PHYSIOLOGICAL REVIEWS Vol. 81, No. 3, July 2001 Printed in U.S.A.

Short-Chain Fatty Acids and Human Colonic Function: Roles of Resistant Starch and Nonstarch Polysaccharides
DAVID L. TOPPING AND PETER M. CLIFTON Commonwealth Scientic and Industrial Research Organization, Health Sciences and Nutrition, Adelaide, Australia

I. Introduction II. Modes of Action of Fiber in the Gastrointestinal Tract III. Large Bowel Microora, Fermentation, and Short-Chain Fatty Acid Production A. Large bowel microora B. Fermentation and large bowel SCFA C. Measurement of large bowel SCFA in humans IV. Metabolic Effects of Short-Chain Fatty Acids in the Large Bowel A. Luminal effects of SCFA and fermentable carbohydrates B. Absorption and metabolism of SCFA by colonocytes C. Effects of SCFA on colonic blood ow and muscular activity D. Trophic effects of SCFA and the maintenance of a normal colonic cell phenotype: role for butyrate and propionate V. Nutrition and Large Bowel Short-Chain Fatty Acids A. Fermentable carbohydrate supply and SCFA VI. Small Intestinal Polysaccharide Digestion and Large Bowel Carbohydrate Supply A. Starch digestion B. Classication of RS C. Determination of RS in foods VII. Resistant Starch in the Large Bowel: Comparisons With Nonstarch Polysaccharides A. Relative contributions of RS and NSP to large bowel carbohydrate supply B. RS and fecal bulking C. Fermentation and colonic and fecal SCFA D. NSP, RS, SCFA, colonic cell proliferation, and colorectal cancer risk E. Potential adverse reactions: RS as a malabsorbed carbohydrate VIII. Conclusions and Future Directions

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Topping, David L., and Peter M. Clifton. Short-Chain Fatty Acids and Human Colonic Function: Roles of Resistant Starch and Nonstarch Polysaccharides. Physiol Rev 81: 10311064, 2001.Resistant starch (RS) is starch and products of its small intestinal digestion that enter the large bowel. It occurs for various reasons including chemical structure, cooking of food, chemical modication, and food mastication. Human colonic bacteria ferment RS and nonstarch polysaccharides (NSP; major components of dietary ber) to short-chain fatty acids (SCFA), mainly acetate, propionate, and butyrate. SCFA stimulate colonic blood ow and uid and electrolyte uptake. Butyrate is a preferred substrate for colonocytes and appears to promote a normal phenotype in these cells. Fermentation of some RS types favors butyrate production. Measurement of colonic fermentation in humans is difcult, and indirect measures (e.g., fecal samples) or animal models have been used. Of the latter, rodents appear to be of limited value, and pigs or dogs are preferable. RS is less effective than NSP in stool bulking, but epidemiological data suggest that it is more protective against colorectal cancer, possibly via butyrate. RS is a prebiotic, but knowledge of its other interactions with the microora is limited. The contribution of RS to fermentation and colonic physiology seems to be greater than that of NSP. However, the lack of a generally accepted analytical procedure that accommodates the major inuences on RS means this is yet to be established.

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Early observational studies showed that native East Africans, consuming a diet high in unrened cereals, were at lower risk of colorectal cancer, diverticular disease, and constipation than Europeans who ate a diet low in such foods (45, 46). The early link to risk was with the overall diet that was high in starches, but attention became focussed on dietary ber which was then thought to act as an indigestible bulking agent. Fiber is comprised principally of polysaccharides (nonstarch polysaccharides, NSP), and it has been established that, depending on type, they are subject to varying degrees of breakdown on transit in humans. This is effected in the human large bowel by a complex bacterial ecosystem resembling that found in obligate herbivores. It has similar substrates, i.e., complex carbohydrates and end products (short-chain fatty acids, SCFA), mainly acetate, propionate, and butyrate. SCFA contribute to normal large bowel function and prevent pathology through their actions in the lumen and on the colonic musculature and vasculature and through their metabolism by colonocytes. Butyrate, in particular, is thought to play a role in maintaining a normal colonocyte population. In ruminants and other herbivores, SCFA are absorbed and transported via the portal vein to the liver, and the fraction not absorbed is distributed to the other body organs and tissues for metabolism (for a general review, see Ref. 28). In herbivores, peripheral venous SCFA concentrations are high due to comparatively low visceral extraction and high rates of absorption into the circulation. However, human peripheral venous blood concentrations are normally low, and only acetate is present in measurable amounts. This prole reects the lower SCFA production rates and greater visceral extraction in omnivores, meaning that human peripheral venous SCFA are not representative of those in the portal circulation. Human experimentation has been conned largely to fecal measurements, which are also limited as 95% of SCFA are produced and absorbed within the colon. Breath H2 evolution has been used but is extremely limited in value as gas production is not indicative of the SCFA that are produced. Incubation of fermentable carbohydrates with fecal inocula can provide valuable information provided a number of precautions are taken, especially in minimizing donor variability. SCFA in colonic contents have been determined in colostomy patients and post mortem, but these approaches are impractical for large-scale dietary studies. Fecal measures are useful in establishing changes in excretion but not necessarily in production because fecal SCFA can be inuenced by rate of transit alone. Consequently, most experimental data have been obtained from model systems. Animal studies, principally with rats and pigs, have shown that large bowel SCFA are increased by the provision of fermentable carbohydrates. However, rats are

coprophagic and the large bowel differs substantially from humans, and pigs (and possibly dogs) appear to be better models. Although NSP resist digestion by intrinsic human intestinal digestive enzymes completely, their intakes do not account for calculated human SCFA production (the carbohydrate gap). Some of the decit may be lled by oligosaccharides (OS), but starch and products of small intestinal starch digestion are thought to contribute the most. This fraction is termed resistant starch (RS). This review aims to examine the relative contributions of RS and NSP to SCFA production in the context of the epidemiological and other data linking complex carbohydrates to improved colon function and lowered disease risk. In view of the reliance placed on animal models and indirect measures of fermentation, the strengths and limitations of these experimental approaches will be evaluated concurrently. A particular problem is that assay procedures are well-established for ber and/or NSP but not for RS. This means that dietary intakes can be calculated for the former but not the latter, and direct comparison of effects in the body may be difcult. Thus health authorities have been able to make dietary recommendations for ber but not yet for RS (22). In this review, the primary focus is on adults, but some attention is given to infants. The fermentative products in preweaned infants differ considerably from adults, with little butyrate being found. Other acids (e.g., formate) and products (e.g., ethanol) are found in substantial quantities but not in adults. The relationship between weaning to solid foods, the microora, and products of fermentation remains to be elucidated. In adults, the production of individual SCFA in the colon is important as is their distribution along the large bowel. Fermentation predominates in the proximal colon and SCFA transported to distal regions by the fecal stream. Samples from patients with colostomy at various sites support a decline in SCFA levels along the large bowel. The distal colon is the site of greatest organic disease, so the delivery of butyrate to this viscus may be especially important. This distribution of SCFA along the colon is found in other omnivores (e.g., pigs) but not in rodents. The ber hypothesis led to expectations of a strong protective role for it in laxation, and this is well-documented, especially for insoluble ber. This is not so for colorectal cancer, an important malignancy in afuent countries. Experimental work (largely in rats) suggested strong protection by ber against chemically induced large bowel tumors. In contrast, epidemiological studies showed that any protection was weak. Interventions, where human volunteers with polyps or adenomas have consumed ber supplements (usually as cereal brans), have also yielded disappointing data on progression or recurrence. Conversely, studies in rats have indicated no benecial or adverse effects of RS on tumors in genetically or chemically induced tumors, while epidemiologi-

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cal studies show that starch and (projected) RS intakes correlate negatively with colorectal cancer risk. Human interventions have shown that greater RS consumption is associated with diminished risk as shown by various indices (e.g., SCFA, colonocyte proliferation). The paradox may accrue from particular features of rodent digestion (e.g., coprophagy) which limits the value of the data. In vivo and in vitro studies indicate that butyrate produced by RS fermentation may be protective (possibly by promoting apoptosis in tumor cells), but direct proof of such a role for RS in colorectal cancer is absent. RS has been shown to promote colon function by alleviating infectious diarrhea and promoting colonic mineral absorption. Part of the benets in diarrhea may be due to interactions between RS and the microora. One RS type (high amylose maize starch) has been shown to be a prebiotic and promotes the survival of lactic acid bacteria. The interactions of RS with the microora in general remain to be elucidated. Although RS could be regarded as a malabsorbed carbohydrate, there is little evidence of any deleterious effect of RS in humans; rather, it appears that interactions between RS and the colonic microora appear to be of benet to the host in the short and long term. In countries with low intakes of starch and RS, it may be of health benet to increase their intakes, but it is not clear whether it should be as total starch, RS, or starch and RS. II. MODES OF ACTION OF FIBER IN THE GASTROINTESTINAL TRACT The rst systematic links between dietary ber and human health were expressed in terms of its indigestibility (45, 46). Although breakdown of some ber components on passage through the gut was recognized, it was considered largely as a bulking agent (45) and was dened as plant structural and exudative components not digested by human digestive enzymes (275). This has been called the roughage model whereby any protection by ber was due to its dilution or binding of toxins and carcinogens in the intestines through its physical presence (302). The then-current analytical procedures accorded with that concept, with gravimetric measurement of residues after extraction of foods or ingredients with neutral or acid detergent solutions yielding a residue of insoluble ber components (241). Application of this, rather limited, technology to humans showed that the breakdown of ber on transit in humans was surprisingly large for some foods. For example, during passage of wheat bran, only 36% was degraded but only 8% of cabbage ber survived (282). The neutral detergent ber (NDF) procedure used in that study is a substantial underestimate as soluble material, including important ber components, are extracted and so not included in the

ber value (241). Comprehensive analyses have been developed for the major ber components that obviate such losses (e.g., Ref. 293), and their application shows that ber in the human diet is principally NSP and Klason lignin, an insoluble, noncarbohydrate residue (275). Rapid enzymatic-gravimetric methods, involving digestion of foods with enzymes to remove digestible components including starch, protein, and fat, have been developed. One of these methods has been validated and accepted by the Association of Ofcial Analytical Chemists and Food and Agricultural and World Health Organizations (for details of the variants, see Ref. 17). This procedure yields values termed total dietary ber (TDF). There are other components such as oligosaccharides that are also not measured routinely. These can be regarded as components of dietary ber through their indigestibility, but their exact dietary contribution is unknown (274). The contribution of lignin to the diet may be as low as 1 g/day (182), compared with 1520 g/day for NSP (22). Thus NSP could be taken as the principal contributors to dietary ber intakes, as has been done for the purposes of this review. NSP can be subdivided into soluble and insoluble NSP, based on their solubility in aqueous solutions, although not necessarily under physiological conditions (299). It appears that foods high in soluble NSP undergo the greatest losses on transit (302). These losses are accompanied by a greater fecal excretion of bacteria, which increased by 2.3 g/day with wheat bran and 4.8 g/day with cabbage (282). The bacteria are derived from the organisms resident in the human large bowel. It is they which degrade carbohydrates entering the large bowel, a process which has a direct impact on colonic function. III. LARGE BOWEL MICROFLORA, FERMENTATION, AND SHORT-CHAIN FATTY ACID PRODUCTION A. Large Bowel Microora The bacterial population of the human cecum and colon is numerically large with at least 1010 to 1011 cfu/g wet wt, which, with an estimated mass of 250 750 g of digesta, gives a calculated total of 1013 cfu in the whole hindgut (138). Similar values have been reported in other omnivores such as pigs (47). Bacteria comprise 40 55% of solid stool matter (77), and 15 g of fecal bacterial biomass is voided daily in individuals consuming Western-type diets (138). More than 50 genera and over 400 species of bacteria have been identied in human feces (100, 120, 138, 258). The dominant organisms in terms of numbers are anaerobes including bacteroides, bidobacteria, eubacteria, streptococci, and lactobacilli, while others, such as enterobacteria, also may be found, usually in

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fewer numbers. Generally, bacteroides (including those that can utilize a wide range of polysaccharides) are most numerous and can comprise more than 30% of the total. The microora can metabolize proteins and protein degradation products, sulfur-containing compounds, and endogenous and exogenous glycoproteins (120). Some organisms grow on intermediate products of fermentation such as H2, lactate, succinate, formate, and ethanol and convert these to end products including SCFA (177). Other organisms metabolize CO2 either yielding CH4 (199) or converting CO2 to acetate (84). Breath CH4 excretion reects methanogenic bacterial activity in the colon (227) but occurs only in individuals colonized by a particular organism (Methanobrevibacter smithii) at 108 cfu/g dry feces (199). Bacterial numbers, fermentation, and proliferation are greatest in the proximal large bowel where substrates are highest. These substrates are depleted on transit, which is reected by a decline in SCFA production (177, 178). In vitro endogenous production was 250 mmol SCFAkg148 h1 during incubation with proximal colonic inocula falling to 50 mmol SCFAkg148 h1 with distal colonic inocula (177). There may be population (as well as numerical) changes on transit due to changes in substrate supply (258). The intestines of humans and other animals are sterile in utero with colonization by maternal anal or vaginal organisms occurring during birth (197). Colonization is time dependent, with enterobacteria and streptococci predominating during the rst 13 days after birth when fecal concentrations of these organisms peak at 1011 cfu/g feces before declining (201). Bidobacteria appear in feces after 2 or more days after birth and become the dominant species at 4 5 days. Colonization by bidobacteria is signicantly higher in breast-fed babies (47.6% of babies vs. 15% fed by bottle) (250), whereas enterococci predominate in bottle fed infants (7.4 vs. 6.7 log10 cfu/g feces in breast fed infants). On weaning, bidobacteria decrease and a more adult prole develops, presumably reecting dietary change (201). The relationship of dietary NSP and RS to this process is unknown. The colonic microora should change in response to gross nutritional shifts (e.g., weaning), progressive change (such as aging), or variations in food intake. In aged persons, Escherichia coli, streptococci, and clostridia increase and bidobacteria decrease further (201). Some studies have linked increasing age with the number of people colonized by methanogens (129) and their activity (as measured by breath CH4 evolution) (99), but other studies have not (33, 194), suggesting that any association may be weak. Very little is known of the role of heredity. A study of two genetically distinct strains of pig (Chinese and United States domestic) showed that the diet was the primary determinant of the effects of ber on large bowel microora and SCFA (186). Information in these areas is limited because conventional microbiolog-

ical techniques are very labor intensive. Newer molecular biological techniques should make investigation easier, quicker, and more discriminating (290). These technologies also raise some concerns about the reliability of traditional methodologies. In a continuous culture of human feces, plating methods showed that at 21 days, 98% of the total culturable count was bidobacteria and lactobacilli, whereas with genus-specic 16S rRNA oligonucleotide probes, bidobacteria were absent and lactobacilli represented 25% of total 16S rRNA at the same time point (266). The methodological issues in bacterial enumeration are hampering the understanding of relationships between substrate supply, fermentation, and end products. Until they are resolved, indirect indices of bacterial activity (e.g., breath gas evolution or the production of specic SCFA in vitro or in animal models) will remain in widespread use because they provide measures of the metabolic products that actually modulate physiological changes. B. Fermentation and Large Bowel SCFA The basic fermentative reaction in the human colon is similar to that in obligate herbivores: hydrolysis of polysaccharides, oligosaccharides, and disaccharides to their constituent sugars, which are then fermented resulting in an increased biomass (258). Carbohydrate hydrolysis is effected by a number of bacterial cell-associated and secreted hydrolases that can digest a range of carbohydrates which the human host cannot. Fermentation yields metabolizable energy for microbial growth and maintenance and also metabolic end products. Nitrogen for protein synthesis can come either from urea (via the urease reaction), undigested dietary protein, or endogenous secretions. In adult humans, the principal products are SCFA together with gases (CO2, CH4, and H2) and some heat. The general reaction of SCFA production and overall stoichiometry has been summarized for a hexose (73) as follows: 59 C6H12O6 38 H2O 3 60 CH3COOH 22 CH3CH2COOH 18 CH3CH2 CH2 COOH 96 CO2 268 H heat additional bacteria. The balance of products differs for other substrates (e.g., uronic acids and pentoses) but is expected to be generally similar (175). Survey data from various populations show that fecal SCFA are in the order predicted from that equation, i.e., acetate propionate butyrate (77, 103, 144, 211, 265, 289) (Table 1). Other organic acids (e.g., lactate or succinate or branched-chain SCFA generated from amino acids) are found in much smaller amounts. In milk-fed infants, acetate is the major acid in feces. Propionate levels are very low while butyrate is virtually absent in babies fed breast milk but may be found in those fed formula (88, 270) (Table 1). No lactate was found in formula-fed infants, but 13.9 mmol lactate/kg

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1. Fiber intakes and fecal SCFA in adult and infant populations

Study Population and Diet Fiber Acetate Propionate Butyrate

(g/day) Adults Fleming et al. (103) US; self selected, low and high ber 33* 2.75

(mg/g wet feces) 1.22 (mM) 1.64

Muir et al. (211) Takahashi et al. (289)

Simulated Australian Simulated Chinese Japanese; self selected Japanese; controlled

24 14 NR 21.7

56.1 39.9 36.0 45.2

15.0 12.8 22.3 23.6 (mmol/kg wet feces)

18.4 12.2 17.5 19.0

Hoverstad and Bjorneklett (144) Segal et al. (265)

Norwegian; self selected South African blacks South African whites

NR 13.5 NR

37.3 83.6 40.5

12.5 32.3 11.9 (mmol/kg wet feces)

12.4 17.9 12.4

Infants Edwards et al. (88) Siigur et al. (270)

Breast (4 wk) Formula (4 wk) Breast (2 mo) Formula (2 mo)

NA NA

46.8 43.6 53.9 52.6

1.2 13.4 2.9 16.2

0.0 2.2 0.4 2.2

NR, not recorded; NA, not applicable. * As total dietary ber. As nonstarch polysaccharides plus lignin. water values and daily short-chain fatty acid (SCFA) output. As dietary ber.

