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U.S. Department of Agriculture/ARS Children’s Nutrition Research Center and Texas Children’s Hospital, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030
4To whom correspondence should be addressed. E-mail: carlosl{at}bcm.tmc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • barley • stable isotope • ß-glucan • obesity • diabetes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). ß-glucans are structural polysaccharides found in the cell walls of the bran layer and endosperm fractions of the whole seed (2
). Structurally, ß-glucans are linear chains of ß-D-glucopyranosyl units in which 
70% of the units are linked (1
4), but which also consist of ß-D-cellotriosyl and ß-D-cellotetraosyl residues separated by (13) linkages arranged in a random manner (3
). The soluble nature of ß-glucans, in conjunction with their chemical structure, helps to increase the viscosity of foods that contain them (1
). Consumption of a barley-containing meal rich in ß-glucans seems to stimulate reverse cholesterol transport, which may contribute to the cholesterol-lowering ability of barley (4
). There is a cultivar of barley called prowashonupana (PW), 5
whose grains contain elevated levels of ß-glucans (5
). The purpose of this study was to compare in healthy adults the energy use derived from a serving of PW with that derived from a serving of normal barley (barley cultivar 594.35.e (BZ)) using 13C-labeled grains. Breath samples subsequently collected were analyzed for 13CO2 as an indicator of the 13C that was oxidized and hydrogen (H2) as a measure of the carbohydrate that was not absorbed. When carbohydrate is incompletely absorbed by the small bowel it reaches the colon, where it is fermented by intestinal bacteria (6
). A byproduct of this fermentation is H2, which is partially absorbed through the colonic mucosa, transported to the lungs, and exhaled in the breath, where it can be measured. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two conventional cultivars of barley (Hordeum vulgare L.) were grown for grain production: cultivar PW (high ß-glucan content; seeds kindly provided by ConAgra Oat Processing, Omaha, NE) and BZ 594.35.e (normal ß-glucan content; seeds kindly provided by Dr. D. R. Clark, Western Plant Breeders, Bozeman, MT). Seeds were planted (25 per pot) in synthetic soil (Metro-Mix 360; Scotts-Sierra Horticultural Products, Marysville, OH) using 8.4-L plastic pots; planting density was 17 pots/m2. Plants were maintained in a controlled environment chamber at 18°C and 50% relative humidity, and were illuminated continuously (24-h photoperiod) by a combination of incandescent and fluorescent lamps. Light intensity was adjusted to 390 µmol of photons per square meter per sec for the first 3 wk of growth, and 500 µmol of photons per square meter per sec for the remainder of growth. Both cultivars were grown in the same chamber at the same time. Plants were watered daily with a nutrient solution containing 1.2 mmol/L KNO3, 0.8 mmol/L Ca(NO3)2, 0.3 mmol/L KH2PO4 and 0.2 mmol/L MgSO4; pots were watered to achieve full saturation of the soil.
Plants were pulse-labeled with 13CO2 on three occasions during the period of grain fill; labelings occurred at 8, 12 and 16 d after 50% spike emergence. A total of 18.9 mmol of 13CO2 was administered to each pot of plants over the course of the three labelings. For each labeling, groups of 12 or 13 pots were placed in a sealed Plexiglas enclosure (1.2 m3) containing air-mixing fans, and connected in a closed loop with an infrared CO2 gas analyzer (model 225-MK3; Analytical Development, Hertfordshire, UK). Labeling was conducted under a combination of natural lighting and metal halide lamps in a greenhouse.
Spikes with mature grains (8% moisture) were harvested at 6 wk after 50% spike emergence. Grains were removed from spikes and were dehulled by hand; hulls were separated from grains using an air stream.
Analysis of grain carbohydrates
A 10-g sample of labeled barley grains from each cultivar was dried and ground to a fine powder using a Wiley mill (Thomas Scientific, Philadelphia, PA) with a 60-mesh screen. Subsamples (50 mg) were used to determine total starch and total ß-glucan content in the grains of each cultivar, using the techniques of Åman and Graham (3
). Starch was assayed with a starch assay kit (SA-20; Sigma-Aldrich, St. Louis, MO) and ß-glucans were assayed with a mixed-linkage ß-glucan kit (Megazyme International, Wicklow, Ireland). Following the enzymatic assays, glucose monomers derived from the starch or ß-glucan polymers were converted to penta-acetate derivatives (7
). Glucose isotopomers were determined by selected ion monitoring of the methane positive ionization spectrum after gas chromatography/mass spectrometry (model 5989A; Hewlett Packard, Palo Alto, CA). Ions monitored were mass/charge 331–337.
