: erinsymonds{at}yahoo.com.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • resistant starch • breath testing • fermentation • 13CO2
Resistant starch, defined as "the sum of starch and products of starch degradation not absorbed in the small intestine of healthy individuals" (1), is incorporated into a number of foods because it is thought to have fermentable properties that protect against colon cancer (2,3). In the large intestine, resistant starch is fermented and produces gas (including H2, CH4 and CO2) and short-chain fatty acids. However, quantitating the amount of starch that escapes digestion by pancreatic amylase in the small intestine is difficult because the gastrointestinal tract in humans is relatively inaccessible (4). One of the most accurate estimations of starch absorption is by intubating the terminal ileum and aspirating the ileal contents after ingestion of a meal containing starch plus a nonabsorbable marker. Disadvantages, however, include the invasive nature and the possible adverse effect of intubation on the absorptive process (5). Measurement of the amount of resistant starch using in vitro techniques has also proven to be inaccurate compared with in vivo measurements (6). Determining the residual starch in the ileostomy output of patients with colectomies is another way of determining starch digestion and the proportion of resistant starch in foods. However, these patients do not have a normal gastrointestinal system and may be at risk for physiologic adaptation leading to modified digestion (6). These traditional methods are inadequate, thus possibly making breath analysis a more suitable technique.
Hydrogen breath tests with assessment of H2 exhalation can provide semiquantitative estimates of carbohydrate fermentation in the large intestine (7); it is not possible to make exact measurements, however, because the proportion of exhaled H2 is variable and can be used up in alternative pathways during methanogenesis or sulfate reduction (8). Therefore combining the H2 breath test with another marker such as 13CO2 provides a more appropriate test.
Corn, a C4 plant, has a natural enrichment of 13C because it fixes CO2 into a 4-carbon intermediate in the Hatch-Slack pathway, whereas most other plants (C3 plants) fix CO2 into a 3-carbon intermediate through the Calvin-Benson reaction. The 2 pathways differ in their isotopic discrimination against 13C in atmospheric CO2, which can result in large differences in 13C content (9). Small intestinal digestion of starch from corn, followed by absorption and liver metabolism, results in breath excretion of 13CO2, as does colonic fermentation. By simultaneously assessing breath for H2, it is possible to determine the origins of the 13CO2 excretion. Breath tests utilizing corn were used previously to assess starch digestion in health and disease (4,10–13), but to assess fermentation, studies have used mainly 13C-enriched wheat flour (14,15). The disadvantage, however, of using synthetically enriched substrates is the high purchase cost. The current study, therefore, aimed to determine whether corn could be used in a combined 13CO2/H2 breath test to assess both digestion and fermentation of starch. A preliminary study was then performed to determine whether the breath test was able to detect changes in the properties of resistant starch due to cooking, a process previously shown to increase digestibility (16–19).
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Study 1. The subjects performed the breath tests in random order with at least 1 wk separating the tests. After collection of baseline breath samples (via exhaling through a straw into an Exetainer tube, Labco Limited) they consumed 40 g cornstarch (White Wings Foods) with 100 mL water, or 40 g resistant cornstarch (Hi-Maize, Penford Australia) with 100 mL water. The cornstarch and resistant cornstarch differed in the ratio of amylose to amylopectin (28% amylose:72% amylopectin and 80% amylose:20% amylopectin, respectively); the higher the proportion of amylose, the more resistant the starch is to small intestinal digestion. After ingestion of the meal, 10-mL breath samples were collected for 13CO2 analysis every 30 min up to 300 min after cornstarch and up to 720 min after resistant cornstarch. Breath samples (20 mL) were also collected every 30 min for H2 analysis. After the 330-min breath collection point of the resistant cornstarch breath test, subjects were permitted a light lunch that did not contain any substrates naturally enriched with 13C. No other food or liquid was permitted throughout the breath testing period, nor was it permitted at any time during the other breath tests.
Study 2. The subjects performed 2 breath tests in random order with at least 1 wk separating the tests. After baseline breath sample collection, subjects ingested 40 g resistant cornstarch in 100 mL milk or 40 g resistant cornstarch baked into a pancake with 40 mL milk and an egg. Breath samples were collected every 30 min for 13CO2 and H2 until 720 min. A light lunch that did not contain any 13C-enriched substrates was consumed after 330 min.
Analysis.
To determine the 13C abundance of the cornstarch and resistant cornstarch, samples of each were combusted with a bomb calorimeter and the resulting CO2 was analyzed using isotope ratio MS (ABCA 20/20 Europa Scientific). The 13C content of the samples was expressed as the 13C/12C ratio expressed as parts per thousand (
) relative to the Pee Dee Belemnite calcium carbonate international primary standard.
For the breath tests, the 10-mL breath samples were analyzed for 13CO2 content using isotope ratio MS, and analysis for H2 content was performed with a model DP Quintron MicroLyzer GC (Quintron). The 13CO2 results were expressed as the percentage 13C recovery per hour of the initial dose given (%dose/h), and as the cumulative percentage of administered dose of 13C recovered over time, calculated using the 13C substrate enrichment values that were determined from combustion. The CO2 production rate of the subjects was assumed to be 300 mmol/(m2 body surface area · h). Body surface area was calculated by the height-weight formula of Haycock et al. (20). H2 results were expressed as parts per million (ppm).