Calculated from fecal

of feces was found in breast-fed babies. Formate and ethanol have been found in quantity in feces from breastfed babies (333). The SCFA prole may be important in gut development. Data from premature infants (which are maintained in incubators) suggest that there is a very sensitive period between days 14 and 21 of life when fecal butyrate increases by 300%, and its excessive production (or the organisms which produce it) may relate to the development of necrotizing enterocolitis which is a substantial threat in these infants (288). It appears that in healthy infants, fermentation is slower than in adults, and butyrate production is established more slowly than that of acetate and propionate but by 2 years an adult SCFA prole has emerged (198). Presumably, the product prole during milk feeding contributes to the specic metabolic needs at this period of development, but this remains to be established as do the changes in individual SCFA during weaning and maturation. C. Measurement of Large Bowel SCFA in Humans Intubation has been used to determine the intestinal digestibility of carbohydrates (including starch) in humans (56, 97, 283) but not yet for SCFA. SCFA have been determined in human gut contents and portal venous blood at autopsy (78) and in portal venous blood of patients during surgery (76, 79, 224, 272). Clearly, these approaches are limited. Dialysis sacs in gelatin capsules have been used to determine SCFA in situ in normal

subjects (335) and in dietary interventions with different types of ber (110). They have been used clinically in ulcerative colitis (245) where the severity of inammation correlated with high concentrations of butyrate (18.9 vs. 14 mM in controls) and lower pH (6.21 vs. 7.47 in controls) in the patients affected most. Continuous sampling with this method is impractical, and the relationship between transit and SCFA is unclear. SCFA have been measured in the stomal efuent of patients with ileostomy, transverse, or sigmoid colostomy and who were consuming a self-selected diet (200). SCFA excretion was high with transverse colostomy compared with sigmoid colostomy, which is consistent with the expected fall in fermentation on transit. 1. Regional considerations of colonic SCFA metabolism Elsden et al. (90) showed both high concentrations of and a progressive decline in volatile acid along the large bowel of a number of herbivorous and omnivorous animal species. The prole has been conrmed in pigs where the fall can be substantial (20, 32, 78, 124, 183185, 221, 303). Depending on diet, total SCFA concentrations in the proximal colon are 70 140 mM falling to 20 70 mM in the distal colon (Table 2). Neither total SCFA nor the individual acids in the distal colon are predictive of those found proximally (32, 183, 184, 303). Fecal values have been measured but not at the time of sampling of gut contents and show increases in the excretion of total and individ-

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2. Total SCFA and butyrate (in parentheses) in regions of the large bowel of pigs fed various sources of fiber and resistant starch
Large Bowel Region Study Carbohydrate Sources Fiber Cecum Proximal colon Mid colon Distal colon

(g/day) Bach-Knudsen et al. (19)* Cummings et al. (78) Wheat our Rolled oats oat bran Pig grower diet 62 194 NR 100 145 83

(mmol/kg contents) 90 125 NR (mM) 60 90 114 60 60 70

Fleming et al. (102)

Beans Bran

63 66 (g/kg diet)

110 (9) 140 (11)

NR NR

NR NR

NR NR

(mmol/kg contents) 156 112 164 151 123 107 160 104 (mM) 82 (8) 69 (5) 72 (5) 69 (4) 63 (4) 70 (8) 131 (13) 124 (6) 92 (8) 78 (6) 77 (8) 96 (9) 43 (4) 81 (6) 66 (9) 94 (12) 139 (9) 97 (12) 36 (3) 52 (6) 87 (10) 32 (72) 72 (8) 50 (7) 85 (12) 80 (7) 73 (10) 25 (2) 30 (4) 65 (8) 21 (2) 63 (9) 19 (2) 52 (5) 65 (6) 39 (5) 70 77 122 82 54 70 74 57

Glitso et al. (124)

Whole rye Rye pericarp Rye aleurone Rye endosperm

156 177 180 94 (g/day)

Marsono et al. (184)

Bird et al. (32) Topping et al. (303)

White rice White rice rice bran Brown rice White rice rice bran Brown rice Low ber, wheat bran Wheat bran Beans Oat bran

20 43 37 33 33 14 44 45 42 (g/day)

(mmol/kg dry matter) NR NR NR 181 356 458 NR NR NR NR NR NR

Stanogias and Pearce (277)

Wheat bran

75 150 300

The values for ber refer either to the amount fed to each animal per day (g/day) or the amount incorporated into each diet (g/kg diet). NR, not recorded. * Values by interpolation from graphical data. As nonstarch polysaccharides plus lignin. As total dietary ber. As neutral detergent ber.

ual acids in pigs fed high RS diets (32, 43, 301). SCFA availability in the distal colon can change on transit with the loss of water and digesta mass. For example, in pigs fed beans and a low-ber control diet, respective digesta masses were 198 and 103 g in the proximal colon and 30 and 21 g in the distal colon. Corresponding SCFA pools were 22.6 and 5.35 mmol and 1.43 and 0.23 mmol, respectively (303). The relative change was greatest for butyrate. In pigs fed white rice (low RS), the distal colonic butyrate pool was 0.06 mmol compared 0.47 mmol in pigs fed brown rice (high RS) (183). SCFA availability changes with rate of digesta passage independently of rates of production. When humans were given senna or wheat bran, transit was 39 or 41 h, respectively, compared with 74 h with loperamide. Mean total fecal SCFA and butyrate concentrations were 113 and 79 mol/g wet wt (wheat bran), 202 and 59 mol/g wet wt (senna), and 82 and 6 mol/g wet wt (loperamide), respectively (170). There is a curvilinear relationship between transit and fecal total and individual SCFA (especially butyrate) so that at

whole gut transit times 50 h, butyrate cannot be detected (probably due to colonic uptake). This is an additional variable to be considered when analyzing fecal values, especially when some studies have shown greater fermentation (as breath H2 evolution) with consumption of fermentable carbohydrate but no change in fecal variables. Tomlin and Read (298) raised the RS intake (as a breakfast cereal) of human volunteers from 0.86 to 10.3 g/day. Integrated breath H2 production measured over 8 h was raised signicantly from 7,529 to 12,072 ppm/min but fecal SCFA were unchanged, suggesting that any change was localized within the colon. The data from stomal patients (200) provide direct support for an SCFA gradient in humans. Concentrations in sigmoid colostomy uid and feces were 40 50% of those in patients with transverse colostomy. This fall is much larger than in postmortem samples where total SCFA values were 118.6, 105.4, 72.4, and 87.5 mmol/kg in the ascending, transverse, descending, and sigmoid colon/rectum, respectively (78). Regional differences in SCFA have implications for

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large bowel disease, especially cancer (54), which is an important malignancy in terms of numbers affected, particularly in afuent westernized societies (334). In these populations tumors predominate distally with incidence rates of 9.5 and 7.3/100,000 of population, for American men and women, respectively, 8 15 cm from the rectum compared with 2.8 and 3.8 in the ascending colon (70). Other conditions (e.g., ulcerative colitis) where SCFA may have a role also predominate in the distal colon. This means that there are several important questions in human large bowel fermentation: 1) Does the overall rate change with diet? 2) Is the production of individual SCFA altered? 3) What is the distribution of the resulting SCFA along the colon? 2. Methods for assessing fermentation and SCFA production in humans Stable isotope technology, in which labeled carbohydrates are consumed and metabolites monitored in blood or expired air, has been applied in a very limited way to SCFA production in humans and pigs (73). It has yet to be tested thoroughly in human dietary studies. The other techniques in current use in vivo are measurement of breath gas (H2 or CH4) or SCFA in peripheral venous plasma or feces. In vitro SCFA production can be measured using fecal or digesta homogenates, but its relationship to the situation in vivo is equivocal. Breath gas evolution is noninvasive and can be carried out in real time and has been shown to increase under conditions favoring fermentation. Gelissen et al. (117) fed subjects with low (2.6 g) and high (15.7 g) ber test meals and found that evolution was 158 and 167 ppm H2/h on days 3 and 5 of consumption of the low-ber meals and 492 and 554 ppm H2/h with the high-ber meals. Acarbose is a potent -glucosidase inhibitor. It is a pseudo-oligosaccharide consisting of an unsaturated aminocyclitol, a deoxyhexose, and a maltose (for graphical structure, see Ref. 39). Inhibition of small intestinal starch digestion through ingesting this agent raises breath H2 excretion and fecal SCFA excretion in humans (142, 259, 260, 326). Scheppach et al. (259) showed that breath H2 was 81 ppm for a test meal with acarbose compared with a maximum of 32 ppm for the test meal alone. Stool weight rose by 68% with the inhibitor. Weaver et al. (326) noted that bowel movements were increased from 8 9/wk when subjects consumed the placebo to 16/wk at a dose of 200 mg acarbose. Total SCFA excretion with and without the inhibitor was 14.8 and 7.6 mmol/day, respectively (260). Holt et al. (142) reported that these effects persist for up to 1 yr of acarbose treatment. Other feeding trials with RS (230, 313) have shown greater breath H2 evolution with consumption of fermentable carbohydrate. van Munster et al. (313) showed an increment of excretion from 101 to 186 ppm H2/h when subjects consumed an additional 45 g

of high amylose starch. SCFA excretion rose from 7.1 to 9.6 mmol/day. However, the technology is limited by the fact that some individuals do not produce H2 (117), so a stoichiometric relationship between gas evolution and production of total and individual SCFA is impossible. Consumption of some fermentable carbohydrates such as transgalacto-oligosaccharides (36) lowers breath H2 despite in vitro evidence of greater SCFA production. In subjects consuming 10 g of this product for 21 days, CH4 production stayed constant at 800 ml/12 h, whereas H2 values fell from 476 ml/12 h on day 1 to 164, 267, and 206 ml/12 h on days 7, 14, and 21, respectively. Flick and Perman (104) found breath H2 evolution was unchanged in volunteers consuming 40 g of lactulose/day for 1 wk despite evidence in vitro of greater SCFA production through lower pH values with fecal inocula. Breath H2 measurement is relatively easy to use but appears to be rather unreliable and incapable of further development. Peripheral venous blood acetate has been used to monitor large bowel events, but there are only a few published reports of its use. Pomare et al. (229) showed a rapid (90 min) rise and fall in mixed venous acetate after oral consumption of a solution of 50 mmol SCFA (30 mmol acetate and 10 mmol each of propionate and butyrate). This time course is similar to that seen in pigs fed sodium propionate and is consistent with absorption from the stomach (147). The maximum concentration achieved was 194 M against a baseline of 54 M (229), which is in the range noted by Muir et al. (209) and Wolever et al. (332). Increments are slower and more sustained after ingestion of lactulose or pectin, consistent with SCFA production by large bowel fermentation (229). A rise in portal venous SCFA (including propionate and butyrate) was reported in patients at surgery after fermentation was increased by cecal installation of lactulose (224). Peak concentrations after infusion of 10 g of lactulose were 0.24, 0.04, and 0.03 mM for acetate, propionate, and butyrate, respectively. These values are low compared with those recorded in rats and pigs where total portal venous SCFA can exceed 2 mM (e.g., Refs. 60, 147) and may reect the small amount of lactulose that was given. Acetate is the main SCFA in mixed venous blood, and propionate and butyrate concentrations are so low that measurement is difcult without considerable sample concentration (209, 332). Wolever et al. (332) recorded values of 4.5 6.6 and 2.0 3.9 M for propionate and butyrate, respectively. Corresponding values reported by Muir et al. (209) were higher at 17.332.8 and 36.3 65.5 M, respectively. Blood acetate alone is of little value as an indicator of SCFA, especially if the other acids are important metabolically. Data from blood-perfused liver (273) and heart (306) show that both organs buffer blood acetate with uptake above a concentration of 0.25 mM and net release below it. Similar hepatic buffering and equilibrium point have been reported in rats in vivo (44)

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and may occur in humans (262, 272) and would limit the value of changes in blood acetate greatly. Peripheral venous SCFA seem to resemble breath gases; they are general indicators of fermentation but not of changes within the viscera. In vitro fermentation of forage with rumen liquor has been used very successfully to determine its nutritional value for ruminant domestic animals (327). SCFA production from foods and ingredients by human fecal homogenates has been examined in a similar manner. The method has the advantage of avoiding complications due to uptake and utilization by colonocytes. Generally, batch cultures (where inocula are incubated with substrate in bottles) have been used. However, the wide range of incubation times, fecal inocula strength, substrate concentration, buffering of medium, addition of protein and micronutrients, and analytical procedures for the substrate make direct comparison between studies difcult. The inoculum itself can be a major factor with considerable time-dependent variability between donors when the same substrates are fermented (87, 203, 205, 258). For example, control production on 1 day of sampling ranged from 20 to 42 mM/24 h (205). In the same study, production from wheat bran by three subjects measured on three separate days ranged from 59 to 111 mM/24 h. Some subjects (possibly 20% of those sampled) seem not to metabolize substrates such as particular types of RS well in vivo (64, 74) or in vitro (64). To minimize this potential variability, McBurney and Thompson (189) recommended use of a minimum of three donors. Other technical issues, e.g., maintenance of a reducing environment, buffering the medium against a fall in pH (87), and the dilution of the inocula (25), can inuence SCFA production. Protein may have an inuence as it appears to be fermented very rapidly by batch cultures (205). Animal studies suggest that resistant protein (RP, named by analogy with RS) can be an important experimental variable (202). In rats fed a diet containing a highly digestible protein (casein) and 200 g/kg of a high amylose cornstarch, succinate and butyrate were present at 651 and 26 mol/cecum, respectively. Partial replacement of casein with an RP (autoclaved egg white) lowered succinate to 381 mol/cecum and raised butyrate to 111 mol/cecum. Substrate concentration is important and varies with ber source. Mortensen et al. (203) showed that the molar proportion of butyrate rose from 9 to 20% by increasing the concentration of pectin from 2.5 to 30 mg/ml. With isphagula, the molar proportion of butyrate rose from 8 to 11%. One large European collaborative study involving ve centers has evaluated fermentation under standardized conditions of incubation and NSP analysis (25). This offers promise of making direct comparison between studies easier. However, the technique remains an intrinsically limited means of studying dietary inuences on SCFA production.

Changes in SCFA excretion with diet have been examined in colostomates (4, 222). Pant et al. (222) found that wheat bran raised SCFA excretion, whereas it was lowered by consumption of oat bran. However, the feeding time in this study was very short (5 days). Ahmed et al. (4) compared SCFA excretion with a high and low RS diet and found that it was signicantly higher with the former (183 vs. 116 mmol/kg dry fecal wt). These limited data conrm that human colonic SCFA seem to respond to change in fermentable substrate as they do in model animal species. 3. Animal models for human large bowel SCFA metabolism A priori, the model species should be as close to humans as possible, i.e., omnivorous with appropriate food intakes, nutrient requirements, and gastrointestinal system. The dog appears to be particularly suitable (315) because its large bowel contributes 14% to total digestive tract volume compared with 17% in humans, 48% in pigs, and 61% in rats. Relationships between fermentable carbohydrates and SCFA have been studied in intact (e.g., Ref. 287) and surgically modied dogs (212) for the purpose of improving canine, not human, nutrition. Compared with pigs, dogs have found relatively little use, probably for social reasons. The pig appears to be optimal especially when considering such issues and the fact that it consumes human foods readily. The relatively large fractional volume of the porcine colon (due to greater length, rather than cross-sectional area) necessitates intakes of dietary ber (80 100 g/day for a 60-kg animal) to maintain laxation, which are higher than those for humans but do not appear to compromise the data. Pigs have been used to examine the effects of numerous human foods and food ingredients including beans (102, 303), rye (124), rice (31, 184), oats (21, 303), starches (185, 301), tagatose (163), and wheat bran (20, 277, 303) and its fractions (20) on large bowel SCFA. All of these studies showed greater large bowel SCFA after consumption of fermentable carbohydrates (Table 2). By implication, SCFA production was increased. In pigs with cecal or proximal colon cannulae, SCFA were increased by feeding of navy beans (102) or wheat bran (277). Electron microscopic examination of native starch granules (Fig. 1A) and those recovered in human ileostomy efuent (Fig. 1B) and porcine cecal contents (Fig. 1C) showed substantial pitting and etching (301). However, the patterns were similar in both digesta samples, and these and the other experimental data suggest a good degree of similarity between pigs and humans. Pigs are also useful models for clinical conditions such as infant necrotizing enterocolitis (83) but not for colon carcinoma. Injection of carcinogens such as dimethylhydrazine (DMH) or azoxymethane (AOM) into rats induces intestinal cancers that can be

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3. Prevention of coprophagy and cecal SCFA in rats fed a control (nonpurified) diet or diets containing oat or wheat bran
Diet Coprophagy Total SCFA, mM Butyrate, mM

Nonpuried Oat bran Wheat bran

Yes No Yes No Yes No

188 101 224 197 48 33

51 29 37 6 48 33

[Modied from Jackson and Topping (149).]

modied by diet, but in pigs DMH produces hepatic necrosis without any intestinal tumors (330). There is no porcine equivalent of rodent models such as the multiple intestinal neoplasms (Min) mouse (208) or Smad3 mutant mouse (345) that have a genetic predisposition to intestinal cancer. These limitations plus the relative cheapness, small size, and ready availability of rodents explains their wide experimental use. However, it overlooks the fact that (unlike pigs and humans) rats are coprophagic cecal fermenters with a complex musculature that ensures selective retention of liquid digesta in that viscus while solid material is voided (284, 315). Rats reingest the feces produced by cecal fermentation specically. This has very important implications for the digestion of RS but not necessarily of ber. The fermentation of insoluble ber differs little between rats in which coprophagy is allowed or not (71), and losses of neutral NSP in wheat bran, apple, cabbage, and carrot are similar in humans and rats (216). In contrast, 17% of starch in aked barley appeared to resist small intestinal digestion in humans but only 1% in rats (242). In these studies , the starch-to-NSP ratio was 0.89 in humans and 0.02 in unrestrained rats (where coprophagy was allowed). Only one study seems to have examined abolition of coprophagy and SCFA where cecal values were changed substantially, depending on ber source (149) (Table 3). Coprophagy is a very important variable, especially for RS and SCFA, and limits the value of rat data. It seems preferable to use studies in humans wherever possible and, failing that, use more suitable species such as pigs or dogs.

FIG. 1. Scanning electron micrographs of native high-amylose starch granules (A) and granules isolated from human ileostomy efuent (B) and the large bowel of a pig with a cannula inserted into the cecum and proximal large bowel (C). Native (ungelatinized) granules of a highamylose (70% of total) starch were examined by scanning electron microscopy and show the characteristic irregular shape of these granules. After passage through the small intestine, the granules were recovered from the terminal human ileum and proximal porcine large bowel. They have undergone amylolysis by small intestinal -amylase, and both exhibit similar patterns of etching and pitting. The starch residue remaining after amylolysis is believed to be high in amylose and is known to be fermented by the large bowel microora. [Modied from Brown et al. (43).]

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IV. METABOLIC EFFECTS OF SHORT-CHAIN FATTY ACIDS IN THE LARGE BOWEL Carbohydrates entering the large bowel can alter colonic physiology in two ways: physical presence and fermentation. Undigested mono-, di-, and oligosaccharides induce osmotic diarrhea if consumed to excess (73). Fecal bulking was an important component of the ber hypothesis (302) and is a recognized attribute of foods such as cereal brans (23, 116, 152, 164, 165). Fiber analogs such as plastic bran akes also speed transit and promote laxation (171), indicating the importance of the roughage effect of ber. The actions of SCFA are wider in scope and more signicant to the colon, and this review focuses on the major acids found in adults, acetate, propionate, and butyrate, about which most is known. Effects of SCFA can be divided into those occurring in the lumen and those arising from their uptake and metabolism by the cells of the large bowel wall. A. Luminal Effects of SCFA and Fermentable Carbohydrates SCFA are the principal luminal anions in humans and other omnivores. They are relatively weak acids with pKa values 4.8, and raising their concentrations through fermentation lowers digesta pH. Food supply is an important variable. In rats fed a nonpuried diet ad libitum, cecal and distal colonic pH values were 6.14 and 6.87, respectively, compared with 7.40 and 7.37 in rats starved for 24 h (49). When rats were restricted to 15 and 19 g of nonpuried diet/day, cecal pH was 7.40 and 7.11, respectively, compared with 6.47 in animals fed ad libitum (148). In rats fed NSP of low fermentability (e.g., cellulose or wheat pericarp-seed coat) at levels of 50 150 g/kg diet, cecal pH is high, with values of 6.7 8.2 (51, 63, 85, 286, 307, 338, 340). Cellulose is a commonly used reference ber, and its effects depend on dietary level. Increasing cellulose from 50 to 100 g/kg diet has no effect on pH (7.55 and 7.70, respectively) but lowers SCFA concentrations signicantly from 102 to 70 mM (307). When rats are fed fermentable carbohydrates such as OS, NSP, or RS or fermentable cereal fractions such as wheat aleurone or whole our, cecal pH is lowered by 12 units (60, 63, 145). Some authors (e.g., Refs. 51, 338) have reported a strong negative relationship between cecal SCFA and pH, but in other studies (e.g., Ref. 307) the relationship is absent. This may reect the buffering by the gut contents or the presence of other dietary components, e.g., calcium which can modify pH (341). The lower limit of pH in these studies seems to be 5. pH values are lower in pigs fed diets that raise large bowel SCFA (20, 124, 185, 303). However, pH values change along the porcine large bowel differently to rats.