Preparation of cooked barley
Servings (35 g) of the dehulled grains (8% moisture) of each cultivar were weighed. Each serving was cooked 1 d before feeding using a rice steamer (Flavor Scenter Handy Steamer, model HS800; Black & Decker, Towson, MD) with deionized water. Barley and water were added to the cooking bowl in a 1:1 ratio (v/v); water also was added to the base of the steamer per the manufacturer’s instructions. Total cooking time for the barley was 3 h (PW) or 2.5 h (BZ). After cooking, the barley was refrigerated in the cooking bowl overnight.
One serving of each cultivar was used for determination of total carbon content and 13C enrichment using isotope ratio mass spectrometry techniques (see below). The total serving was dried and ground to a fine powder (0.25-mm sieve size) using a cutting mill (model SM1; Brinkmann Instruments, Westbury, NY) to homogenize each sample. Six subsamples of each cultivar were analyzed and their values were averaged to determine the dose of 13C administered to each study subject.
Subjects and study design
The protocol was approved by the Baylor College of Medicine Institutional Review Board for Human Research and complied with the Helsinki Declaration as revised in 1983. Participants were 10 healthy adults (four male and six female; age range, 25–42 y; weight range, 52–77 kg) who had not received antibiotics for at least 15 d before the test. Subjects signed an informed consent and underwent a screening test to determine whether they were capable of producing H2 following the ingestion of a nonabsorbable carbohydrate (lactulose). For that purpose, subjects were instructed by a dietitian to ingest a low-fermentable dinner. After an overnight fast, they entered the Metabolic Research Unit of the Children’s Nutrition Research Center. A breath sample was obtained by having each subject blow into a breath collection bag. Subjects then ingested a 25-g serving of cooked rice to which 15 g of lactulose syrup containing 10 g of a nonabsorbable carbohydrate (Duphalac; Solvay Pharmaceuticals, Marietta, GA) had been added. Breath samples were collected every 30 min during the ensuing 240 min. Subjects who mounted a H2 response of 20 ppm or greater remained in the study and returned to the Metabolic Research Unit 7–10 d later. Subjects entered a whole-body indirect calorimeter where, under similar experimental conditions, 3–7 d apart, they ingested, in random order, a 35-g serving of one of the two barley cereals and 240 mL water. CO2 production was measured continuously by the calorimeter. Breath samples were collected at baseline and intermittently for 450 min for measurement of H2 and 13CO2 abundance.
Calorimetry measurements
Oxygen consumption (O2), production of carbon dioxide (CO2) and the resultant respiratory quotient, defined as CO2/O2, were measured continuously in a room calorimeter for 450 min. The operation, calibration and performance of the calorimeters have been described previously in detail (8
). Each chamber has its own microprocessor-based gas analyzers for CO2 (Ultramat 5E; Siemens, Karlsruhe, Germany) and O2 (Oxymat 5E; Siemens) that enable continuous data collection, recorded at 1-min intervals. Thermal mass controllers (Sierra Instruments, Monterey, CA) regulate airflow through the chambers to maintain constant CO2 concentration (0.45%) and gauge pressure (0.1 mm Hg). Errors from 24-h gas infusions averaged 0.34 ± 1.24% for O2 and 0.11 ± 0.98% for CO2. Calorimeters were calibrated before each test. Calorimeter temperature and relative humidity were controlled between 23 and 25°C and 40–60%, respectively.
Breath sample analysis for 13CO2 and H2
Breath samples (Becton Dickinson, Franklin Lakes, NJ) were transferred from the bag and stored in air-tight Vacutainers and later analyzed by isotope ratio mass spectrometry using a RoboprepG attached to a 20:20 isotope ratio mass spectrometer (Europa Scientific, Franklin, OH) at the Children’s Nutrition Research Center.
Samples were analyzed for H2 content using a Quintron DP Microlyzer (Quintron, Menomonee Falls, WI) within 3 h of collection. Results are expressed as parts per million. Peak breath H2 levels were calculated by subtracting from the highest breath H2 level obtained during the test the lowest level. Peak breath H2 levels of 10 ppm over baseline were considered indicative of carbohydrate malabsorption (9
).
Calculations and data analysis
The percentage of the 13C dose that was recovered in breath was calculated after correction for CO2 production according to the following formulas: micromoles of CO2 per min = (mean VCO2 x 106)/22.4; micromoles of 13C excess = (micromoles of CO2 per min x APE enrichment)/100; and percent dose excess per min = (micromoles of 13C excess x 10)/13C dose administered, where 22.4 is the gas constant at standard temperature pressure to convert liters to micromoles and VCO2 is the volume of CO2 produced.