Statistical analysis. Data are expressed as means ± SEM because all were normally distributed (Kolmogorov-Smirnov test). Comparison of gas excretion patterns after ingestion of the different starches was performed with the paired Student’s t test, with significant difference taken as P < 0.05. All statistical analyses were performed with SigmaStat for Windows (version 2.03).
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RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and the resistant cornstarch had a 13C abundance of –8.52 ± 0.09, as shown by combustion of the samples. This 13C enrichment with a given dose of 40 g was great enough to significantly increase the 13CO2 excretion above the previously determined levels of standard healthy adults (9). After ingestion of the resistant cornstarch, there a was double-peaked 13CO2 excretion curve (Fig. 1A). The first increase in 13CO2 excretion began at 55 ± 9 min, which occurred at the same time as the 13CO2 excretion after cornstarch ingestion at 68 ± 9 min. Little H2 excretion occurred in the 300-min breath test after cornstarch ingestion (data not shown). The second rise in the 13CO2 excretion curve after ingestion of resistant cornstarch, defined as an increase in %dose/h for 2 consecutive samples after the first peak, began at 432 ± 15 min. This coincided with an increase in breath H2 that began at 428 ± 48 min (Fig. 1B).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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After ingestion of cornstarch and resistant cornstarch there were 2 peaks of 13CO2 excretion. The 13CO2 significantly increased (i.e., >2 SD) above standard levels present in basal breath that our research department (data not shown) and others (11,21) determined previously. This shows that the natural enrichment of corn is sufficient to be used as a breath test substrate even in Australian subjects, a population that has high 13C baseline values due to high daily intake of 13C enriched-food products (including corn and sugar cane).
The first increase in 13CO2 excretion after ingestion of resistant cornstarch occurred at the same time as the increase after cornstarch ingestion, and was not associated with elevated H2 excretion, thus confirming that the 13CO2 signal was from digestion and metabolism rather than bacterial fermentation. The time at which it occurred was also the same as in previous findings (11,12,22). Starch may be digested in the upper gut under the enzymatic control of salivary and pancreatic 
-amylase, followed by brush border enzyme digestion to produce glucose, which is absorbed, transported to the liver and oxidized to CO2 (23). Starch was shown to be suitable in breath tests to assess digestion because starch hydrolysis and not monosaccharide absorption is the rate-limiting step, and patients with pancreatic disease have a decreased 13CO2 recovery after starch ingestion (12). The finding in the current study that there was small intestinal digestion of resistant cornstarch is consistent with previous studies that found digestible components of resistant starch (4) and should be expected because the proportion of amylose to amylopectin was 80:20.
After ingestion of resistant cornstarch, there was a second increase in breath 13CO2 excretion. This was not due to the light lunch consumed because subjects avoided food with any 13C abundance, and instead consumed cheese sandwiches or rice. These have been shown to alleviate hunger without affecting breath 13C abundance (9,24). The increase in 13CO2 excretion occurred at the same time as breath H2 elevation which was indicative of the arrival of the substrate in the colon and bacterial fermentation because colonic fermentation is the only source of breath H2 (25). The second increase in breath 13CO2, however, did not occur at the same time as that reported in previous studies (10,22). These differences could be due to the incorporation of lactulose in the starch meal in the other studies(10), which would accelerate transit time (26), and allowing the subjects to sleep during the breath test (22) because both sleep and being positioned in the supine position can delay intestinal transit time (27–29).
The ability of the breath test to detect changes in the properties of starches was assessed by comparison of 13CO2 excretion after ingestion of cooked and uncooked resistant cornstarch. All starches exhibit disorganization of the semicrystalline structure of the starch granules during gelatinization (heating in the presence of water) (30). This process increases access for -amylase (31), increases the digestibility in the small intestine, and decreases the levels of resistant starch. The breath test showed significant changes in the 13CO2 excretion profile after ingestion of cooked resistant cornstarch; these were not due to differences in gastrointestinal transit of the different meals because transit times were the same, but instead were due to an increase in the digestibility. Because many foods now contain resistant starch for the health benefits for the colon [due to its high production of butyrate with fermentation (32)], the combined 13CO2/H2 breath test will be a useful tool to determine how changes to the physicochemical properties (e.g., bread containing resistant starch, consumed hot) affect the degree of fermentation.
In conclusion, this study showed that a combined 13CO2/H2 breath test can be used to assess small intestinal digestion and colonic fermentation of resistant starch. This will be a useful test because assessment of starch digestion has remained difficult due to the relative inaccessibility of the gastrointestinal tract. The breath test requires a long breath sampling procedure because breath samples are collected at 30-min intervals for a total of 720 min, but the hunger of the subjects can be alleviated by a light lunch of foods that do not produce H2 or affect 13CO2 excretion (9,33). When performing the test, it is important to consider that certain factors can influence breath CO2 and H2 exhalation including exercise, smoking, and eating. This study also showed that the breath test can be used to show changes in the properties of resistant starch with cooking. The combined 13CO2/H2 breath test can be used in further studies to determine whether the digestibility of resistant starch is altered in different physiologic and pathologic conditions.
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FOOTNOTES |
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Manuscript received 7 November 2003. Initial review completed 14 December 2003. Revision accepted 16 February 2004.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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