In rats, proximal colonic pH may be higher than in the cecum and distal colon. In pigs, pH values are higher in the cecum, equal or lower in the proximal colon, with a continuous gradient of rising values toward the distal colon. Importantly, distal colonic or fecal pH is not necessarily predictive of conditions in the proximal bowel of pigs. In animals fed low-ber diets (20 30 g NSP/kg), proximal colonic pH is 7.1, rising to 7.5 in the distal colon. Values are lower when diets containing additional ber (21, 124, 303), high RS foods (303), or high RS starches RS (185) are fed. The gradient was maintained in these trials, and one study failed to nd any effect of an RS on SCFA or pH, but that may reect the rapid fermentation in that experiment (301). Data from human interventions are limited largely to fecal values. When volunteers have consumed fermentative substrates such as lactulose (104), wheat bran (165, 170), oat bran (154), RS (214), partially hydrolyzed guar gum (289), or high-ber diets (196), pH values are lowered signicantly. Other studies with fructo-oligosaccharides (36, 312) or RS (298, 314) have failed to show such lowering. The actual pH values recorded vary by study. For example, Takahashi et al. (289) showed that control pH values were 6.17 6.25, falling to 5.4 5.5 when subjects were fed partially hydrolyzed guar gum. In contrast, Noakes et al. (214) found that mean pH was 6.18 when RS was fed and 6.40 with a low-RS diet. One of the contributory reasons for these differences may be the absolute value of SCFA as Segal et al. (265) recorded a strong (r 0.704) negative relationship between fecal SCFA and pH. This means that the absolute SCFA concentrations may override other inuences on pH (e.g., buffering by gut contents). Lower pH values (and raised SCFA) are believed to prevent the overgrowth of pH-sensitive pathogenic bacteria, although this is based largely on in vitro incubation studies. For example, propionate or formate have been shown to kill E. coli or Salmonella at high (pH 5) acidity (62). Some animal studies support such ndings, with greater SCFA production having been reported to lower the numbers of potential pathogens (such as Salmonella) in swine (231). However, the rapid weaning of piglets to diets high in fermentable carbohydrate (RS and NSP) leads to raised large bowel SCFA, colonization with a bacterium (Serpulina hyodysenteriae), and appearance of clinical symptoms including diarrhea (228). The syndrome seems to result from the commercial practice of very abrupt introduction of solid food rather than any adverse effect of SCFA (31). There are relatively few studies in human diarrhea, but it appears that SCFA can assist in the management of antibiotic-induced and infectious diarrhea. Fecal SCFA are lower during the active phase of cholera disease, while their elevation (through feeding of RS at 40 g/l of oral rehydration solution) diminishes uid loss substantially and speeds remission by up to 50% (236). Diarrhea has been shown in rats when they

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are fed puried diets containing very high levels (10 15%) of water-soluble polysaccharides such as gum arabic (307). This same reaction occurs in humans when the load of water-holding carbohydrate exceeds the fermentative capacity of the microora. Normally it seems that SCFA production and absorption from RS and NSP is associated with diminished uid loss. Fermentable carbohydrates can alter the microbial ecology greatly by acting as substrates or supplying SCFA. Much attention has been directed toward the study of specic benecial lactic acid bacteria, i.e., probiotics (usually bidobacteria or lactobacilli) rather than the ora as a whole. These organisms are unlikely to change the major SCFA in the colon (31). Probiotic numbers have been enhanced by prebiotics that are dened as nondigestible food ingredients that benecially affect the host by selectively stimulating the growth and (or) activity of one or a limited number of bacterial species already resident in the colon, and, thus, improve host health (120). Consumption of lactosucrose (218) or inulin (157) increases the fecal counts of bidobacteria in human volunteers. Kleessen et al. (157) found that at a dose of 40 g/day, inulin consumption increased bidobacteria numbers from 7.9 to 9.2 log10 cfu/g dry feces, but total bacterial counts remained unchanged. These changes were unrelated to any change in SCFA and pH. Similar bacteriological data have been reported in rats fed indigestible OS (oligofructose or xylooligosaccharides) where cecal bidobacteria numbers were higher than in controls (51). Feeding trials in pigs with RS have shown greater fecal numbers of bidobacteria after their oral ingestion (43). When a high amylose starch was fed, the fecal numbers were 8.91 log10 cfu/g wet wt compared with 8.12 log10 cfu/g wet wt when a low amylose starch was fed. Some of the interaction between RS and bacteria appears to be physical, with the organisms adhering to modied or unmodied starch granules (31). This adhesion has been studied relatively little as has the interaction between RS, SCFA, and the large bowel microora. New technologies for bacterial enumeration will facilitate development of a fuller understanding of these relationships. B. Absorption and Metabolism of SCFA by Colonocytes Less than 5% of bacterially derived SCFA appear in feces due to colonic uptake (195, 246, 252) which is responsible for the major decline in concentrations along the large bowel. Intubation studies have shown that SCFA are taken up from the perfused human large bowel in a concentration-dependent manner (252). In the guinea pig proximal and distal colon, this uptake was not saturable up to a concentration of 120 mM (237). At least 60% of that

uptake is effected by simple diffusion of protonated SCFA involving hydration of luminal CO2, while the remainder occurs by cellular uptake of ionized SCFA involving cotransport of Na or K (101). SCFA uptake is associated with a transport of water that appears to be greater in the distal than the proximal colon (38). The reduction in SCFA levels in antibiotic-associated colitis may be responsible for some of the diarrhea because SCFA stimulate colonic uid and electrolyte transport (66), although an inhibition of NSP breakdown (with attendant laxation) is possible (204). Enhanced uid transport helps to explain the accelerated remission from cholera seen with feeding RS. Na and K were thought to be the principal cations absorbed (91). However, the feeding of oligosaccharides to rats prevents osteopenia following gastrectomy (219) and increases the apparent absorption of Ca2 and Mg2 (338). A human study has shown greater calcium retention after consumption of fermentable carbohydrate, inulin and beet ber (72). Apparent colonic absorption was increased signicantly by inulin (33.7 vs. 21.3% in controls), but there was no change in Mg2, Zn2, or Fe2 retention. Studies in humans in which SCFA have been infused into the rectum support a direct stimulation of Ca2 absorption (308). A recent study in pigs has shown that apparent colonic Ca2 absorption was increased by 20% with consumption of RS (32). This increment was largely in the proximal colon, i.e., where SCFA are highest. The major SCFA are absorbed at similar rates in various regions of the large bowel (91). Acetate, propionate, and butyrate are absorbed at comparable rates in humans (252) and rat cecum (311) and colon (101). There are regional differences in guinea pigs, with acetate clearance being high in the proximal colon and low in the cecum and distal colon (91). Under the pH conditions (5.57.5) thought to apply normally in the human colon, 50% of SCFA would be present in the dissociated form. However, experimentally induced changes in pH within this range affect absorption rates relatively little. This may be due to a putative unstirred layer where reassociation may occur (91), which suggests that any regional differences are due to colonocyte metabolism and not the local luminal environment. SCFA are metabolized rapidly by colonocytes and are major respiratory fuels and trophic to the small bowel and colon (331, 339). Their oxidation supplies some 60 70% of the energy needs of isolated colonocytes (244) and suppresses that of glucose (15, 244) and spares pyruvate (48). Of the three major SCFA, butyrate is the major intestinal fuel even when competing substrates such as glucose and glutamine are available (243). There is a hierarchy of oxidation, with butyrate apparently being oxidized more in the proximal than distal colon. This, coupled with lower levels and slower absorption, may be important in human distal ulcerative colitis where it has been hypothesized that

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there is a defect in butyrate metabolism. Inhibition of fatty acid -oxidation in rats through rectal infusion of 2-bromo-octanoate causes the symptoms of colitis (247). Diversion colitis occurs in human patients in those segments isolated from the fecal stream and the supply of SCFA (89). Patients with ulcerative colitis have low fecal butyrate (and pH) and high lactic acid levels during exacerbations (318). Intracolonic infusion of SCFA preparations reduce the degree of inammation of the defunctioned segment in humans (3, 130), although this has not been conrmed (126). Butyrate enemas induced remission of ulcerative colitis (e.g., Ref. 263), but later reports have yielded inconclusive results (e.g., Ref. 278). Proliferation of cells in the upper 40% of the crypt, measured with proliferating cell nuclear antigen (PCNA), is reduced by treatment with butyrate or SCFA in patients with ulcerative colitis (261). The ratio h is a measure of the location of the proliferative zone in the crypt and is as follows: [labeled cells in upper 40% of crypt]/[labeled cells in whole crypt]. Any increase in humans is thought to predispose to cancer risk. A lowering of h by butyrate delivery could be of benet, especially in the distal colon, which is the region most at risk of pathology. C. Effects of SCFA on Colonic Blood Flow and Muscular Activity In vitro studies have shown that incubation with SCFA (as the sodium salts) at concentrations as low as 3 mM dilate precontracted colonic resistance arterioles in isolated human colonic segments (204). Acetate and propionate were most effective. Rectal infusion of SCFA into human surgical patients leads to 1.5- to 5.0-fold greater splanchnic blood ow (203). Greater colonic blood ow has been observed with infusion of acetate, propionate, or butyrate (separately or as a mixture) into the denervated canine large bowel (162). When acetate, propionate, and butyrate were infused at 75, 30, or 30 meq/l, respectively, blood ow rose by 18.1 and 3.1% for acetate and propionate, respectively, but fell by 3.4% when butyrate was infused. The mechanism of action of SCFA on blood ow does not involve either prostaglandins or - or -adrenoreceptor-linked pathways (204). The presence of SCFA (as the sodium salts) in rat colon incubated in vitro leads to increased contraction that persists for 1 min after application of SCFA solutions of up to 10 mM (337). The maximal effect was achieved at 0.1 mM with an order of effectiveness of acetate butyrate propionate. At higher concentrations (100 mM), contractile activity was abolished (276). The mechanisms of action may involve local neural networks as well as chemoreceptors together with direct effects on smooth muscle cells (61). SCFA produced in the colon and entering the portal circulation seem to inuence the upper gut musculature. Manometric

studies in humans have shown a decrease in gastric tone giving an expansion of volume after ingestion of fermentable carbohydrate (lactulose) or rectal infusion of lactose or SCFA (249). The decrease was not obviously linked to circulating peptides of intestinal origin (glucagon-like peptide 1, oxyntomodulin-like immunoreactivity, or peptide YY). SCFA appear to activate the ileocolonic brake directly in a dose-dependent manner. This effect was assayed by increases in volume in a barostat bag inserted in the volunteers stomach with a greater rise in volume showing slower transit. The integrated changes in volume with time were 56 103 and 84 103 ml/min for 54 and 90 mmol SCFA, respectively, compared with 5 103 ml/min when the control solution was infused. These actions are important for the maintenance of the function of the whole gastrointestinal system, not just the colon. Slowing of upper gastrointestinal passage of food would be expected to improve nutrient digestion, whereas more rapid transit of food through the colon is thought to improve laxation. It is expected that greater blood ow enhances tissue oxygenation and transport of absorbed nutrients. D. Trophic Effects of SCFA and the Maintenance of a Normal Colonic Cell Phenotype: Role for Butyrate and Propionate In rats, SCFA stimulate the growth of colorectal and ileal mucosal cells when they are delivered colorectally or intraperitoneally (159, 254). Other animal studies have shown that SCFA supplementation of total parenteral nutrition (TPN) infusions retards the mucosal atrophy seen after massive bowel resection in rats (158). Feeding of diets high in fermentable carbohydrates to rats promotes ileal growth and raises ileal and cecal glucagon-like peptide-1 mRNA levels (238). In addition to promoting growth, the major SCFA (especially butyrate) appear to lower the risk of malignant transformation in the colon. In normal rats, butyrate at concentrations of 10 and 25 mM enhance proliferation only at the crypt base resulting in a fall in h. This effect is blocked by 5 mM deoxycholic acid (316), although the cotreatment does not reverse deoxycholate-induced increases in colon weight and indices of cell proliferation (317). Secondary bile acids are cytotoxic, and in rats fed deoxycholate plus cholesterol, cell proliferation as measured by incorporation of [3H]thymidine was increased (167). When the diet contained 0.15% deoxycholate plus 1% cholesterol, incorporation was 81.4 versus 43.4 dpm/g DNA in controls. This change appears to be associated with greater susceptibility to the development of cancer (82, 173, 292). Normal mucosa from colorectal carcinoma patients resists bile-acid induced apoptosis, implying that high levels of bile acids may select for cells resistant to apoptosis (223). Some of the

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effects of SCFA may be due to low intra-colorectal pH rather than any specic SCFA. At a pH of 6, bile acids are largely protonated and insoluble and so would not be taken up by colonocytes (235), and lower pH inhibits the bacterial conversion of primary to secondary bile acids (176, 213) and so would lower their carcinogenic potential. Tumorigenesis is a multistep process with a progression from a hyperproliferative epithelium to preinvasive and metastatic carcinoma via formation of aberrant crypts and various stages of dysplasia (342). Genetic alterations are believed to occur at each step, but their determinants are uncertain and it is unclear whether butyrate opposes any or all of them in vivo. The greater proliferation with butyrate is a paradox in that it could be expected to increase risk of tumor formation. The answer may lie in the differential effects of butyrate on apoptosis in normal and tumor cell lines. In the absence of butyrate, normal colonic cells in culture undergo apoptosis within 150 min, paralleled by a vefold increase in Bax protein gene expression (131). In contrast, butyrate leads to growth arrest, differentiation, and apoptosis in tumor cell lines (18, 81, 115, 121, 127, 128, 328). In these studies, differentiation was assessed by increased expression of the brush-border glycoproteins, alkaline phosphatase, and carcinoembryonic antigen. Normal colonic cells show decreased expression of these markers after incubation with 1 4 mM butyrate (121). SW620 cells became arrested at G0-G1 and G2-M within 12 h of exposure to butyrate with apoptosis 4 h later that was related to abnormalities in the mitochondrial electron chain (133). Siavoshian et al. (268) demonstrated changes in the cell cycling factors p21 and D in HT-29 human colonic adenocarcinoma cells. Heerdt et al. (132) demonstrated elevated alkaline phosphatase in cancer cells treated with butyrate, particularly in the oating apoptotic cells, suggesting that programmed cell death occurred subsequent to differentiation. One of the possible mechanisms for differentiation in tumor cells in vitro is a reduction in nuclear levels of the protooncogene c-myc (68, 291), a factor which is important in control of tumor growth. Treated cells have a signicant loss in colony-forming capacity in soft agar (156) which is related to the lowering of c-myc (291). Butyrate treatment also reduces cytoskeleton-associated tyrosine protein kinase activity (264), which is important in cellular responses to cytokines such as transforming growth factor-1 that promote growth in HT-29 tumor cells (24). Inhibition of DNA synthesis may occur through inhibition of histone deacylase (160) as removal of histones is an important rst step in DNA replication. Apoptosis may be pivotal in the progression from colon adenomas to colon carcinomas as mutations in genes, such as p53, which control programmed cell death are often seen in tumors (251, 256). Apoptosis is enhanced by butyrate but not through the p53 gene (128, 132). Propionate and

acetate also induce apoptosis but to a lesser extent than butyrate (127) and at higher concentrations (40 mM), although these can be achieved in colonic digesta in vivo. This accords with the differential effects of the three fatty acids in inhibiting proliferation and inducing differentiation (115, 328). Additionally, propionate induces apoptosis in adherent cells, but butyrate induces it only in oating cells (127, 128, 132), suggesting differential metabolic effects of the two SCFA. Cells may lose their responsiveness to butyrate (30, 329), with some cancer cell lines resisting butyrate-induced apoptosis (127). Butyrate and acetate (but not isobutyrate or propionate) also appear to inhibit DNA oxidative damage due to H2O2 in isolated rat distal colon cells at concentrations of 6.25 M (1). Surprisingly, a mixture of the major SCFA did not oppose the genotoxicity. It has been shown in rats treated with a carcinogen (AOM) that apoptosis was increased in aberrant crypt foci when large bowel butyrate was increased through feeding it in slow release pellets (50). Cecal and colonic butyrate concentrations were 85 and 9 mM after the treatment, and apoptosis (measured by TUNEL) was increased from 0.12 in controls to 0.81. Apoptosis measured morphologically was rather higher in controls and lower in the butyrate-treated group but was still signicantly different. Neither cell proliferation nor aberrant crypt foci induction were changed. One study has shown oral butyrate stimulates tumor promotion in rats treated with dimethylhydrazine (111). Plasma butyrate levels were not measured, so it is unknown if the transformed cells were exposed to a higher butyrate concentration. However, if oral butyrate were absorbed like propionate (as appears likely), then most would be absorbed via the upper gut and cleared by the liver (147). Inter alia, the experimental data support a role for SCFA in promoting a normal phenotype in colonocytes that is beyond the provision of metabolic substrate. It appears that butyrate and (to a lesser extent) propionate act to prevent the development of abnormal cell populations. A direct role for these SCFA in the prevention of human colorectal carcinoma remains to be established. V. NUTRITION AND LARGE BOWEL SMALL-CHAIN FATTY ACIDS The inuence of weaning and gender (as well as heredity) on SCFA have yet to be explored in detail. Fecal data suggest that gender may be an important factor for NSP and RS. Lampe et al. (165) have shown that the digestibility of dietary ber (measured as NDF) in wheat bran was 43% in women and 37% in men when they consumed 30 g of wheat bran ber/day. Fecal bulking was lower and mouth to anus transit was longer in women than in men and varied with ber type (vegetable vs.

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wheat) and level of intake (10 or 30 g/day). In women consuming a low-ber diet, the loss of starch into the colon (as measured by breath H2) after a standard test meal is 30% lower during the luteal phase of the menstrual cycle than during the follicular phase (188). The respective calculated mean values were 9.7 and 6.6 g/50 g starch, and stool weight was also lower during the former phase. These differences warrant further investigation. A. Fermentable Carbohydrate Supply and SCFA Of the techniques available and applied widely, only in vitro fermentation gives an estimate of SCFA production. Breath gas evolution is an indicator of fermentation while measurements in human or animal feces or digesta measure increases or decreases in concentrations or pools. These are taken to reect altered production, which appears to be reasonable (but imprecise) when ones considers studies in cannulated animals that show greater portal venous SCFA concentrations and transport after feeding of fermentable carbohydrates (123, 147, 239). 1. Total SCFA
A) IN VITRO STUDIES. Of the methods used widely, only in vitro incubation offers a means of assessing SCFA production. Animal and human studies generally report changes in concentration or pools and take these to be an index of formation. Increased SCFA production by human fecal inocula has been demonstrated with ber-rich foods including bran fractions from wheat, oats, barley, corn and rice, soybean ber, vegetable extracts, and pea ber (25, 73, 189, 207). Puried preparations (such as glucose, cellulose, guar gum, pectin, and starch) and isolates (e.g., from vegetables) also have been examined (25, 73, 189, 207, 324). Some puried carbohydrates (such as cellulose) are fermented slowly and incompletely while glucose is fermented quickly and completely. This quality is referred to generally as fermentability, a term which combines the rate and extent of carbohydrate degradation. It is highly variable with 97% of pectin and only 6 7% of cellulose and maize bran being fermentable (25, 37). Less than 50% of wheat bran components are fermented, whereas estimates for psyllium fall in the range 20 50% (285) and 96% of oat bran is lost (58). High fermentability relates to greater SCFA production in vitro. Fermentation of 30 mg glucose, pectin, or cellulose/ml yielded concentrations of 220, 172, and 23 mmol total SCFA/l, respectively, in the incubation uid (203). Additionally, greater fermentability may be associated with a more rapid fermentation (25). The large European interlaboratory study that examined a number of ber sources showed a close relationship between NSP degradation and SCFA production (25). Similar relationships between

RS degradation and SCFA production have been noted with pig fecal inocula (185). The European ber study was of sufcient size to allow for the variables that can impact on fermentation and showed clearly the wide range of values obtained in ve laboratories (Table 4). A similar range could be expected for RS, and appropriate steps can be taken to minimize it. B) ANIMAL STUDIES. Cecal and colonic SCFA fall in starved rats or rats fed restricted amounts of a nonpuried diet (49, 148), with the greater fall occurring in the colon (49) (Table 5). These changes were reversed by restoration of unrestricted feeding, although the distal colonic values were slowest to recover. There have been numerous studies in rats with measures generally conned to the cecum. These experiments conrm that enrichment of the diet with NSP, RS, and OS leads to elevation of large bowel SCFA. Feeding diets containing complex ber mixtures (187) or ber-enriched cereal fractions such as wheat bran (60, 107, 191, 193, 248), oat bran (146, 344), barley bran (191), wheat aleurone (60), or rice bran (107) leads to higher concentrations of SCFA in large bowel digesta. For example, concentrations of 50 70 mM have been recorded in rats fed diets low in fermentable carbohydrate (cellulose or wheat pericarpseed coat) with values as high as 180 M in rats fed fermentable carbohydrate (wheat aleurone) at levels of 100 g/kg diet (60). Similar increases have been shown with polysaccharide isolates including pectin (143, 346), inulin (248), guar gum (86), gum karaya (86), xanthan gum (86), and gum arabic (286, 305, 307). The feeding of OS (51, 219) and RS (125, 187, 341) also increases cecal SCFA. In many of these reports, the weight of large bowel digesta was increased leading to cecal SCFA pools, which were 100 300% higher than in controls depending on carbohydrate source and dietary inclusion level. Assuming that absorption rates were not changed greatly, pool size gives a rough measure of SCFA production rates that indicate substantial enhancement by RS, OS, and ber.

TABLE

4. In vivo and in vitro substrate fermentability and production of SCFA

Fiber Source Incubation Time, h Fiber Degradation, % SCFA Produced, mM

Pectin Sugarbeet ber Soybean ber Maize bran Cellulose

10 24 10 24 10 24 10 24 10 24

89 (69 to 112) 97 (83 to 117) 32 (1 to 70) 60 (14 to 98) 72 (55 to 95) 91 (80 to 98) 0 (28 to 21) 6 (21 to 27) 9 (9 to 38) 7 (15 to 45)

62 (45 to 83) 68 (46 to 97) 26 (6 to 54) 45 (29 to 72) 51 (29 to 70) 64 (41 to 79) 5 (2 to 11) 12 (3 to 26) 2 (1 to 4) 3 (0 to 7)

Values in parentheses indicate range. [Modied from Barry et al. (25).]