Statistical methods
The area under the breath H2 curve was calculated using a computer program (Ms-Dos Q-basic; Microsoft, Redmond, CA). Results of the 13CO2 in breath were compared by repeated measures ANOVA, with adjusted paired t tests to determine at which time points the means differed. Results are expressed as means ± SD. Differences with a value of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The modified barley was well tolerated. There was no difference in CO2 production or oxygen consumption following the ingestion of the two test cereals. The overall percent dose of 13C oxidized was higher after BZ than after PW intake (P < 0.05) (Fig. 1
). Breath 13CO2 in samples obtained within 120 min of the ingestion of the cereals was significantly greater after consumption of BZ than of PW (Fig. 1)
. The area under the curve for H2 was higher (P < 0.05) after PW intake (8658 ± 6582) than after BZ (5178 ± 4759). The breath H2 levels following PW and BZ intakes differed in the samples obtained between 120 and 210 min (Fig. 2
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,11
). Intrinsic labeling of plants with stable isotopes constitutes a safe way to study absorption and utilization of nutrients (12
). The labeling of all organic molecules can be achieved though the introduction of 13CO2 to the plant because carbon fixed in the photosynthetic processes is ultimately passed on to all biosynthetic pathways (13
). The primary requirement of a 13CO2 labeling system is a closed, relatively gas-tight chamber to house the plants and to isolate the enriched atmosphere. In the past, we have used 13C-enriched rice to determine its absorption in infants with diarrhea (14
).
In the present study, we used two indicators: one of oxidation (digestion and utilization) and one of malabsorption. For the former, we measured utilization of C from barley by measuring 13CO2 excreted in breath. For the latter, we measured breath H2, an indicator of malabsorbed carbohydrate. Breath 13CO2 in samples obtained in the first 120 min following ingestion of the cereals differed, indicating a difference in the rate of utilization by the subjects. As time elapsed, 13CO2 generated in the colon by fermentation of malabsorbed barley and recirculation of 13C through the bicarbonate pool resulted in 13CO2 outputs that were not different from one another (15
). The breath H2 levels after PW and BZ ingestion were significantly different in the samples obtained between 120 and 210 min. Before that time, the difference in the amount of the two types of barley that had reached the colon was not large enough to be detected. These results reflect the approximate time it takes for the cereal to arrive in the colon and be fermented by colonic bacteria. The combination of the two tests used in this study indicates lower oxidation of PW due to decreased absorption of this type of barley compared with BZ. De Vries et al. (16
) used barley groats to determine the mouth to cecum transit times. They found that H2 was detected in breath in all subjects by 8 h postingestion and in some at 2 h and 45 min. In our study H2 was detected in breath at much earlier times. This difference can be attributed to the amount of cereal ingested (much lower in our study) and/or the fact that we used cooked barley whereas De Vries et al. (16
) used barley just softened in water. Behall et al. (17
), using a serving size of oats similar to the one administered by De Vries et al. (16
), did not observe a difference between cooked and uncooked cereal in the amount of H2 detected in breath but did not report the mouth to cecum transit time (17
).
A previous study demonstrated that ingestion of PW resulted in a significantly lower postprandial glycemic response compared with that of Sustacal (Ross Laboratories, Columbus, OH) or oatmeal (18
). Our results support the conclusion of Battilana et al. (19
), who stated that the lowered postprandial glycemic response following a meal containing ß-glucans is related not to changes in carbohydrate or lipid metabolism but to delayed or decreased absorption.
In summary, PW is less well absorbed and used than BZ. The modified barley thus could serve as a nutritionally appropriate food item for patients with diabetes or obesity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This project has been funded in part with federal funds from the U.S. Department of Agriculture/ARS under Cooperative Agreement No. 58–6250-600l and was supported in part by ConAgra (Omaha, NE).
3 Presented in part at the Experimental Biology meeting [Lifschitz, C., Grusak, M. & Butte, N. (2000) Decreased digestion of carbohydrates from a ß-glucans-rich barley. FASEB J. 14: A212 (abs.)].
5 Abbreviations used: ANOVA, analysis of variance; APE, atoms percent excess; BZ, barley cultivar BZ 594.35.e; PW, prowashonupana.
Manuscript received 15 February 2002. Initial review completed 15 March 2002. Revision accepted 26 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. BeMiller, J. N. & Whistler, R. L. (1996) Carbohydrates. Fennema, O. R. eds. Food Chemistry 1996:157-223 Marcel Dekker New York, NY. .