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5. Changes in large bowel SCFA in rats with starvation and refeeding with a nonpurified diet
SCFA, mM Region Acetate Propionate Butyrate Total

Time of starvation, h 0

56

Cecum Proximal colon Distal colon Cecum Proximal colon Distal colon Cecum Proximal colon Distal colon Cecum Proximal colon Distal colon

55 20 43 47 0 4 38 27 18 72 58 39

27 6 13 10 0 0 17 10 6 23 16 12

33 6 15 5 0 0 11 6 3 14 11 4

122 32 73 59 0 5 63 42 21 106 89 56

Time of refeeding, h 5

15

[Modied from Butler et al. (49).]

When intakes of dietary water-soluble carbohydrates exceed the fermentative capacity of the microora, SCFA fall due to osmotic diarrhea secondary to their presence in the digesta. In pigs, feeding ber or RS elevates large bowel SCFA concentrations and pools. When pigs were fed a westerntype diet (i.e., high in fat and protein and low in ber), increasing ber intake from 14 to 42 g NSP/day by feeding wheat bran raised total digesta from 255 to 498 g and SCFA pools from to 12.7 to 32.2 mmol (303). However, NSP were not predictive of changes in digesta mass and SCFA, so when pigs were fed 40 g NSP as navy beans, these were 655 g and 60 mmol, respectively. The disparity appears to be due to the RS present in these foods, which can add considerably to their fermentable carbohydrate content. Fleming et al. (102), using cannulated pigs, noted a similar expansion when beans were fed. Broadly similar data have been obtained with white rice plus rice bran and brown rice with the latter resulting in a disproportionate rise in digesta and SCFA (32, 184). C) HUMAN STUDIES. Controlled dietary studies in humans are few and generally limited to fecal measurements. One such trial showed that consumption of an additional 10 30 g of fermentable carbohydrate/day (as wheat bran or vegetable ber) raised fecal SCFA (110). However, these supplements also raised fecal bulk and shortened transit time (164, 165), which could have raised fecal SCFA without any change in production. Other studies in which there was no change in laxation indicate that greater intake of fermentable carbohydrate results in higher SCFA production (e.g., Refs. 214, 314). Fecal SCFA concentrations and excretion have been shown to be higher with feeding of some NSP such as partially hydrolyzed guar gum (289) but not others (oat bran) (214). Various sources of RS (74, 153, 214, 225, 267, 314) and acarbose (142, 259, 326) raise fecal SCFA. These increases

have been reported as higher concentrations, excretion, or both, which may reect passage of the fecal stream. This is likely to be the reason for the lack of effect of OS and RS at low doses on fecal SCFA as they would be expected to be fermented relatively rapidly, and the products of fermentation could be absorbed in the more proximal colon. 2. Individual SCFA
A) IN VITRO STUDIES. Acetate is the most abundant SCFA in fecal and digesta samples and formed in vitro. Fecal inocula from adults and children produce lesser amounts of propionate and butyrate, whereas inocula from breastfed infants produce little or no butyrate. In the latter, acetate, ethanol, propionate, and formate are the main products (333). Rodent studies suggest that propionate and butyrate production are related inversely. Fiber sources (such as oat bran) that lower plasma cholesterol through enhancing steroid excretion raise the contribution of propionate relative to butyrate, whereas wheat bran has the opposite actions (59). The relationship seems to be secondary to changes in large bowel steroids because feeding of diets containing cholesterol plus cholic acid to rats lowers cecal butyrate to 1 mM (96). Moreover, in rats fed wheat fractions, there is a negative correlation between cecal butyrate and steroids (total and individual bile acids and neutral sterols) and a positive correlation between the latter and propionate (145). The correlations were strong between deoxycholate and butyrate (r 0.66, P 0.001) and propionate (r 0.55, P 0.01) (Fig. 2). Whether such relationships occur in humans is not established. The in vitro data indicate that substrate inuences propionate and butyrate production considerably. Barry et al. (25) showed that 24-h production of total SCFA and

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FIG. 2. Relationship between the pools of short-chain fatty acids (SCFA) and deoxycholic acid in the cecal contents of rats. Rats were fed puried diets containing milled whole wheat or wheat bran, wheat pollard (a bran-rich our), or white wheat our obtained from that our. SCFA and bile acids and neutral sterols were measured in cecal contents at the end of the feeding trial. Each data point represents the mean values for each of the four dietary groups and shows that there were strong negative correlations between the total pool of deoxycholate (a secondary bile acid) and the pool of butyrate. Conversely, there was a strong positive relationship between deoxycholate and the pool of propionate. [Modied from Illman et al. (145).]

butyrate was high with pectin, intermediate with soybean ber, and low with sugarbeet ber (Table 4). However, this study and others (e.g., Ref. 203) have shown strong time and concentration dependencies that limit data interpretation. The latter may be particularly important in relation to human diets. For example, at 2.5 mg pectin/ml, acetate and butyrate contributed 81 and 9% of total SCFA, respectively, but at 30 mg/ml, the corresponding values were 74 and 20%. B) ANIMAL STUDIES. Starvation or restricted feeding of rats leads to a fall in acetate that is reversed on restoration of ad libitum feeding (Table 5). Conversely, when SCFA have been elevated through feeding of foods high in NSP, OS, and RS or isolates of those carbohydrates, concentrations and (where measured) pools have been increased. Propionate and butyrate fall after starvation or restricted feeding of rats, with butyrate falling most and being restored more slowly on refeeding, especially in the distal colon (Table 5). This difference could be due either to its apparent preferential utilization by colonocytes or the time dependence observed in vitro. Fermentable carbohydrates, including ber-rich fractions, puried NSP, OS, and RS raise the concentrations and pools of propionate and butyrate in rats. However, it is hard to extrapolate these data to humans and also to link the fermentation of individual carbohydrates with particular SCFA proles. Analysis of NSP monosaccharides suggests that the uronic acid content correlates strongly with production of acetate, while xylose content is weakly related to butyric acid production (255). Dietary NSP mixtures are more fermentable and produce quite different SCFA proles (with higher butyrate levels) than when the NSP are

fed alone (305). For example, total cecal SCFA were 171 and 157 mM and butyrate was 22 and 32 mM in rats fed either cellulose or gum arabic at 140 g/kg diet. When these NSP were fed as a 50/50 mixture, total SCFA and butyrate were 237 and 53 mM, respectively. Extrapolation of these data from rats to other model species and to humans eating mixed diets needs caution. When ber-rich foods (wheat bran and oat bran) were fed to pigs, large bowel propionate and butyrate were similar (303) despite reported differences in rats. Portal venous butyrate concentrations were higher in pigs fed oat bran than in those fed wheat bran (304), and Bach Knudsen et al. (21) showed in pigs that oat -glucan raised large bowel butyrate. In this species, low-ber diets depress butyrate and propionate in pigs, especially in the distal colon (184, 303). When pigs were fed physiological RS as either beans or brown rice, the distribution of SCFA was quite different. Butyrate was raised more with brown rice (especially in the distal colon), whereas with beans the increase was greater for propionate in the proximal bowel. Martin et al. (185) have noted similar distributional differences between RS types. C) HUMAN STUDIES. The limited human data are contradictory. Greater fecal excretion of butyrate and propionate has been observed with consumption of wheat bran compared with vegetable ber (110). In contrast, feeding of partially hydrolyzed guar gum resulted in greater fecal excretion of all three major acids, but no change in the concentration of propionate and butyrate and a decline in their relative contribution (289). The data for RS are more consistent, and several studies have shown that its consumption raises fecal butyrate (214, 225, 314). Given the putative importance of butyrate in maintaining large bowel function, these (and other experimental and epidemiological ndings) have focused attention on RS as a fermentative substrate for the colonic microora. VI. SMALL INTESTINAL POLYSACCHARIDE DIGESTION AND LARGE BOWEL CARBOHYDRATE SUPPLY NSP and non-starch-derived oligosaccharides are virtually completely resistant to digestion by the intrinsic enzymes of the human stomach and small intestine. The reported recovery of ingested ber components in ileal efuent of ileostomates consuming whole foods was 97% (151). The excretion of inulin and oligofructose in ileostomates was close to 100% (11), whereas that of uronic acids (alginate) was 95% (257). The (133),(134)--Dglucans found principally in oat and barley grains at concentrations of 312 g/100 g total seed wt appear to be exceptional. At low intakes (1.8 g -glucan/day), 80% was degraded, and at higher intakes (16 g/day), 30% was lost on transit from the mouth through the small intestine of human ileostomates (8, 9). Similar data have been ob-

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tained with pigs cannulated in the terminal ileum (20). Twenty-ve to 36% of oat -glucans was recovered at this site compared with 82104% of wheat NSP. -Glucans could be limited substrates for human and porcine -amylase. However, ileostomy efuent contains measurable quantities of SCFA (200), so the degradation in humans and pigs may be bacterial. This is supported by the fact that the last few centimeters of the ileum appear to take on the appearance of the cecum in patients with stable ileostomy (140). Normally, ileal NSP digestibility is virtually zero. A. Starch Digestion Small intestinal starch hydrolysis is effected rst by -amylases that release maltodextrins that are then hydrolyzed by membrane-associated maltases to free glucose which is absorbed. RS is determined by the factors that control this small intestinal digestion. These include starch structure, the presence of other food components (NSP, lipids), cooking, industrial processing, and individual physiological inuences such as chewing and transit (13). 1. Starch structure and ileal digestibility NSP are an extremely diverse group of homo- and heteropolymers. In contrast, starch is a glucose homopolymer found in two forms: amylose and amylopectin. The former is an essentially linear structure where the glucose units are joined by (134) glycosidic links while amylopectin consists of linear (134) linked chains with (136) linked branch points. Amylose has degrees of polymerization (DP) of 100 10,000 monomer units, whereas the molecular weight of amylopectin can exceed 107 (for general reviews, see Refs. 13, 22, 31, 114). In raw foods, starch is present as crystalline granules with two main forms (A and B) that differ in the relative proportions of amylose and amylopectin. A-form starches have chain lengths of 2329 glucose units and are found in cereals. Tuber and amylose-rich starches are B form with 30 44 glucose units. Both have similar molecular arrangements with left-hand parallel double helices, but the B form has more associated water. A third form (C) is found in legumes and appears to be a mixture of A and B forms that resists digestion as do B-form starches. The positions of the branch points relative to the linear regions of amylopectin dictate its cluster and structural properties in the crystalline region of the starch granule. The majority of the amylose exists in the amorphous regions of the starch granules, while the amylopectin gives starch its crystalline structure. When raw starches are heated with limited water, they melt, which leads to loss of order in the crystalline region and can take up the V form in which starch becomes associated with lipid. These V-form com-

plexes appear to be digested incompletely in the small intestine of dogs (212). When starches are cooked with water, the granules become gelatinized to a degree that depends on temperature, proportion of water, and time of cooking, but above 90C much of the crystallite order is lost through breaking of the intermolecular forces allowing penetration of water (69). Gelatinized starches are digested much more rapidly than raw ones. Starch gels are unstable, and B-type crystallites form on standing at cool temperatures. This process is termed retrogradation and results from the reassociation of the linear regions of the polymers to form insoluble crystallites containing short linear segments of -(134)-linked glucose units (122) that resist enzymatic hydrolysis (269). Retrogradation occurs when foods are cooked and then cooled. Generally, there is a negative relationship between the amylose content of a starch and the onset and peak minimum temperature of gelatinization (109). High amylose starches (such as those from various maize cultivars) require higher temperatures and pressures fully than high amylopectin starches to be gelatinized (69). Thus high amylose starches are intrinsically more resistant than those higher in amylopectin and retrograde more readily. Starch processing is important as repeated autoclaving and cooling increases the amount of resistant starch and slows starch digestion overall (108). B. Classication of RS The name resistant starch was coined to describe the incomplete digestion in vitro of starch in foods that had been cooked and then cooled (29) but now includes all starch and starch degradation products that resist small intestinal digestion and enter the large bowel in normal humans (16). It is dened strictly in terms that exclude the small intestine. Small intestinal amylolysis can occur at different rates, but only incompletely digested starch can contribute to RS (310). RS was originally classied into three main types (94), but a fourth has been added (42) (Table 6). RS1 includes that trapped within whole plant cells and food matrices (e.g., partly milled grains and seeds) where there is a physical barrier

TABLE

6. Nutritional classification of resistant starches

Examples of Occurrence

Types of Resistant Starch

RS1; physically inaccessible RS2; resistant granules RS3; retrograded RS4; chemically modied

Partly milled grains and seeds Raw potato, green banana, some legumes, and high-amylose starches Cooked and cooled potato, bread, and cornakes Etherized, esteried, or cross-bonded starches (used in processed foods)

[As classied by Brown et al. (42) and Englyst et al. (94).]

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to amylolysis. The presence of intact cell walls contributes to the RS content of legumes. More extensive milling (and chewing) can make these starches more accessible and less resistant. RS2 comprises those granules from certain plants that are gelatinized poorly and hydrolyzed slowly by -amylases (e.g., raw potato and green banana, high amylose maize starch). RS3 comprises retrograded starches, and examples are cooked and cooled rice or potato. RS4 comprises the chemically modied starches (e.g., ethers or esters) that are used by food manufacturers to improve the functional characteristics of the starch. Although these modied starches are found very widely in processed foods, neither their contribution to RS intakes nor their physiological effects have been studied widely. Studies in vitro with puried pancreatic amylases have shown little effect of chemical modication on rate of hydrolysis of tapioca starch but a lower degree of hydrolysis of waxy maize corn distarch adipate (168). Several long-term studies (80, 141, 297) suggested that modied starches could function as RS as judged by increased cecal digesta masses, consistent with escape of carbohydrate into the colon. In vitro incubations have shown that modied starches resist amylolysis in proportion to the degree of substitution (336). More recent studies by Raben et al. (234) have examined the blood glucose responses in humans after consumption of starches modied by -cyclodextrinisation or by acetylation. They found lower values with the latter compared with control starches, suggesting diminished small intestinal hydrolysis. A preliminary report has shown that acylated starches function as RS and raise large bowel SCFA in rats through release of the esteried acid and fermentation of the starch (13). However, the type of modication may be important because Ebihara et al. (85) reported that hydroxypropylated starch raised cecal digesta but not SCFA. Evidently, determining the effects of the different types of RS (including RS4) in whole foods and mixed diets is important. C. Determination of RS in Foods RS has been measured chemically as that starch not hydrolyzed after 2 h of incubation at 37C with pancreatin (containing -amylase plus proteolytic and lipolytic enzymes), amyloglucosidase, and invertase (94). These conditions approximate those in the human small intestine. The assay has been validated in extensive ileostomy studies, although there is individual variation of 74 126% of RS starch fed, depending on the food source (95, 271). Berry (29) and Muir and ODea (210) used incubation at 37C for 1516 h, with the latter providing in vivo validation (through parallel measurement in ileostomists). As yet, there is no physiologically relevant measure of RS for foods as they are eaten, and any analysis needs to allow

for the presence of other inhibitory food components (amylase inhibitors, lipids, NSP, etc.) as well as physiological variables including chewing and individual variation in transit (13, 55, 271). Sample pretreatment through milling, grinding, or chewing can all inuence the nal outcome. One way to encompass these factors is to use standardized mincing (94). When this is done, the results correlate highly with the amount of starch recovered in ileostomy efuent (which is one of the few means of assessing starch digestion in humans in vivo). However, the method is labor intensive and gives poor reproducibility unless conducted by highly trained personnel and so has not been adopted widely outside Britain. Muir and co-workers (209, 210) have developed a more physiological procedure that mimics chewing and gastric digestion. Their in vitro data correlate reasonably well with the starch recovered in ileostomy efuent. The range of foods they have examined was quite small, the amount of resistant starch fed was low, and the number of volunteers used was restricted. kerberg et al. (5) have modied the Muir procedure (to standardize the chewing step) and also extended the range of foods examined and obtained good agreement (r 0.97) between values measured in foods in vitro and in vivo. Clearly, such a suitable methodology to measure RS needs to be applied widely so that the RS content of foods and the RS intakes of individuals and groups can be estimated with condence. VII. RESISTANT STARCH IN THE LARGE BOWEL: COMPARISONS WITH NONSTARCH POLYSACCHARIDES A. Relative Contributions of RS and NSP to Large Bowel Carbohydrate Supply 1. Evidence for incomplete starch digestion in humans and estimates of RS intake The carbohydrate gap is the discrepancy between NSP intakes and calculations of bacterial activity of the large bowel microora (281) and supports a signicant contribution by RS. Individuals in afuent westernized countries may consume up to 28 g NSP/day (22). However, much larger quantities, possibly as much as 80 g, of fermentable carbohydrate are needed to sustain the biomass and account for SCFA production (281), and NSP may only provide 25% of that requirement (139). With the use of portal venous SCFA data from patients and estimates of human portal venous blood ow, production rates of 163 mmol/day (fasting) and 353 mmol/day (postmeal) have been estimated (73). Livesey and Elia (175) calculated a maximal yield of 0.6 g SCFA/g carbohydrate fermented. Using this value, Cummings (73) estimated that 32 42 g of carbohydrate would need to be fer-

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mented daily to produce 300 400 mmol of SCFA, which would contribute 2 4% of daily energy. More direct measures are possible in animals. Using an electromagnetic meter to measure portal venous blood ow, SCFA were shown to provide 30% of the total energy requirements of a 62-kg pig fed twice daily (239). When pigs were fed single meals sufcient to provide 50% of their daily energy needs, net SCFA absorption was 800 1,429 mmol/ day (123, 239). This translates to 120 g carbohydrate/ day, which is higher than those estimated in humans. In pigs fed a diet containing 6% cellulose as the ber source, the quantity of SCFA transported (1.18 mol) was considerably greater than that which could be supplied by fermentation of the 48 g of ber in the diet. In humans, RS and OS could close the carbohydrate gap (274), but consumption of OS appears to be selflimiting due to osmotic effects and may contribute only 510 g/day. Direct evidence that a physiologically signicant amount of starch reached the terminal ileum (and could enter the colon) was shown in intubated volunteers (283). Substantial quantities of starch (and other macronutrients) were found in ileal efuent after consumption of certain foods such as beans and high amylose starch. Thus, in a highly digestible food such as white bread, only 2.8% of available carbohydrate (i.e., starch) appeared in the efuent compared with 13.8% with lentils and 22.6% with high amylose bread (279). The ber content of the food was found to be an important determinant of digestibility, and greater ber content also increased ileal protein losses. Muir et al. (209) compared high and low RS meals and showed that of meals containing 52 g of starch, 4% (1 g) was undigested with low RS food and 48% (25 g) with high RS food. Silvester et al. (271) showed that RS intakes varied from 0.4 to 34.8 g/day, depending on dietary starch content and type. Each type of RS classication has discrete inuences on the quantity entering the large bowel. For RS1, which is starch physically entrapped within the food, the degree of milling is an important factor (172, 210). The comparison made by Muir et al. (209) was of a mixture of bread cooked with high-amylose starch, uncooked green banana our, and coarsely ground uncooked wheat (high RS) versus cooked, low-amylose starches (low RS). RS13 are subject to a range of inuences that could change their levels in processed foods. Cooking conditions are very important in determining the amounts of RS2 and RS3 through gelatinization and retrogradation. Chewing also decreases the amount of RS present by reducing particle size (210). Rapid small intestinal transit time is likely to deliver more starch into the large bowel (271), and smaller particles have slower rates of small intestinal passage than do large ones (137). The importance of this variable has been conrmed in cannulated pigs in which starch concentrations were equally higher when they were fed coarse brown or white rice as opposed to ne brown or white

rice (32, 184). Even though high levels of NSP (as occurs in brown rice) appear to inhibit small intestinal amylolysis (279), the pig data suggest that particle size is as important. Modied starches (RS4) appear to survive the conditions that affect the other forms of RS (336), but this is yet to be established in vivo. Although the absolute quantity of dietary NSP entering the normal human cecum and colon via the ileocecal valve can be calculated from intakes, this is not possible for RS. The RS content of most foods has not been determined, and precise values of intake for individuals consuming mixed diets are not available. Brighenti et al. (40) have made one such estimate and calculated that Italian diets provided only 8.5 g RS/ day, which appears to be a low value relative to other estimates. Overall, it seems that as more starch is eaten, more enters the colon (57, 283), and it is thought that 10% of total dietary starch may escape digestion in the human small intestine (10, 92, 105, 106, 279, 283). Clearly, the lack of any quantitative estimate of RS limits comparison between it and NSP. B. RS and Fecal Bulking Dietary ber accelerates transit, promotes laxation (171), relieves constipation (23), and protects against diverticular disease (7). This is achieved through fecal bulking, which is also associated with diminished risk of colorectal carcinoma. Cummings et al. (75) reported that death rates from colonic cancer ranged from 20/100,000 (age standardized) in Scotland to 0.4/100,000 in Uganda. Corresponding daily fecal weights were 7293 g and 470 g. Wheat bran is a most effective fecal bulking agent and raises stool mass by up to 4.9 g/g consumed. Less than 50% of wheat bran is fermented (216), and the passage of fermented plus unfermented material may account for its effectiveness (282). RS is of generally high fermentability, so its fecal bulking action is rather variable and much less than that of wheat bran. Increased stool weight and/or altered bowel habit have been observed in some human studies with starch (e.g., Refs. 74, 153, 267, 225, 314) or acarbose (142, 259, 326). Data from human interventions in which effects of RS on fecal bulking, pH, and SCFA have been investigated are consolidated in Table 7. Holt et al. (142) reported that over 1 yr fecal wet weight was increased from 155 to 221 g/day in individuals consuming acarbose. Other studies have failed to show any effect (106, 298). In part, these inconsistencies may reect the relatively low incremental effect of RS on fecal bulk with 11.7 g additional stool/g RS consumed (74, 214, 225, 314). The variability may reect either the effect of dosage or differences between RS types, while RS fermentation may spare that of NSP (225), which could also contribute to some of the variability through laxation. Cummings et al.

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7. Changes in fecal variables in human interventions with acarbose or RS

Authors Study Design RS Type Diets Fecal Mass, g pH Fecal Total SCFA Fecal Butyrate

(mmol/g wet wt) Scheppach and co-workers (259, 260) Double-blind crossover (n 12 for mass, 11 for SCFA) Acarbose Placebo Acarbose 124a 208a NR 57.6 65.8 12.4a 19.6a

(mmol/g dry wt) Holt et al. (142) Placebo controlled, 1 yr (n 24) Acarbose Placebo Acarbose 155 221a
a

7.0 6.3a

452 435 (mM)

76 76

van Munster et al. (314) Noakes et al. (214) Low-amylose starch

Sequential (n 11) Crossover (n 23)

RS2 RS2

Control RS2 High-amylose starch Oat bran

119 147a 108 100 109

6.6 6.7 6.18a 6.22b 6.40ab

87.8 89.6 119.2* 101.1 100.6

9.1 10.4 31.1ab 23.5a 20.1a

(mmol/kg wet wt) Cummings et al. (74) Randomized crossover (n 12, 510 individuals/treatment) RS2 RS3 R SDS Bran Potato RS2 Banana RS2 Wheat RS3 Maize RS3 110 201 151 161 153 161 NR 98.9a 77.1abc 99.7b 97.5c 83.4 85.7 (mM) Phillips et al. (225) Jenkins et al. (153) Randomized crossover (n 11) Randomized crossover (n 24) RS2 RS2 and RS3 Low RS High RS Low ber Wheat bran RS 138 197a 163a 258ab 185b
a

15.0a 15.8b 18.4abc 15.2c 17.0 14.6

6.3 6.9a NR

79.0a 99.5a 102.8 107.9 108.1

19.0a 26.2a 19.2a 21.3a 22.7b

NR, not recorded. * As sum of acetate, propionate, and butyrate reported by authors. R SDS rapidly slowly digestible abc starch. Values sharing a common superscript are signicantly different (P 0.05, minimum).

(74) reported that raw potato and green banana (both RS2) increased stool wet weight by 1.6 and 1.7 g/g, respectively. The effects of RS appear to be highly dependent on source, with green banana RS actually increasing transit time. RS3 fed as wheat or high-amylose maize starch gave increments of 2.4 and 2.7 g/g, respectively. These effects were much less than for NSP, which increased both wet and dry matter while RS increased dry matter only. Overall, RS appears to rank alongside other highly fermentable carbohydrates such as legume NSP and pectin (73), fructo-oligosaccharides (119), polydextrose (2), or inulin (119) in its effectiveness. C. Fermentation and Colonic and Fecal SCFA There are two important issues in relation to RS and SCFA production: the absolute contribution and the acids that are formed. 1. In vitro studies Some studies show that starch favors butyrate production (64, 271, 324, 326). However, individuals differ

with inocula from some consistently producing more propionate than butyrate (323) and from others being incapable of metabolizing some types of RS (74). Martin et al. (185) examined the fermentation of various types of RS with pig inocula and found complete fermentation of wheat and maize starch and nearly complete fermentation of pea and potato starch within 24 h. However, only 87 and 57%, respectively, of an RS2 and the same starch after retrogradation (RS3) were fermented. Signicantly more SCFA were recovered from maize starch (340 mM) than from wheat starch (297 mM) despite complete fermentation. Substantially more butyrate was formed from potato starch (25% of total SCFA) and the RS2 (23%) than from the RS3 (14%) and maize starch (14%). All of these data show that a blanket assumption that fermentation of starches is complete and yields more butyrate is unjustied. The data may need to be revisited because some of the incubations were with starch alone. Feeding trials (especially with humans and pigs) are conducted with RS plus NSP, and it is known that SCFA prole obtained with mixtures differs from that with individual carbohydrates. The role of altered bile acid excretion also needs to be determined.

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2. Human breath gas measurements There have been relatively few reports of RS and breath gas evolution, and they are equivocal and tend to dissociate breath gases from other colonic events. Achour et al. (2) showed a rise in breath H2 that correlated with an increase in plasma acetate in humans consuming RS. Conversely, Flourie et al. (106) found that the amount of breath H2 produced was not related to the amount of starch infused into the cecum in human volunteers. Tomlin and Read (298) fed humans with corn akes containing 3% RS3 and found a substantial increase in breath H2 evolution but no change in fecal variables. Van Munster et al. (313) showed that when healthy volunteers consumed 28 g RS2, 24-h integrated production of breath H2 was increased relative to a control. The increment was greater in methane nonproducers, but methane evolution was increased also in methane producers. Poppitt et al. (230) examined the relationship between the amount of resistant starch ingested and the amount of hydrogen and methane excreted. Healthy men were fed a diet containing either 16 or 38 g NSP together with 16 or 19 g RS. H2 production increased nonsignicantly, but CH4 decreased. As noted, South African blacks consuming a high maize corn diet (likely to be a mixture of RS2 and RS3) had substantially higher breath gas evolution than whites with a higher ber intake (220). However, studies with a similar mixture in a population of North American volunteers showed no change in breath gases (153). This may reect the quantity of RS consumed, indicating that dose responses need to be established, preferably coupled with measures of SCFA production. However, this is not the only reason because high-amylose starches (known to raise colonic SCFA in animals and fecal SCFA in humans) did not raise breath H2 in volunteers (221). These discrepancies underscore the unsatisfactory nature of breath gas tests in estimating fermentation and show that the absence of greater gas evolution after RS consumption does not indicate unchanged SCFA production. 3. Human fecal measurements With one exception (298), RS consumption increased fecal SCFA and butyrate concentration and/or excretion. For example, Scheppach et al. (260) showed an increase in both butyrate concentration (Table 7) and excretion with consumption of acarbose. In studies with RS2, van Munster et al. (314) showed only the former, and Noakes et al. (214) the latter. Again, the disparity may reect either the dose or type of RS and also differences in transit (which could inuence absorption within the colon). Time of adaptation may also be important as Holt et al. (142) showed in an extended feeding trial with acarbose. Quite different values for fecal SCFA were obtained at 6 and 12 mo. Furthermore, it is not clear whether the

presentation of fecal values as concentrations or excretion rates is more appropriate. In a study where several forms of RS were consumed by volunteers, fecal bulk was increased by raw potato starch, green banana our (RS2), retrograded high-amylose maize starch and retrograded wheat starch (RS3), and by fully digestible starch fed with wheat bran (control). Maize RS3 did not alter fecal total SCFA concentrations from controls with respective mean values of 85.7 and 80.2 mmol/kg (74). If the data were to be expressed on daily output, these would diverge to 8.6 and 13.8 mmol/day, respectively. A similar difference emerges in the study by Holt et al. (142) where SCFA output increased with acarbose treatment while fecal SCFA concentrations remained unchanged. In the study by van Munster et al. (314), indices of fecal water cytotoxicity and colonocyte proliferation were lowered by the RS diet with no change in butyrate concentration. Total fecal excretion may be the more relevant measure, especially as greater fecal bulk is associated with lower risk of colorectal cancer. A contribution by RS to fermentation and not direct fecal bulking is supported by the increased excretion of bacterial dry mass from 6.3 g/day in controls to 11.3 g/day with acarbose (259). RS resembles highly fermentable NSP and OS and contributes to greater fecal microbial mass and SCFA. RS (as RS2) may also increase fecal butyrate, but the effect of the other forms needs to be investigated systematically. Only one published study has investigated effects of a high starch and RS diet (as opposed to specic foods containing RS). Total starch and resistant starch were increased by three- and sixfold with a simulated Chinese diet compared with a simulated Australian diet (211). This did not lead to putatively benecial fecal outcomes, but the converse. Fecal mass (86 vs. 141 g/day) and SCFA concentrations were lower (Table 7), and fecal ammonia and phenols were higher on the Chinese diet (211). This study needs to be compared with that of Takahashi et al. (289), which showed that when Japanese subjects ate a similar high-starch diet, fecal SCFA were higher than on a self-selected diet (Table 7). The difference may reect the relatively short duration of such interventions which last for 3 4 wk, which may be insufcient time for microbial adaptation. This adaptation is important for rice as fecal outputs and starch fall with time in pigs fed brown rice, a change attributed to the microora (32). During the rst week of feeding a simulated Indonesian diet, fecal wet weight and starch excretion were 365 and 2.5 g/day, respectively, when the diet was high in RS (as brown rice). Corresponding values were 247 and 0.8 g/day during week 2. Stool output (226 and 228 g/day, weeks 1 and 2) and starch excretion (0.8 and 0.8 g/day, weeks 1 and 2) stayed constant in pigs fed white rice at the same level of ber, present as rice bran.

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Animal studies have shown conclusively that feeding of RS of all types raises large bowel total SCFA. In rats, this has been shown through the feeding of potato, pulse, high-amylose corn, and chemically modied starches where cecal SCFA concentrations and cecal bulk and SCFA pools are increased. The role of microora has been conrmed by the low SCFA values recorded in germ-free rats fed RS (12). Studies in pigs have shown similar results with greater large bowel SCFA concentrations and pools. Most studies show relative increases in butyrate, but an increase in propionate has been reported in rats fed a high-amylose maize starch (12) and in pig feces (43, 301). Greater fecal SCFA excretion has been shown in pigs fed RS (32, 43, 301). In keeping with the large bowel data, the increase was in butyrate excretion in pigs fed brown rice (32) and propionate in pigs fed high-amylose maize starch (43, 301). The latter also suggested that RS2 could be fermented rapidly when compared with other types of RS. If so, this could be an important determinant of SCFA distribution along the colon. The effects of RS on total SCFA in these experiments are comparable to those of puried NSP and ber-rich foods, while wheat bran is the product that raises butyrate consistently. However, there are data which show also that RS types differ and RS3 may be more effective than RS2 in promoting expansion of the biomass in pigs (134). A preliminary report has shown that starches acylated with a specic SCFA raise that acid preferentially in the large bowel of rats (13). This offers the opportunity to target delivery of individual acids to the colon. D. NSP, RS, SCFA, Colonic Cell Proliferation, and Colorectal Cancer Risk Further epidemiological studies have supported the earlier studies in Africans and shown that overall cereal consumption protects against colorectal cancer (139). However, evidence of a discrete benet of ber in colorectal cancer is not strong (139). Trock et al. (309) reviewed 39 epidemiological studies of diet and colorectal cancer risk and found that ber intake was protective in only 50% of them. Of the latter, the effect achieved significance in only 45% of studies, conclusions similar to those of Cassidy et al. (53). Relative risk of colorectal cancer did not vary with energy-adjusted ber intakes from 9.8 to 24.9 gperson1day1 in a large (88,000) cohort of American women followed over 16 yr (113). Human intervention studies also have failed to show consistent effects. In one trial (The Australian Polyp Prevention Project), subjects were asked to follow a low-fat, high-ber diet, but there was no difference in polyp recurrence compared with those eating a normal western diet, although the former diet inhibited the transition from smaller to larger

adenomas (180). A recent intervention study in which patients were given a supplement of wheat bran ber at 13.5 or 2 g/day showed no difference in the recurrence of adenomas with recurrence in 47% of the former and 51% of the latter (6). Kashtan et al. (154) fed wheat bran or oat bran to 45 polyp patients and 49 polyp-free volunteers and found no change in thymidine colorectal crypt-cell labeling before and after intervention. Whether patients with colorectal adenomas or carcinomas have enhanced proliferation is not established rmly despite several claims of positive association (82, 174, 292). A study using PCNA as a measure of proliferation found no differences between mucosa from normal patients and mucosa from patients with neoplasia (112). In ascending colon biopsies incubated in vitro, 10 mM sodium butyrate, ammonium butyrate, and butyric acid increased crypt labeling indices. The index was lowered with calcium butyrate, and deoxycholic acid-induced proliferation was abolished by sodium butyrate (26). Starch is an important nutrient, and intakes vary widely across countries ranging from 150 gperson1 day1 in afuent westernized countries to 350 gperson1day1 in societies consuming traditional agrarian diets (41, 53). Analysis of nutrient intakes indicates a negative relationship between total starch consumption and large bowel neoplasia (280, 296, 309). A meta-analysis of studies across 12 countries worldwide showed no relationship between dietary NSP and large bowel cancer risk (r 0.23, NS, men and women combined) (53). In contrast, there was a strong inverse correlation (r 0.70, P 0.001) between starch intake and risk that extended to calculated intakes of RS (r 0.52, P 0.05). These data accord with the ndings that low-risk populations such as the Japanese (161) or South African blacks (220) eat relatively little ber, whereas high-risk populations (e.g., Australians) have quite high NSP intakes but eat relatively little starch (22). South African blacks ate only 43% of the recommended daily intakes of ber but consumed substantially more maize starch than high-risk whites living in the same region (220). Fecal SCFA values were similar in both groups, but fasting and peak postmeal breath H2 and CH4 evolution was higher in blacks. The respective mean fasting values for both gases were 11.5 and 18.3 ppm in blacks and 6.2 and 8.0 ppm in whites. Rectal mucosal biopsies also showed lower epithelial cell proliferation in the blacks. The protective mechanism of starch (or RS) is presumed to be through SCFA production due to its high fermentability. RS is trophic in rats as measured by greater cecal wall weight in animals fed RS2 compared with animals fed a highly digestible starch (12, 125). However, data from pigs suggest that RS types may vary with longer colons in pigs fed RS2 (301) compared with controls or animals fed RS1 (32, 184, 303). However, the exact epidemiological relationship between these variables and

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human colorectal carcinomas and adenomatous polyps is unclear due to the absence of systematic dietary RS and SCFA information. Data from cancer patients and interventions also are inconclusive. A brief report by Vernia et al. (319) indicated that fecal acetate was signicantly lower in cancer patients (59.7 mM) than in those with polyps (79.6 mM) or in controls (89.2 mM). Butyrate concentrations were not signicantly different between those with cancer (12.5 mM) and the other two groups (20.9 and 19.3 mM). Weaver et al. (322) showed that there were no consistent differences in SCFA between patients presenting for sigmoidoscopy for various reasons (including cancer) except for a higher molar contribution by butyrate in the controls (17.3%) compared with polyp/ colon cancer patients (12.3 and 11.2%). Kashtan et al. (154) found no difference in fecal butyrate between controls and postpolypectomy patients except that fecal butyrate was lower in the latter following adaptation to a wheat bran supplement (16 vs. 6 mM). Thornton et al. (294) reported than in such patients 6 g starch/day reached the colon as opposed to 10 g/day in controls. This could explain both the variable human SCFA data and the lower in vitro butyrate production rates by fecal inocula from these patients (65). However, this difference in starch digestion has not been conrmed (215). A decreased incidence of bowel cancer with lower pH has been reported (181, 320) but not conrmed (232). Upper crypt proliferative indices were lower in biopsies taken from humans consuming acarbose (in whom breath H2 and fecal butyrate were raised) compared with those consuming placebo (142). h was unaltered by treatment, but there was a strong negative correlation between butyrate levels and rectal crypt proliferation. Further support for lowered indices of risk came when consumption of RS2 by humans lowered fecal secondary bile acid concentration (314). Colonic mucosal proliferation in rectal biopsies (measured with PCNA) decreased from 6.7 to 5.4%, while in vitro fecal water cytotoxicity also was lowered. The relationship between steroids, large bowel SCFA, and cancer risk may be important because secondary bile acids (such as deoxycholate) are thought to promote neoplasia. If lower bile acid excretion were to favor butyrate production and lower the cytotoxic potential of bile acids, there could be a stronger effect than for either change alone. RS increases fecal butyrate, and RS3 lowers cholic acid excretion by 42% in human ileostomists (166). Lower tumor numbers and/or burdens have been observed in rats treated with carcinogens when they were fed insoluble NSP such as cereal brans (107, 191, 192, 343, 346). These reductions were not necessarily related to large bowel SCFA. In contrast, enhancement of carcinogen-induced tumor growth has been seen with soluble and more fermentable bers such as carageenan, pectin, guar, and alfalfa (27, 150, 240), although others have observed a decrease in tumors with guar and pectin (135,

325). In the latter study there was a correlation between butyrate production in the azoxymethane-treated rats and protection from tumors with hydrolyzed guar. In rats exposed to azoxymethane there was a signicant reduction in the number of aberrant crypt foci with a raw potato starch diet (and also in energy intake) compared with a basic, sucrose, or cornstarch diet (295). Contrary evidence was found by Young et al. (343) who demonstrated hyperproliferation, greater density of aberrant crypt foci, and enhanced tumor production with dimethylhydrazine (DMH) using rats fed 20% of carbohydrate as raw potato starch. Wheat bran in addition to the potato starch reduced the enhanced tumor production but not the hyperproliferation. Sakamoto et al. (253) found no effect of resistant starch (10% by weight from high-amylose maize starch hydrolyzed with pancreatin) on tumor volume in the same model even though butyrate levels rose 1.5-fold in the cecum and 2-fold in feces. In these animals, cellulose halved cancer volume with no fecal SCFA changes, as was reported earlier by Heitman et al. (136). Cassand et al. (52) found that RS (type unspecied) reduced aberrant crypt foci in the DMH rat model. In this experiment there were large changes in fecal and cecal weight, with cecal butyrate being 2.6-fold higher and fecal butyrate 4.5-fold higher. In the Min mouse, most of the tumors are located in the small intestine, which raises questions about its relevance to humans. Despite this and the fact that it resembles only those humans who develop 1% of total human colorectal tumors, its tumors are susceptible to cyclooxygenase inhibition (with sulindac) (35) as is human colorectal carcinogenesis (118). In Min mice, neither wheat bran nor RS (from retrograded highamylose starch treated with amyloglucosidase) reduced colorectal tumors, although oligofructose did (226). However, Quesada et al. (233) have shown in the APC gene 1309 knockout mouse that acarbose treatment did not alter whole gut tumor multiplicity signicantly. Gastric and large bowel tumor numbers were lowered by acarbose, but only the former was signicant. Acarbose treatment decreased the number of tumors of diameter 3 mm from 3.78 (controls) to 2.36. Pierre et al. (226) examined effects of diet on intestinal tumors in Min mice and found most were in the small intestine and 7% in the large bowel. Neither wheat bran nor RS altered large bowel tumor numbers (2.1, 3.0, and 1.5 in control, RS, and wheat bran, respectively). Only OS reduced the number signicantly to 0.7/mouse. These ndings suggest that extrapolation from small intestinal tumors (which are very rare in humans) to colorectal tumors is not justied. This may apply also to the data obtained with carcinogens (AOM and DMH) because substantial numbers (0 50% of total, depending on diet) of tumors can occur in the small intestine (191), possibly reecting reingestion of the carcinogen. There is also the possibility that such treatment could alter the microbial population and so inuence the