2. Newman, R. K. & Newman, C. W. (1991) Barley as a food grain. Cereal Foods World 36:800-805.
3. Aring;man, P. & Graham, H. (1987) Analysis of total and insoluble mixed-linked (13), (14)-ß-D-glucans in barley and oats. J. Agric. Food Chem. 35:704-709.
4. Bourdon, I., Yokoyama, W., Davis, P., Hudson, C., Backus, R., Richter, D., Knuckles, B. & Schneeman, B. O. (1999) Postprandial lipid, glucose, insulin, and cholecystokinin responses in men fed barley pasta enriched with ß-glucan Am. J. Clin. Nutr. 69:55-63.
5. Newman, R. K., Klopfsenstein, C. F., Newman, C. W., Guritno, N. & Hofer, P. J. (1992) Comparison of the cholesterol-lowering properties of whole barley, oat bran, and wheat red dog in chicks and rats. Cereal Chem. 69:240-244.
6. Levitt, M. D. (1969) Production and excretion of hydrogen gas in man. N. Engl. J. Med. 281:122-127.
7. Katz, J., Lee, W.-N. P, Wals, P. A. & Bergner, E. A. (1989) Studies of glycogen synthesis and the Krebs cycle by mass isotopomer analysis with [U-13C]glucose in rats. J. Biol. Chem. 264:12994-13001.
8. Moon, J. K., Vohra, F. A., Valerio Jimenez, O. S., Puyau, M. R. & Butte, N. F. (1995) Closed-loop control of carbon dioxide concentration and pressure improves response of room respiration calorimeters. J. Nutr. 125:220-228.
9. Bond, J. H. & Levitt, M. D. (1972) Use of pulmonary hydrogen measurements to quantitate carbohydrate absorption. J. Clin. Invest. 51:1219-1225.
10. Tappy, L., Gugolz, E. & Wursch, P. (1996) Effects of breakfast cereals containing various amounts of ß-glucan fibers on plasma glucose and insulin responses in NIDDM subjects. Diabetes Care 19:831-834.[Abstract]
11. Wursch, P. & Pi-Sunyer, F. X. (1997) The role of viscous soluble fiber in the metabolic control of diabetes: a review with special emphasis on cereals rich in ß-glucan. Diabetes Care 20:1774-1780.[Abstract]
12. Weaver, C. L. (1984) Intrinsic labeling of edible plants with stable isotopes. Turnlund, J. R. Johnson, P. E. eds. Stable Isotopes in Nutrition 1984:61-75 American Chemical Society Washington, D.C. .
13. Grusak, M. A. (1997) Intrinsic stable isotope labeling of plants for nutritional investigations in humans. Nutr. Biochem. 8:164-171.
14. Lifschitz, C. H., Torun, B., Chew, F., Boutton, T. W., Garza, C. & Klein, P. D. (1991) Absorption of carbon 13C-rice in milk by infants during acute gastroenteritis. J. Pediatr. 118:526-530.[Medline]
15. Irving, C. S., Wong, W. W., Wong, W. M., Boutton, T. W., Shulman, R. J., Lifschitz, C. H., Malphus, E. W., Helge, H. & Klein, P. D. (1984) Rapid determination of whole body bicarbonate kinetics by use of a summed primed, exponential, and constant infusion protocol. Am. J. Physiol. 247:R709-R716.
16. De Vries, J. J., Collin, T., Bijleveld, C. M., Kleibeuker, J. H. & Vonk, R. J. (1988) The use of complex carbohydrates in barley groats for determination of the mouth-to-cecum transit time. Scand. J. Gastroenterol. 23:905-912.[Medline]
17. Behall, K. M., Scholfield, D. J., van der Sluijs, A. M. & Hallfrisch, J. (1998) Breath hydrogen and methane expiration in men and women after oat extract consumption. J. Nutr. 128:79-84.
18. Rendell, M., Vanderhoof, J., Venn, M., Shehan, M. A., Arndt, E. & Rao, C. S. (1998) Reduced blood sugar response to a genetically modified grain in diabetes. J. Am. Coll. Nutr. 17:A111.
19. Battilana, P., Ornstein, K., Minehira, K., Schwarz, J. M., Acheson, K., Schneiter, P., Burri, J., Jequier, E. & Tappy, L. (2001) Mechanisms of action of ß-glucan in postprandial glucose metabolism in healthy men. Eur. J. Clin. Nutr. 55:327-333.[Medline]
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