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results. Maciorowski et al. (179) showed that the injection of AOM altered the fecal bacterial population leading to higher anaerobe counts than controls when they were fed pectin. The opposite occurred with cellulose. The issue of the difference between rodents and other species may be involved because SCFA are associated positively with cecal but not distal colonic proliferation in rats (344). Based on some of these negative or inconclusive studies, it has been suggested that the safety of RS may be doubtful (95, 98). This is a radical response to inconclusive animal data, especially given the epidemiological data showing negative associations between starch intake and colorectal cancer rates and human studies which show generally favorable changes in risk indices, including proliferation. The evidence from animal studies is more consistently negative for soluble bers and cancer risk. Many of these NSP lower cholesterol by enhancing bile acid excretion (299), and Wasan and Goodlad (321) have even suggested that they may increase cancer risk in humans. Altered bile acid excretion could contribute to the data in rodents. In rats, RS2 lowers plasma cholesterol through enhancing fecal steroid excretion (187), which would be expected to enhance experimental tumor formation. However, RS2 does not affect plasma cholesterol in humans (153, 214) and actually lowers steroid excretion. On the basis that RS raises butyrate and lowers steroid excretion, it would not be expected to contribute to human colorectal cancer risk. E. Potential Adverse Reactions: RS as a Malabsorbed Carbohydrate By analogy with lactose, undigested starch could be regarded as a malabsorbed carbohydrate (13). It appears that in underdeveloped areas such as Myanmar (formerly Burma), starch malabsorption (measured through breath H2 evolution) is common, with up to 70% of children being affected (34). In these areas, hygiene is inadequate and it appears that small intestinal bacterial overgrowth is a contributor to the syndrome, with 33% of rice malabsorbers being affected compared with 4.5% of absorbers (155). In contrast to malabsorbed lactose and similar sugars, there appears to be no adverse impact of RS on gastrointestinal function in well-nourished people with adequate food and personal hygiene. Moreover, in children with diarrheal disease, RS has been shown to promote recovery. VIII. CONCLUSIONS AND FUTURE DIRECTIONS Figure 3 provides an overview of the relationships between transit through the human gastrointestinal tract and the digestion of nutrients. Comminuted food (i.e., food that has been rendered more digestible by processing, chewing in the mouth, and wetting in the stomach)

enters the small intestine where enzymatic digestion occurs. Unabsorbed food components enter the large bowel into a zone of high fermentative activity where SCFA levels are high. On passage of the digesta stream, fermentative substrate becomes depleted, and SCFA values fall due to absorption. Voided feces contain undigested food components, endogenous secretions, and biomass. Comparison of the actions of NSP and RS should be viewed in the context of this passage, which is a balance between passage and fermentability, which helps to explain why their effects are markedly different in some important respects. Fiber-rich foods, especially those high in insoluble NSP, are less fermentable than RS and are wellestablished laxating agents. Experimental studies have shown that they protect against chemically induced tumors in rodents. Paradoxically, the epidemiological data for a protective role for NSP in human large bowel cancer are weak, and any protection afforded at the population level is not great; water-soluble NSP may promote risk. In contrast, effects of RS on fecal bulking and laxation are inconsistent except when starch digestion is impaired specically by acarbose. Some NSP and most RS are highly fermentable by the large bowel microora, and there are data suggesting that RS fermentation favors butyrate production, but other results indicate that this is not uniformly true. The greatest difference between RS and NSP lies in relation to cancer, where the protection by RS in animals with chemically induced cancer is weak but the supportive epidemiological evidence is stronger. Indeed, it appears that some of the low-risk African populations that gave rise to the ber hypothesis (67, 300) consume diets low in ber but high in starch. If this is so, then large-scale interventions with high-starch (or highRS) diets may be needed to ascertain if they are protective against cancer in patients at risk. Animal and human interventions with RS usually show improvements in indices of risk, while population studies associate greater starch (but not necessarily RS) intake with substantially lower disease incidence. A major conclusion lies in the area of analytical technology that is well advanced for NSP with convenient enzymatic-gravimetric measures for ber in which the foods are digested with enzymes (amylase, protease) to digest nonber components (17). Recent developments, including the application of liquid chromatography, offer to make this procedure more rapid (217). Interestingly, enzymatic-gravimetric methods measure a fraction of RS that is included in the overall ber gure. This means that, depending on analytical procedure, some of the reported effects of ber include a contribution by RS. Appropriate analyses (including steps to mimic mastication) have been developed for RS but need to be applied widely. Measures of intake at the population level remain inadequate, and it is a priority to develop the methodology further so that it can be applied widely. Any analytical

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FIG. 3. An overview of the relationship between transit of food through the human gastrointestinal tract and the digestion of nutrients in the small intestine and fermentation in the cecum and colon. Foods are rendered digestible (comminuted) by processing (milling, cooking) and by mastication in the mouth. Digestion is also enhanced by wetting and macerating in the stomach. In the small intestine, digestion occurs through the action of intrinsic enzymes, and nutrients are absorbed. Food components and endogenous secretions not absorbed in that viscus pass through the ileocecal valve and are fermented. Fermentation is high in the proximal large bowel as is the SCFA production. Absorption of SCFA and of water and minerals (including calcium) is high in this viscus. On passage of the fecal stream, fermentation declines through substrate depletion, and SCFA values fall. The distal large colon and rectum are the regions of the large bowel with the most limited supply of SCFA and are the site of most pathology. Bacteria and unfermented components of low fermentability are voided in the feces.

procedure needs to accommodate the different types of RS, and only when this has been done will it be possible to assess how much RS is actually being consumed by animals and humans. Then, this will enable a realistic assessment of the relationships between RS, colonic metabolism, and disease risk. The methodology to measure RS and NSP fermentation in vitro by digesta and fecal inocula seems to be established, and a major European collaboration has shown one way to maximize resources to determine SCFA production from different substrates and by different population groups. The factors that control starch fermentation and the products in vitro remain to be identied. Human experimentation is constrained by the ethical and logistic problems associated with accessing the human large bowel, so direct measures are difcult to obtain. Ileal starch digestibility can be determined in human ileostomists, but in situ determination of SCFA metabolism by intubation seems to be too difcult for routine application. Fecal measures of SCFA and other variables are valid but do not allow for continuous sampling and have yet to be applied at the population level. Indirect measures of SCFA production (breath gas evolution, peripheral blood SCFA) can be obtained in real time but are not very informative beyond indicating overall

changes in fermentation. Animal experimentation remains necessary and, although rodents are used commonly, some of the data obtained appear of questionable relevance to humans. Other omnivores such as pigs and dogs seem to be more appropriate but pose ethical questions. Greater standardization of the experimental conditions is desirable. A major deciency is lack of knowledge of the relationships between diet, SCFA production, and the large bowel microora, especially in relation to the distribution of SCFA in the colon and risk of disease. This is crucial because effects of RS appear to be largely through fermentation products and not physical bulking. Some types of RS can enhance production of specic SCFA, including butyrate, but the relationship needs to be dened more thoroughly. Current evidence suggests that a signicant number of individuals lack the capacity to ferment RS1, RS2, and RS3. Limited data indicate that rates of SCFA production and the molar ratios of the major acids may be characteristic of an individual and are not inuenced by diet. These issues need to be claried. Quantitative fecal cultures should be combined with measures of fermentation to elucidate which organisms are in low numbers or missing when some forms of RS (e.g., RS3) are not fermented. The advent of newer, less labor-intensive tech-

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nologies for bacterial identication and enumeration should enable this information to be gathered. There is no evidence that RS4 (or any other form of RS) has any adverse effects on fermentation or large bowel physiology, and acylated starches have the potential to deliver specic SCFA to the colon. Through the production of SCFA, RS has specic benets, e.g., management of infectious diarrhea, but the actual effectiveness needs to be established. This is especially relevant in the long-term prevention of serious diseases such as colorectal cancer. The suggestion that RS may increase large bowel cancer risk needs investigation, although it is not supported well by the epidemiological or human intervention data (which point to a protective role). Indeed, some of the doubts about RS and cancer risk may only be a consequence of the choice of experimental models (rats and mice). Other areas needing investigation include the transition from milk (breast or formula) to solid foods, especially adaptation to starches. It is not clear if low doses of resistant starch (10 20 g/day) affect colonic metabolism enough to modify disease risk. Clearly, dose-response studies are required, especially at higher levels of intake than those studied hitherto and whether any increase in intakes should be as total starch, RS or RS plus starch. Interventions (including the feeding of mixtures of RS of different types) would also show whether RS has a fecal bulking action at high levels of intake. Although recommendations have been made for ber intake, it does not seem possible to do so for RS at present. In making such recommendations, it will be useful to make comparison between RS and NSP. This could be achieved by dening of RS in terms of ber equivalents (67, 300), i.e., expressing actions of RS relative to ber preparations of established effectiveness at known dose levels, e.g., wheat bran for bulking or isolates such as pectin for fermentation.
Drs. Tony Bird, Ivor Dreosti, and Ian Brown as well as Roger King made very helpful suggestions to the writing of this review. The contribution by D. L. Topping is in affectionate memory of Raymond A. Weller, who did a great deal to develop and sustain interest in this important subject. Address for reprint requests and other correspondence: D. L. Topping, CSIRO Health Sciences and Nutrition, PO Box 10041, Kintore Ave., Adelaide BC 5000, Australia (E-mail: [email protected]).

REFERENCES
1. ABRAHAMSE SL, POOL-ZOBEL BL, AND RECHKEMMER G. Potential of short chain fatty acids to modulate the induction of DNA damage and changes in the intracellular calcium concentration by oxidative stress in isolated rat distal colon cells. Carcinogenesis 20: 629 634, 1999. B, BRIET F, FRANCHISSEUR C, BORNET F, CHAMP M, 2. ACHOUR L, FLOURIE RAMBAUD JC, AND MESSING B. Metabolic effects of digestible and partially indigestible cornstarch: a study in the absorptive and postabsorptive periods in humans. Am J Clin Nutr 66: 11511159, 1997.

3. AGARWAL VP AND SCHIMMEL EM. Diversion colitis: a nutritional deciency syndrome? Nutr Rev 47: 257261, 1989. 4. AHMED R, SEGAL I, AND HASSAN H. Fermentation of dietary starch in humans. Am J Gastroenterol 95: 10171020, 2000. 5. KERBERG AKE, LILJEBERG HGM, GRANFELDT YE, DREWS AE, AND RCK IME. An in vitro method, based on chewing, to predict BJO resistant starch content in foods allows parallel determination of potentially available starch and dietary ber. J Nutr 128: 651 660, 1998. 6. ALBERTS DS, MARTINEZ ME, ROE DJ, GUILLEN-RODRIGUEZ JM, MARSHALL JR, VAN LEEUWEN JB, REID ME, RITENBAUGH C, VARGAS PA, BHATTACHARYYA AB, EARNEST DL, AND SAMPLINER RE. Lack of effect of a high-ber cereal supplement on the recurrence of colorectal adenomas. N Engl J Med 342: 1156 1162, 2000. 7. ALDOORI WH, GIOVANNUCCI EL, ROCKETT HR, SAMPSON L, RIMM EB, AND WILLETT WC. A prospective study of dietary ber types and symptomatic diverticular disease in men. J Nutr 128: 714 719, 1998. 8. MAN P, PETTERSSON P, ZHANG JX, TIDEHAG P, AND HALLMANS G. Starch and dietary ber components are excreted and degraded to variable extents in ileostomy subjects consuming mixed diets with wheat- or oat-bran bread. J Nutr 125: 23412347, 1995. 9. AMAN P, ZHANG JX, HALLMANS G, AND LUNDIN E. Excretion and degradation of dietary ber constituents in ileostomy subjects consuming a low ber diet with and without brewers spent grain. J Nutr 124: 359 363, 1994. 10. ANDERSON IH, LEVINE AS, AND LEVITT MD. Incomplete absorption of the carbohydrate in an all purpose wheat our. N Engl J Med 304: 891 892, 1981. 11. ANDERSSON HB, ELLEGARD LH, AND BOSAEUS IH. Nondigestibility characteristics of inulin and oligofructose in humans. J Nutr 129: 1428S1430S, 1999. 12. ANDRIEUX CE, PACHECO ED, BOUCHET B, GALLANT S, AND SZYLIT O. Contribution of the digestive tract microora to amylomaize starch degradation in the rat. Br J Nutr 67: 489 499, 1992. 13. ANNISON G AND TOPPING DL. Nutritional role of resistant starch: chemical structure versus physiological function. Annu Rev Nutr 14: 297320, 1994. 14. ANNISON G, TOPPING DL, ILLMAN RJ, TRIMBLE RP, AND MCGRATH L. Novel food ingredients for delivering specic short chain fatty acids to the large bowel (Abstract). Proc Annu Meet Nutr Soc Aust 22nd Adelaide Australia 1998, p. 91. 15. ARDAWI MSM AND NEWSHOLME EA. Fuel utilization in colonocytes of the rat. Biochem J 231: 713719, 1985. 16. ASP NG. Resistant starch. Eur J Clin Nutr 46, Suppl 2: S1, 1992. 17. ASP NG. Enzymatic gravimetric methods. In: CRC Handbook of Dietary Fiber in Human Nutrition, edited by Spiller GA. Boca Raton, FL: CRC, 1993, p. 37. 18. AUGERON C AND LABOISSE CL. Emergence of permanently differentiated cell clones in a human colonic cancer cell line after treatment with sodium butyrate. Cancer Res 44: 39613969, 1984. 19. BACH KNUDSEN KE AND HANSEN I. Gastrointestinal implications in pigs of wheat and oat fractions. 1. Digestibility and bulking properties of polysaccharides and other major constituents. Br J Nutr 65: 217232, 1991. 20. BACH KNUDSEN KE, JENSEN BB, ANDERSEN JO, AND HANSEN I. Gastrointestinal implications in pigs of wheat and oat fractions. 2. Microbial activity in the gastrointestinal tract. Br J Nutr 65: 233248, 1991. 21. BACH KNUDSEN KE, JENSEN BB, AND HANSEN I. Oat bran but not -glucan-enriched oat fraction enhances butyrate production in the large intestine of pigs. J Nutr 123: 12351247, 1993. 22. BAGHURST PA, BAGHURST KI, AND RECORD SJ. Dietary bre, nonstarch polysaccharides and resistant starch: a review. Food Aust 48, Suppl: S3S35, 1996. 23. BAGHURST KI, HOPE AK, AND DOWN EC. Dietary bre intake in a group of institutionalized elderly and the effects of a bre supplementation program on nutrient intake and weight gain. Community Health Stud 9: 99 108, 1985. 24. BARNARD JA AND WARWICK G. Butyrate rapidly induces growth inhibition and differentiation in HT-29 cells. Cell Growth Differ 4: 495501, 1993. 25. BARRY JL, HOEBLER LC, MACFARLANE GT, MACFARLANE S, MATHERS S,

July 2001

SHORT-CHAIN FATTY ACIDS AND HUMAN COLONIC FUNCTION

1057

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40. 41. 42.

43.

44. 45.

REED KA, MORTENSEN PB, NORDGAARD I, ROWLAND IR, AND RUMNEY CJ. Estimation of the fermentability of dietary ber in vitro: a European interlaboratory study. Br J Nutr 74: 303322, 1995. BARTRAM HP, SCHEPPACH W, SCHMID H, HOFMAN A, DUSEL G, RICHTER F, AND KASPER H. Proliferation of human colonic mucosa as an intermediate biomarker of carcinogenesis: effects of butyrate, deoxycholate, calcium, ammonia and pH. Cancer Res 53: 32833288, 1993. BAUER HG, ASP NG, DAHLQVIST A, FREDLUND PE, NYMAN M, AND OSTE R. Effect of two kinds of pectin and guar gum on 1,2-dimethylhydrazine initiation of colon tumors and on fecal beta-glucuronidase activity in the rat. Cancer Res 41: 2518 2523, 1981. BERGMAN EN. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev 70: 567590, 1990. BERRY CS. Resistant starch: formation and measurement of starch that survives exhaustive digestion with amylolytic enzymes during the determination of dietary bre. J Cereal Sci 4: 301314, 1986. BERRY RD AND PARASKEVA C. Expression of carcinoembryonic antigen by adenoma and carcinoma derived epithelial cell lines: possible marker of tumor progression and modulation of expression by sodium butyrate. Carcinogenesis 9: 447 450, 1988. BIRD AR, BROWN IL, AND TOPPING DL. Starches, resistant starches, the gut microora and human health. Curr Issues Intest Microbiol 1: 2537, 2000. BIRD AR, HAYAKAWA T, MARSONO Y, GOODEN JM, CORRELL RL, AND TOPPING DL. Coarse brown rice increases fecal and large bowel short-chain fatty acids and starch but lowers calcium in the large bowel of pigs. J Nutr 130: 1780 1787, 2000. BJORNEKLETT A AND JENSEN E. Relationships between hydrogen (H2) and methane (CH4) production in man. Scand J Gastroenterol 17: 985992, 1982. BOLIN TD, MYO-KHIN SOE-AUNG GENGE JR, AND DUNCOMBE VM. Correlation of hydrogen and methane production to rice carbohydrate malabsorption in Burmese (Myanmar) children. J Pediatr Gastroenterol Nutr 22: 144 147, 1996. BOOLBOL SK, DANNENBERG AJ, CHADBURN C, MARTUCCI C, GUO XJ, RAMONETTI JT, ABREU-GORIS M, NEWMARK HL, LIPKIN ML, DECOSSE JJ, AND BERTAGNOLLI MM. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res 56: 2556 2560, 1996. B, DAGAY-ABENSOUR L, POCHART P, GRAMET G, BOUHNIK Y, FLOURIE DURAND M, AND RAMBAUD JC. Administration of transgalacto-oligosaccharides increases fecal bidobacteria and modies colonic fermentation metabolism in healthy humans. J Nutr 127: 444 448, 1997. BOURQUIN LD, TITGEMEYER EC, GARLEB KA, AND FAHEY GC JR. Shortchain fatty acid production and ber degradation by human colonic bacteria: effects of substrate and cell wall fractionation procedures. J Nutr 122: 1508 1520, 1992. BOWLING TE, RAIMUNDO AH, GRIMBLE GK, AND SILK DB. Reversal by short-chain fatty acids of colonic uid secretion induced by enteral feeding. Lancet 342: 1266 1268, 1993. BRUNKHORST C, ANDERSEN C, AND SCHNEIDER E. Acarbose, a pseudooligosaccharide, is transported but not metabolized by the maltosemaltodextrin system of Escherichia coli. J Bacteriol 181: 2612 2619, 1999. BRIGHENTI F, CASIRAGHI MC, AND BAGGIO C. Resistant starch in the Italian diet. Br J Nutr 80: 333341, 1998. BRITISH NUTRITION FOUNDATION. Complex Carbohydrates in Foods. London: Chapman and Hall, 1990, p. 22. BROWN IL, MCNAUGHT KJ, AND MOLONEY E. Hi-maizeTM: new directions in starch technology and nutrition. Food Aust 47: 272275, 1995. BROWN IL, WARHURST M, ARCOT J, PLAYNE M, ILLMAN RJ, AND TOPPING DL. Fecal numbers of bidobacteria are high in pigs fed Bidobacterium longum with a high amylose (amylomaize) starch than with a low amylomaize starch. J Nutr 127: 18221827, 1997. BUCKLEY BM AND WILLIAMSON DH. Origins of blood acetate in the rat. Biochem J 166: 539 545, 1977. BURKITT DP. Relationship as a clue to causation. Lancet 2: 1237 1240, 1970.

46. BURKITT DP. Some diseases characteristic of western civilisation. Br Med J 2: 274 276, 1973. 47. BUTINE TJ AND LEEDLE JA. Enumeration of selected anaerobic bacterial groups in cecal and colonic contents of growing-nishing pigs. Appl Environ Microbiol 55: 11121116, 1989. 48. BUTLER RN, STAFFORD I, TRIANTAFILLOS E, ODEE CD, JARRETT IG, FETTMAN MJ, AND ROBERTS-THOMSON I. Pyruvate sparing by butyrate and propionate in proliferating colonic epithelium. Comp Biochem Physiol B Biochem 97: 333337, 1990. 49. BUTLER RN, TOPPING DL, ILLMAN RJ, GOLAND G, LAWSON MJ, AND ROBERTS-THOMSON I. Effects of starvation-refeeding on volatile fattyacid distribution in the large bowel of the rat. Nutr Res 10: 9198, 1990. 50. CADERNI G, LUCERI C, LANCIONI L, TESSITORE L, AND DOLARA P. Slowrelease pellets of sodium butyrate increase apoptosis in the colon of rats treated with azoxymethane, without affecting aberrant crypt foci and colonic proliferation. Nutr Cancer 30: 175181, 1998. 51. CAMPBELL JM, FAHEY GC JR, AND WOLF BW. Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal shortchain fatty acids, pH and microora in rats. J Nutr 127: 130 136, 1997. 52. CASSAND P, MAZIERE S, CHAMP M, MEFLAH K, BORNET F, AND NARBONNE JF. Effects of resistant starch- and vitamin A-supplemented diets on the promotion of precursor lesions of colon cancer in rats. Nutr Cancer 27: 5359, 1997. 53. CASSIDY A, BINGHAM SA, AND CUMMINGS JH. Starch intake and colorectal cancer risk: an international comparison. Br J Cancer 69: 937942, 1994. 54. CATS A, DE VRIES EG, MULDER NH, AND KLEIBEUKER JH. Regional differences of physiological functions and cancer susceptibility in the human large intestine. Int J Oncol 9: 10551069, 1996. 55. CHAMP M. Determination of resistant starch in foods and food products: interlaboratory study. Eur J Clin Nutr 46 Suppl 2: S51 S62, 1992. B, BORNET F, PELLIER P, COLONNA P, 56. CHAMP M, MOLIS C, FLOURIE GALMICHE JP, AND RAMBAUD JC. Small intestinal digestion of partially resistant cornstarch in healthy subjects. Am J Clin Nutr 68: 705 710, 1998. 57. CHAPMAN RW, SILLERY JK, GRAHAM MM, AND SAUNDERS DR. Absorption of starch by healthy ileostomates: effect of transit time and of carbohydrate load. Am J Clin Nutr 41: 1244 1248, 1985. 58. CHEN HL, HAACK VS, JANECKY CW, VOLLENDORF NW, AND MARLETT JA. Mechanisms by which wheat bran and oat bran increase stool weight in humans. Am J Clin Nutr 68: 711719, 1998. 59. CHEN WJL, ANDERSON JW, AND JENNINGS D. Propionate may mediate the hypocholesterolemic effects of certain soluble plant bers in cholesterol-fed rats. Proc Soc Exp Biol Med 175: 215218, 1984. 60. CHENG BQ, TRIMBLE RP, ILLMAN RJ, STONE BA, AND TOPPING DL. Comparative effects of dietary wheat bran and its morphological components (aleurone and pericarp-seed coat) on volatile fatty acid concentrations in the rat. Br J Nutr 57: 69 76, 1987. 61. CHERBUT C. Effects of short-chain fatty acids on gastrointestinal motility. In: Physiological and Clinical Aspects of Short-Chain Fatty Acids, edited by Cummings JH, Rombeau JL, and Sakata T. Cambridge, UK: Cambridge Univ. Press, 1995, p. 191. 62. CHERRINGTON CA, HINTON M, PEARSON GR, AND CHOPRA I. Short-chain organic acids at pH 5.0 kill Escherichia coli and Salmonella spp without causing membrane perturbation. J Appl Bacteriol 70: 161 165, 1991. 63. CHOCT M, ILLMAN RJ, BIEBRICK DA, AND TOPPING DL. White and wholemeal ours from wheats of low and higher apparent metabolisable energy differ in their nutritional effects in rats. J Nutr 128: 234 238, 1998. 64. CHRISTL SU, KATZENMAIER U, HYLLA S, KASPER H, AND SCHEPPACH W. In vitro fermentation of high-amylose cornstarch by a mixed population of colonic bacteria. J Parenter Enteral Nutr 21: 290 295, 1997. 65. CLAUSEN MR, BONNEN H, AND MORTENSEN PB. Colonic fermentation of dietary ber to short chain fatty acids in patients with adenomatous polyps and colonic cancer. Gut 32: 923928, 1991. 66. CLAUSEN MR, BONNEN H, TVEDE M, AND MORTENSEN PB. Colonic fermentation to short-chain fatty acids is decreased in antibioticassociated diarrhea. Gastroenterology 101: 14971504, 1991. 67. COBIAC L AND TOPPING DL. Dietary bre: potential role in the aeti-

1058

DAVID L. TOPPING AND PETER M. CLIFTON

Volume 81

68.

69.

70. 71.

72.

73. 74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

ology of disease. In: Encyclopaedia of Human Nutrition, edited by Sadler M, Caballero B, and Strain S. London: Academic, 1999, p. 546. COLLINS JF, HERMAN P, SCHUCH C, AND BAGBY GC JR. c-Myc antisense oligonucleotides inhibit the colony-forming capacity of Colo 320 colonic carcinoma cells. J Clin Invest 89: 15231527, 1992. COLONNA P AND MERCIER C. Gelatinization and melting of maize starches with normal and high amylose phenotypes. Phytochemistry 24: 16671674, 1985. CORREA P AND HAENSZEL W. The epidemiology of large-bowel cancer. Adv Cancer Res 26: 1141, 1978. CREE TC, WADLEY DM, AND MARLETT JA. Effect of preventing coprophagy in the rat on neutral detergent ber digestibility and apparent calcium absorption. J Nutr 116: 1204 1208, 1986. ME SY C, VERMOCOUDRAY C, BELLANGER J, CASTIGLIA-DELAVAUD C, RE REL M, AND RAYSSIGUIER Y. Effects of soluble or partly soluble dietary bres supplementation on absorption and balance of calcium, magnesium, iron and zinc in healthy young men. Eur J Clin Nutr 51: 375380, 1997. CUMMINGS JH. The Large Intestine in Nutrition and Disease. Brussels: Institute Danone, 1997. CUMMINGS JH, BEATTY ER, KINGMAN SM, BINGHAM SA, AND ENGLYST HN. Digestion and physical properties of resistant starch in the human large bowel. Br J Nutr 75: 733747, 1996. CUMMINGS JH, BINGHAM SA, HEATON KW, AND EASTWOOD MA. Fecal weight, colon cancer risk, and dietary intake of nonstarch polysaccharides. Gastroenterology 103: 17831789, 1992. CUMMINGS JH, GIBSON GR, AND MACFARLANE GT. Quantitative estimates of fermentation in the hind gut of man. In: International Symposium on Comparative Aspects of the Physiology of Digestion in Ruminant and Hindgut Fermenters, edited by Skadhauge E and Norgaard P. Copenhagen, Denmark: Acta Vet Scand, 1989, vol. 85, p. 76 82. CUMMINGS JH AND MACFARLANE GT. The control and consequences of bacterial fermentation in the human colon. J Appl Bacteriol 70: 443 459, 1991. CUMMINGS JH, POMARE EW, BRANCH WJ, NAYLOR CP, AND MACFARLANE GT. Short chain fatty acids in human large intestine, portal, hepatic and venous blood. Gut 28: 12211227, 1987. DANKERT J, ZIJLSTRA JB, AND WOLTHERS BG. Volatile fatty acids in human and peripheral and portal blood: quantitative determination vacuum distillation and gas chromatography. Clin Chim Acta 110: 301307, 1981. DE GROOT AP, TIL HP, FERON VJ, DREEF-VAN DER MEULEN HC, AND WILLEMS MI. Two-year feeding and multigeneration studies in rats on ve chemically modied starches. Food Cosmetics Toxicol 12: 651 663, 1974. DENG G, LIU G, LU L, GUM JR JR, AND KIM YS. Transcriptional regulation of the human placental-like alkaline phosphatase gene and mechanisms involved in its induction by sodium butyrate. Cancer Res 52: 3378 3383, 1992. DESCHNER EE, LONG FC, HAKISSIAN M, AND HERRMAN SL. Differential susceptibility of AKR, C57BL/6J, and CF1 mice to 1,2-dimethylhydrazine-induced colonic tumor formation predicted by proliferative characteristics of colonic epithelial cells. J Natl Cancer Inst 70: 279 282, 1983. DI LORENZO M, BASS J, AND KRANTIS A. An intraluminal model of necrotizing enterocolitis in the developing neonatal piglet. J Pediatr Surg 30: 1138 1142, 1995. DURAND M AND BERNALIER A. Reductive acetogenesis in animal and human gut. In: Physiological and Clinical Aspects of Short-chain Fatty Acids, edited by Cummings JH, Rombeau JL, and Sakata T. Cambridge, UK: Cambridge Univ, Press, 1995, p. 107. EBIHARA K, SHIRAISHI R, AND OKUMA K. Hydroxypropyl-modied potato starch increases fecal bile acid excretion in rats. J Nutr 128: 848 854, 1998. EDWARDS CA AND EASTWOOD MA. Caecal and faecal short-chain fatty acids and stool output in rats fed on diets containing non-starch polysaccharides. Br J Nutr 73: 773781, 1995. EDWARDS CA, GIBSON GT, CHAMP M, JENSEN BB, MATHERS JC, NAGENGAST F, RUMNEY C, AND QUEHL A. In vitro method for quantication of the fermentation of starch by human faecal bacteria. J Sci Food Agric 71: 209 217, 1996.

88. EDWARDS CA, PARRETT AM, BALMER SE, AND WHARTON BA. Faecal short chain fatty acids in breast-fed and formula-fed infants. Acta Paedatr 83: 459 462, 1994. 89. EDWARDS CM, GEORGE B, AND WARREN BF. Diversion colitis: new light through old windows. Histopathology 35: 86 87, 1999. 90. ELSDEN SR, HITCHCOCK MWS, MARSHALL RA, AND PHILLIPSON AT. Volatile acid in the digesta of ruminants and other animals. J Exp Biol 22: 191202, 1946. 91. ENGELHARDT WV. Absorption of short-chain fatty acids from the large intestine. In: Physiological and Clinical Aspects of ShortChain Fatty Acids, edited by Cummings JH, Rombeau JL, and Sakata T. Cambridge, UK: Cambridge Univ. Press, 1995, p. 149. 92. ENGLYST HN AND CUMMINGS JH. Digestion of the polysaccharides of some cereal foods in the human small intestine. Am J Clin Nutr 42: 778 787, 1985. 93. ENGLYST HN AND CUMMINGS JH. Digestion of the carbohydrates of banana (Musa paradisiaca sapientum) in the human small intestine. Am J Clin Nutr 44: 4250, 1986. 94. ENGLYST HN, KINGMAN SM, AND CUMMINGS JH. Classication and measurement of nutritionally important starch fractions. Eur J Clin Nutr 46, Suppl 2: S33S50, 1992. 95. ENGLYST HN, KINGMAN SM, HUDSON GJ, AND CUMMINGS JH. Measurement of resistant starch in vitro and in vivo. Br J Nutr 75: 749 755, 1996. 96. EVANS AJ, HOOD RL, OAKENFULL DG, AND SIDHU GS. Relationship between structure and function of dietary bre: a comparative study of the effects of three galactomannans on cholesterol metabolism in the rat. Br J Nutr 68: 217229, 1992. 97. FAISANT N, GALLANT DJ, BOUCHET B, AND CHAMP M. Banana starch breakdown in the human small intestine studied by electron microscopy. Eur J Clin Nutr 49: 98 104, 1995. 98. FERGUSON LR AND HARRIS PJ. Do resistant starches as well as dietary bers protect against colorectal cancer? J Environ Pathol Oncol 16: 335341, 1997. 99. FERNANDES R, WOLEVER TMS, AND RAO AV. Increased serum cholesterol in healthy human methane producers is confounded by age. J Nutr 128: 1349 1354, 1998. 100. FINEGOLD SM, SUTTER VL, AND MATHISEN GE. Normal indigenous intestinal ora. In: Human Intestinal Microora in Health and Disease, edited by Hentges DJ. London: Academic, 1983, p. 3. 101. FLEMING SE, CHOI YS, AND FITCH DM. Absorption of short-chain fatty acids from the rat cecum in vivo. J Nutr 121: 17871797, 1991. 102. FLEMING SE, FITCH DM, AND CHANSLER MW. High-ber diets: inuence on characteristics of cecal digesta including short-chain fatty acid concentrations and pH. Am J Clin Nutr 50: 9399, 1989. 103. FLEMING SE, ODONNELL A, AND PERMAN JA. Inuence of frequent and long-term bean consumption on colonic function and fermentation. Am J Clin Nutr 41: 909 918, 1985. 104. FLICK JA AND PERMAN JA. Nonabsorbed carbohydrate: effect on fecal pH in methane-excreting and nonexcreting individuals. Am J Clin Nutr 49: 12521257, 1989. B, FLORENT C, JOUANY JP, THIVEND P, ETANCHAUD F, AND 105. FLOURIE RAMBAUD JC. Colonic metabolism of wheat starch in healthy humans. Effects on fecal outputs and clinical symptoms. Gastroenterology 90: 111119, 1986. B, LEBLOND A, FLORENT C, RAUTUREAU M, BISALLI A, AND 106. FLOURIE RAMBAUD JC. Starch malabsorption and breath gas excretion in healthy humans consuming low- and high-starch diets. Gastroenterology 95: 356 363, 1988. 107. FOLINO M, MCINTYRE A, AND YOUNG GP. Dietary bers differ in their effects on large bowel epithelial proliferation and fecal fermentation-dependent events in rats. J Nutr 125: 15211528, 1995. RK I, ANDERSSON R, LILJEBERG H, SILVERIO J, 108. FREDRIKSSON H, BJO ELIASSON AC, AND MAN P. Studies on -amylase degradation of retrograded starch gels from waxy maize and high-amylopectin potato. Carb Polymers 43: 81 87, 2000. 109. FREDRIKSSON H, SILVERIO J, ANDERSSON R, ELIASSON AC, AND MAN P. The inuence of amylose and amylopectin characteristics on gelatinization and retrogradation properties of different starches. Carb Polymers 35: 119 134, 1998. 110. FREDSTROM SB, LAMPE JW, JUNG HJ, AND SLAVIN JL Apparent ber digestibility and fecal short-chain fatty acid concentrations with

July 2001

SHORT-CHAIN FATTY ACIDS AND HUMAN COLONIC FUNCTION

1059

111.

112.

113.

114.

115.

116. 117.

118.

119.

120.

121.

122.

123.

124.

125.

126.

127.

128.

129. 130.

131.

ingestion of two types of dietary ber. J Parenter Enteral Nutr 18: 14 19, 1994. FREEMAN HJ. Effects of differing concentrations of sodium butyrate on 1,2 dimethylhydrazine-induced rat intestinal neoplasia. Gastroenterology 91: 596 602, 1986. FROMMEL TO, MOBARHAN S, DORIA M, HALLINE AG, LUK GD, BOWEN PE, CANDEL A, AND LIAO Y. Effect of beta-carotene supplementation on indices of colonic cell proliferation. J Nat Cancer Inst 87: 17811787, 1995. FUCHS CS, GIOVANNUCCI EL, COLDITZ GA, HUNTER GA, STAMPFER MJ, ROSNER B, SPEIZER FE, AND WILLETT WC. Dietary ber and the risk of colo-rectal cancer and adenoma in women. N Engl J Med 340: 169 176, 1999. GALLANT DJ, BOUCHET B, BOULEON A, AND PEREZ S. Physical characteristics of starch granules and susceptibility to enzymatic degradation. Eur J Clin Nutr 46, Suppl 2: S3S16, 1992. ME SY C, AND MURAT JC. GAMET L, DAVIAUD D, DENIS-POUXVIEL C, RE Effects of short-chain fatty acids on growth and differentiation of the human colon-cancer cell line HT29. Int J Cancer 52: 286 289, 1992. GEAR JS, BRODRIBB AJ, WARE A, AND MANN JI. Fibre and bowel transit times. Br J Nutr 45: 77 82, 1981. GELISSEN I, ALLGOOD GS, AND EASTWOOD MA. Reproducibility of the breath hydrogen measurement after a low and high ber meal. Eur J Clin Nutr 48: 266 272, 1994. GIARDIELLO FM, OFFERHAUS JA, TERSMETTE AC, HYLIND LM, KRUSH AJ, BRENSINGER JD, BOOKER SV, AND HAMILTON SR. Sulindac induced regression of colorectal adenomas in familial adenomatous polyposis: evaluation of predictive factors. Gut 38: 578 581, 1996. GIBSON GR, BEATTY ER, WANG X, AND CUMMINGS JH. Selective stimulation of bidobacteria in the human colon by oligofructose and inulin. Gastroenterology 108: 975982, 1995. GIBSON GR AND ROBERFROID MB. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr 125: 14011412, 1995. GIBSON PR, MOELLER I, KAGELARI O, FOLINO M, AND YOUNG GP. Contrasting effects of butyrate on the expression of phenotypic markers of differentiation in neoplastic and non-neoplastic colonic epithelial cells in vitro. J Gastroenterol Hepatol 7: 165172, 1992. GIDLEY MJ, COOKE D, DARKE AH, HOFFMANN RA, RUSSELL AL, AND GREENWELL P. Molecular order and structure in enzyme-resistant retrograded starch. Carbohydrate Polymers 28: 2331, 1995. GIUSI-PERIER A, FISZLEWICZ M, AND RERAT A. Inuence of diet composition on intestinal volatile fatty acid and nutrient absorption in unanaesthetized pigs. J Anim Sci 67: 386 402, 1989. GLITSO LV, BRUNSGAARD G, HOJSGAARD S, SANDSTROM B, AND BACH KNUDSEN KE. Intestinal degradation in pigs of rye dietary bre with different structural characteristics. Br J Nutr 80: 457 68, 1998. GOODLAD JS AND MATHERS JC. Large bowel fermentation in rats given diets containing raw peas (Pisum sativum). Br J Nutr 64: 569 587, 1990. GUILLEMOT F, COLOMBEL JF, NEUT C, VERPLANCK N, LECOMTE M, ROMOND C, PARIS JC, AND CORTOT A. Treatment of diversion colitis by short-chain fatty acids. Prospective and double blind study. Dis Colon Rectum 34: 861 864, 1991. HAGUE A, ELDER DJ, HICKS DJ, AND PARASKEVA C. Apoptosis in colorectal tumor cells: induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate. Int J Cancer 60: 400 406, 1995. HAGUE A, MANNING AM, HANLON KA, HUSCHTSCHA I, HART D, AND PARASKEVA C. Sodium butyrate induces apoptosis in human colonic tumor cell lines in a p53-independent pathway: implications for the possible role of dietary ber in the prevention of large bowel cancer. Int J Cancer 55: 498 505, 1993. HAINES AP, IMESON JD, AND WIGGINS HS. Relation of breath methane with obesity and other factors. Int J Obesity 8: 675 680, 1984. HARIG JM, SOERGEL KH, KOMOROWSKI RA, AND WOOD CM. Treatment of diversion colitis with short-chain-fatty acid irrigation. N Engl J Med 320: 2328, 1989. HASS R, BUSCHE R, LUCIANO L, REALE E, AND ENGELHARDT WV. Lack of butyrate is associated with induction of Bax and subsequent apoptosis in the proximal colon of guinea pig. Gastroenterology 112: 875 881, 1997.

132. HEERDT B, HOUSTON MA, AND AUGENLICHT LH. Potentiation by specic short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cells. Cancer Res 54: 3288 3293, 1994. 133. HEERDT B, HOUSTON MA, AND AUGENLICHT LH. Short-chain fatty acid-initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to mitochondrial function. Cell Growth Differ 8: 523532, 1997. 134. HEIJNEN ML AND BEYNEN AC. Consumption of retrograded (RS3) but not uncooked (RS2) resistant starch shifts nitrogen excretion from urine to feces in cannulated piglets. J Nutr 127: 1828 1832, 1997. 135. HEITMAN DW, HARDMAN WE, AND CAMERON IL. Dietary supplementation with pectin and guar gum on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats. Carcinogenesis 13: 815 818, 1992. 136. HEITMAN DW, ORD VA, HUNTER KE, AND CAMERON IL. Effect of dietary cellulose on cell proliferation and progression of 1,2-dimethylhydrazine-induced colon carcinogenesis in rats. Cancer Res 49: 5581 5585, 1989. 137. HELLER SN, HACKLER LR, RIVERS JM, VAN SOEST PJ, ROE DA, LEWIS BA, AND ROBERTSON J. Dietary ber: the effect of particle size of wheat bran on colonic function in young adult men. Am J Clin Nutr 33: 1734 1744, 1980. 138. HILL MJ. Bacterial fermentation of complex carbohydrates in the human colon. Eur J Cancer Prevent 4: 353358, 1995. 139. HILL MJ. Cereals, cereal bre and colorectal cancer risk: a review of the epidemiological literature. Eur J Cancer Prevent 6: 219 225, 1997. 140. HILL MJ. Composition and control of ileal contents. Eur J Cancer Prevent 7 Suppl: S75S78, 1998. 141. HODGKINSON A, DAVIS D, FOURMAN J, ROBERTSON WG, AND ROE F. A comparison of the effects of lactose and of two chemically modied waxy maize starches on mineral metabolism in the rat. Food Chem Toxicol 20: 371382, 1982. 142. HOLT PR, ATILLASOY E, LINDENBAUM J, HO SB, LUPTON JR, MCMAHON D, AND MOSS SF. Effects of acarbose on fecal nutrients, colonic pH and short-chain fatty acids and rectal proliferative indices. Metabolism 45: 1179 1187, 1996. 143. HOVE EL AND KING S. Effects of pectin and cellulose on growth, feed efciency, and protein utilization, and their contribution to energy requirement and cecal VFA in rats. J Nutr 109: 1274 1278, 1979. 144. HOVERSTAD T AND BJORNEKLETT A. Short-chain fatty acids and bowel functions in man. Scand J Gastroenterol 19: 1059 1065, 1984. 145. ILLMAN RJ, STORER GB, AND TOPPING DL. White wheat our lowers plasma cholesterol and increases cecal steroids relative to whole wheat our, wheat bran and wheat pollard in rats. J Nutr 123: 1094 1100, 1993. 146. ILLMAN RJ AND TOPPING DL. Effects of dietary oat bran on faecal steroid excretion, plasma volatile fatty acids and lipid synthesis in the rat. Nutr Res 5: 839 846, 1985. 147. ILLMAN RJ, TOPPING DL, MCINTOSH GH, TRIMBLE RP, AND TAYLOR MN. Hypocholesterolaemic effects of dietary propionate: studies in whole animals and perfused rat liver. Ann Nutr Metab 32: 97107, 1988. 148. ILLMAN RJ, TOPPING DL, AND TRIMBLE RP. Effects of food restriction and starvation-refeeding on volatile fatty acid concentrations in the rat. J Nutr 116: 1694 1700, 1986. 149. JACKSON KA AND TOPPING DL. Prevention of coprophagy does not alter the hypocholesterolaemic effects of oat bran in the rat. Br J Nutr 70: 211219, 1993. 150. JACOBS LR. Inuence of soluble bers on experimental colon carcinogenesis. In: Dietary Fiber: Chemistry, Physiology and Health Effects, edited by Kritchevksy D, Boneld C, and Anderson JW. New York: Plenum, 1990, p. 389. 151. JENKINS DJ, CUFF D, WOLEVER TM, KNOWLAND D, THOMPSON L, COHEN L, AND PROKIPCHUK E. Digestibility of carbohydrate in an ileostomate: relationship to dietary ber, in vitro digestibility, and glycemic response. Am J Gastroenterol 82: 709 717, 1987. 152. JENKINS DJ, PETERSON RD, THORNE MJ, AND FERGUSON PW. Wheat ber and laxation: dose response and equilibration time. Am J Gastroenterol 82: 1259 1263, 1987. RSCH P, JEFFCOAT R, WARING 153. JENKINS DJ, VUKSAN V, KENDALL CW, WU S, MEHLING CC, VIDGEN E, AUGUSTIN LS, AND WONG E. Physiological effects of resistant starches on fecal bulk, short chain fatty acids, blood lipids and glycemic index. J Am Coll Nutr 17: 609 616, 1998.

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154. KASHTAN H, STERN HS, JENKINS DJ, JENKINS AL, THOMPSON LU, HAY K, MARCON N, MINKIN S, AND BRUCE WR. Colonic fermentation and markers of colo-rectal cancer risk. Am J Clin Nutr 55: 723728, 1992. 155. KHIN-MAUNG-U, BOLIN TD, DUNCOMBE VM, MYO-KHIN, NYUNT-NYUNTWAI, PEREIRA SP, AND LINKLATER JM. Epidemiology of small bowel bacterial overgrowth and rice carbohydrate malabsorption in Burmese (Myanmar) village children. Am J Trop Med Hyg 47: 298 304, 1992. 156. KIM YS, TSAO D, SIDDIQUI B, WHITEHEAD JS, ARNSTEIN P, BENNETT J, AND HICKS J. Effect of sodium butyrate and dimethylsulfoxide on biochemical properties of human colon cancer cells. Cancer 45: 11851192, 1980. 157. KLEESSEN B, SYKURA B, ZUNFT HJ, AND BLAUT M. Effects of inulin and lactose on fecal microora, microbial activity, and bowel habit in elderly constipated persons. Am J Clin Nutr 65: 13971402, 1997. 158. KORUDA MJ, ROLANDELLI RH, ZIMMARO-BLISS D, HASTINGS J, AND SETTLE RG. Parenteral nutrition supplemented with SCFA: effect on the small bowel mucosa in normal rats. Am J Clin Nutr 51: 685 689, 1990. 159. KRIPKE SA, FOX AD, BERMAN JM, SETTLE RG, AND ROMBEAU JL. Stimulation of intestinal mucosal growth with intracolonic infusion of short chain fatty acids. J Parent Enteral Nutr 13: 109 116, 1989. 160. KRUH J. Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Mol Cell Biochem 42: 65 82, 1982. 161. KURATSUNE M, HONDA T, ENGLYST HN, AND CUMMINGS JH. Dietary ber in the Japanese diet as investigated in connection with colon cancer risk. Jap J Cancer Res 77: 736 738, 1986. 162. KVIETYS PR AND GRANGER DN. Effect of volatile fatty acids on blood ow and oxygen uptake by the dog colon. Gastroenterology 80: 962969, 1981. 163. LAERKE HN AND JENSEN BB. D-Tagatose has low small intestinal digestibility but high large intestinal fermentability in pigs. J Nutr 129: 10021009, 1999. 164. LAMPE JW, FREDSTROM SB, SLAVIN JL, AND POTTER JD. Sex differences in colonic function: a randomised trial. Gut 34: 531536, 1993. 165. LAMPE JW, WETSCH RF, THOMPSON WO, AND SLAVIN JL. Gastrointestinal effects of sugarbeet ber and wheat bran in healthy men. Eur J Clin Nutr 47: 543548, 1993. RCK I, ASP NG, AND ANDERSSON H. 166. LANGKILDE AM, EKWALL H, BJO Retrograded high-amylose corn starch reduces cholic acid excretion from the small bowel in ileostomy subjects. Eur J Clin Nutr 52:790 795, 1998. 167. LAPRE JA AND VAN DER MEER L. Diet-induced increase in colonic bile acids stimulates lytic activity of fecal water and proliferation of colonic cells. Carcinogenesis 13: 41 44, 1992. 168. LEE PC, BROOKS SP, KIM O, HEITLINGER LA, AND LEBENTHAL E. Digestibility of native and modied starches: in vitro studies with human and rabbit pancreatic amylases and in vivo studies with rabbits. J Nutr 115: 93103, 1985. ME SY C, AND DEMIGNE C. Effect of soybean 169. LEVRAT MA, BEHR SR, RE bre on cecal digestion in rats previously adapted to a ber-free diet. J Nutr 121: 672 678, 1991. 170. LEWIS SJ AND HEATON KW. Increasing butyrate concentration in the distal colon by accelerating intestinal transit. Gut 41: 245251, 1997. 171. LEWIS SJ AND HEATON KW. The intestinal effects of bran-like plastic particles: is the concept of roughage valid after all? Eur J Gastroenterol Hepatol 9: 553557, 1997. RCK I. Metabolic responses to 172. LILJEBERG H, GRANFELDT Y, AND BJO starch in bread containing intact kernels versus milled our. Eur J Clin Nutr 42: 561575, 1992. 173. LIPKIN M, BLATTNER WE, FRAUMENI JF JR, LYNCH HT, DESCHNER E, AND WINAWER S. Tritiated thymidine (p, h) labeling distribution as a marker for hereditary predisposition to colon cancer. Cancer Res 43: 1899 1904, 1983. 174. LIPKIN M, ENKER WE, AND WINAWER SJ. Tritiated-thymidine labeling of rectal epithelial cells in non-prep biopsies in individuals at increased risk for colonic neoplasia. Cancer Lett 37: 153161, 1987. 175. LIVESEY G AND ELIA M. Short chain fatty acids as an energy source in the colon: metabolism and clinical implications. In: Physiological and Clinical Aspects of Short-Chain Fatty Acids, edited by

176.

177.

178.

179.

180.

181.

182.

183.

184.

185.

186. 187.

188.

189.

190. 191.

192.

193.

194. 195. 196.

197.

198.

Cummings JH, Rombeau JL, and Sakata S. Cambridge, UK: Cambridge Univ. Press, 1995, p. 427. MACDONALD IA, SINGH G, MAHONY DE, AND MEIER CE. Effect of pH on bile salt degradation by mixed fecal cultures. Steroids 32: 245256, 1978. MACFARLANE GT AND GIBSON GR. Microbiological aspects of the production of short-chain fatty acids in the large bowel. In: Physiological and Clinical Aspects of Short-Chain Fatty Acids, edited by Cummings JH, Rombeau JL, and Sakata S. Cambridge, UK: Cambridge Univ. Press, 1995, p. 87. MACFARLANE GT, GIBSON GR, AND CUMMINGS JH. Comparison of fermentation reactions in different regions of the human colon. J Appl Bacteriol 72: 57 64, 1992. MACIOROWSKI KG, TURNER ND, LUPTON JR, CHAPKIN RS, SHERMER CL, HA SD, AND RICKE SC. Diet and carcinogen alter the fecal microbial population in rats. J Nutr 127: 449 457, 1997. MACRAE F. Wheat bran ber and development of adenomatous polyps: evidence from randomized, controlled clinical trials. Am J Med 106, Suppl: 38S 42S, 1999. MALHOTRA SL. Faecal urobilinogen levels and pH of stools in population groups with different incidence of cancer of the colon, and their possible role in its aetiology. J R Soc Med 75: 709 714, 1982. MARLETT JA AND CHEUNG TF. Database and quick methods of assessing typical dietary ber intakes using data for 228 commonly consumed foods. J Am Diet Assoc 97: 1139 1148, 1997. MARSONO Y. Complex Carbohydrates and Lipids in Rice Products: Effects on Large Bowel Volatile Fatty Acids and Plasma Cholesterol in Animals (PhD thesis). Bedford Park: Flinders Univ. of South Australia, 1995. MARSONO Y, ILLMAN RJ, CLARKE JM, TRIMBLE RP, AND TOPPING DL. Plasma lipids and large bowel volatile fatty acids in pigs fed on white rice, brown rice and rice bran. Br J Nutr 70: 503513, 1993. MARTIN LJM, DUMAN HJW, AND CHAMP MMJ. Production of shortchain fatty acids from resistant starch in a pig model. J Sci Food Agric 77: 71 80, 1998. MAY TR, MACKIE RI, AND FAHEY GC JR. Fiber digestion in the hindgut of Chinese and domestic pigs (Abstract). FASEB J 7: A740, 1993. ME SY C, GUEUX E, LEVRAT MA, AND DEMIGNE C. Effects of MAZUR A, RE a diet rich in fermentable carbohydrate on plasma lipoprotein levels and on lipid catabolism in rats. J Nutr 120: 10371045, 1990. MCBURNEY MI. Starch malabsorption and stool excretion are inuenced by the menstrual cycle in women consuming low-bre Western diets. Scand J Gastroenterol 26: 880 886, 1991. MCBURNEY MI AND THOMPSON LU. Effect of human fecal donor on in vitro fermentation variables. Scand J Gastroenterol 24: 359 367, 1989. MCBURNEY MI AND THOMPSON LU. Fermentative characteristics of cereal brans and vegetable bers. Nutr Cancer 13: 271280, 1990. MCINTOSH GH, LE LEU RK, ROYLE PJ, AND YOUNG GP. A comparative study of the inuence of differing barley brans on DMH-induced intestinal tumors in male Sprague-Dawley rats. J Gastronterol Hepatol 11: 113119, 1996. MCINTYRE A, GIBSON PR, AND YOUNG GP. Butyrate production from dietary ber and protection against large bowel cancer in a rat model. Gut 34: 386 391, 1993. MCINTYRE A, YOUNG GP, TARANTO T, GIBSON PR, AND WARD PB. Different bers have different regional effects on the luminal contents of rat colon. Gastroenterology 101: 1274 1281, 1991. MCKAY LF, EASTWOOD MA, AND BRYDON WG. Methane excretion in man: a study of breath, atus and feces. Gut 26: 69 76, 1985. MCNEIL NI, CUMMINGS JH, AND JAMES WPT. Short chain fatty acid absorption by the human large intestine. Gut 19: 819 822, 1978. MELCHER EA, LEVITT MD, AND SLAVIN JL. Methane production and bowel function parameters in healthy subjects on low- and highber diets. Nutr Cancer 16: 8592, 1991. MEVISSEN-VERHAGE EAE, MARCELIS JH, DE VOS MN, HARMSEN-VAN AMERONGEN WCM, AND VERHOEF J. Bidobacterium, Bacteroides and Clostridium spp. in fecal samples from breast-fed and bottlefed infants with and without iron supplement. J Clin Microbiol 25: 285289, 1987. MIDTVEDT AC AND MIDTVEDT T. Production of short chain fatty acids by the intestinal microora during the rst 2 years of human life. J Pediatr Gastroenterol Nutr 15: 395 403, 1992.

July 2001

SHORT-CHAIN FATTY ACIDS AND HUMAN COLONIC FUNCTION

AND

1061

199. MILLER TL AND WOLIN MJ. Methanogens in animal and human intestinal tracts. Syst Appl Microbiol 7: 223229, 1986. 200. MITCHELL BL, LAWSON MJ, DAVIES M, KERR-GRANT A, ROEDIGER WEW, ILLMAN RJ, AND TOPPING DL. Volatile fatty acids in the human intestine: studies in surgical patients. Nutr Res 5: 1089 1092, 1985. 201. MITSUOKA T. Intestinal ora and human health. Asia Pacic J Clin Nutr 5: 29, 1996. 202. MORITA T, KASAOKA S, OH-HASHI A, IKAI M, NUMASAKI Y, AND KIRIYAMA S. Resistant proteins alter caecal short-chain fatty acid proles in rats fed high amylose cornstarch. J Nutr 128: 1156 1164, 1998. 203. MORTENSEN FV, HESSOV I, BIRKE H, KORSGAAD N, AND NIELSEN H. Microcirculatory and trophic effects of short chain fatty acids in the human rectum after Hartmanns procedure. Br J Surg 78: 1208 1211, 1991. 204. MORTENSEN FV AND NIELSEN H. In vivo and in vitro effects of shortchain fatty acids on intestinal blood circulation In: Physiological and Clinical Aspects of Short-Chain Fatty Acids, edited by Cummings JH, Rombeau JL, and Sakata T. Cambridge, UK: Cambridge Univ. Press, 1995, p. 391. 205. MORTENSEN PB, CLAUSEN MR, BONNEN H, HOVE H, AND HOLTUG K. Colonic fermentation of ispaghula, wheat bran, glucose and albumin to short-chain fatty and ammonia evaluated in vitro in 50 subjects. J Parenteral Enteral Nutr 16: 433 439, 1992. 206. MORTENSEN PB, HOVE H, CLAUSEN MR, AND HOLTUG K. Fermentation to short-chain fatty acids and lactate in human fecal batch cultures. Intra- and inter-individual variations versus variations caused by changes in fermented saccharides. Scand J Gastroenterol 26: 1285 1294, 1991. 207. MORTENSEN PB AND NORDGAARD-ANDERSEN I. The dependence of the in vitro fermentation of dietary bre to short-chain fatty acids on the contents of soluble non-starch polysaccharides. Scand J Gastroenterol 28: 418 422, 1993. 208. MOSER AR, PITOT HC, AND DOVE WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 247: 322324, 1990. 209. MUIR JG, LU ZX, YOUNG GP, CAMERON-SMITH D, COLLIER GR, AND ODEA K. Resistant starch in the diet increases breath hydrogen and serum acetate in human subjects. Am J Clin Nutr 61: 792799, 1995. 210. MUIR JG AND ODEA K. Measurement of resistant starch: factors affecting the amount of starch escaping digestion in vitro. Am J Clin Nutr 56: 123127, 1992. 211. MUIR JG, WALKER KZ, KAIMAKAMIS MA, CAMERON MA, GOVERS MJ, LU ZX, YOUNG GP, AND ODEA K. Modulation of fecal markers relevant to colon cancer risk: a high-starch Chinese diet did not generate expected benecial changes relative to a Western-type diet. Am J Clin Nutr 68: 372379, 1998. 212. MURRAY SM, PATIL AR, FAHEY GC JR, MERCHEN NR, WOLF BW, LAI CS, AND GARLEB KA. Apparent digestibility of a debranched amylopectin-lipid complex and resistant starch incorporated into enteral formulas fed to ileal-cannulated dogs. J Nutr 128: 20322035, 1998. 213. NAGENGAST FM, HECTORS MP, BUYS WA, AND VAN TONGEREN JH. Inhibition of secondary bile acid formation in the large intestine by lactulose in healthy subjects of two different age groups. Eur J Clin Invest 18: 56 61, 1988. 214. NOAKES M, CLIFTON PM, NESTEL PJ, LEU R, AND MCINTOSH G. Effect of high amylose starch and oat bran on metabolic variables and bowel function in subjects with hypertriglyceridemia. Am J Clin Nutr 64: 944 951, 1996. 215. NORDGAARD I, RUMESSEN JJ, NIELSEN SA, AND GUDMAND-HOYER E. Absorption of wheat starch in patients resected for left-sided colonic cancer. Scand J Gastroenterol 27: 632 634, 1992. 216. NYMAN M, ASP NG, CUMMINGS JH, AND WIGGINS H. Fermentation of dietary bre in the intestinal tract: comparison between man and rat. Br J Nutr 55: 487 496, 1986. 217. OHKUMA K, MATSUDA I, KATTA Y, AND TSUJI K. New method for determining total dietary ber by liquid chromatography. J AOAC Int 83: 10131019, 2000. 218. OHKUSA T, OZAKI Y, SATO C, MIKUNI K, AND IKEDA H. Long-term ingestion of lactosucrose increases Bidobacterium sp. in human fecal ora. Digestion 56: 415 420, 1995. 219. OHTA A, OHTSUKI M, UEHARA M, HOSONO A, HIRAYAMA M, ADACHI T,

220.

221.

222.

223.

224.

225.

226.

227.

228.

229.

230.

231.

232.

233.

234.

235.

236.

237.

238.

239.

240.

HARA H. Dietary fructooligosaccharides prevent postgastrectomy anemia and osteopenia in rats. J Nutr 128: 485 490, 1998. OKEEFE SJ, KIDD M, ESPITALIER-NOEL G, AND OWIRA P. Rarity of colon cancer in Africans is associated with low animal product consumption, not ber. Am J Gastroenterol 94: 13731380, 1999. OLESEN M, RUMESSEN JJ, AND GUDMAND-HOYER E. Intestinal transport and fermentation of resistant starch evaluated by the hydrogen breath test. Eur J Clin Nutr 48: 692701, 1994. PANT I, TOPPING DL, WONG SH, SHEARMAN DJ, AND FARMAKALIDIS E. Fermentation of soluble and insoluble bre in the human colon. In: Proceedings of the XV International Congress of Nutrition, Adelaide 1993. London: Smith-Gordon, 1994, p. 895. PAYNE CM, BERNSTEIN H, BERNSTEIN C, AND GAREWAL H. Role of apoptosis in biology and pathology: resistance to apoptosis in colon carcinogenesis. Ultrastruct Pathol 19: 221248, 1995. PETERS SG, POMARE EW, AND FISHER CA. Portal and peripheral blood short chain fatty acid concentrations after caecal lactulose installation at surgery. Gut 33: 1249 1252, 1992. PHILLIPS J, MUIR JG, BIRKETT A, LU ZX, JONES GP, ODEA K, AND YOUNG GP. Effect of resistant starch on fecal bulk and fermentationdependent events in humans. Am J Clin Nutr 62: 121130, 1995. PIERRE F, PERRIN P, CHAMP M, BORNET F, MEFLAH K, AND MENANTEAU J. Short-chain fructo-oligosaccharides reduce the occurrence of colon tumors and develop gut-associated lymphoid tissue in Min mice. Cancer Res 57: 225228, 1997. PITT P, DE BRUIJN KM, BEECHING MF, GOLDBERG E, AND BLENDIS LM. Studies on breath methane: the effect of ethnic origins and lactulose. Gut 21: 951954, 1980. PLUSKE JR, DURMIC Z, PETHICK DW, MULLAN BP, AND HAMPSON DJ. Conrmation of the role of rapidly fermentable carbohydrates in the expression of swine dysentery in pigs after experimental infection. J Nutr 128: 17371744, 1998. POMARE EW, BRANCH WJ, AND CUMMINGS JH. Carbohydrate fermentation in the human colon and its relation to acetate concentrations in venous blood. J Clin Invest 75: 1448 1454, 1985. POPPITT SD, LIVESEY G, FAULKS RM, ROE M, PRENTICE AM, AND ELIA M. Circadian patterns of total 24-h hydrogen and methane excretion in humans ingesting non starch polysaccharide (NSP) diets and the implications for indirect calorimetry and D218O methodologies. Eur J Clin Nutr 50: 524 534, 1996. PROHASZKA L, JAYARAO BM, FABIAN A, AND KOVACS S. The role of intestinal volatile fatty acids in the Salmonella shedding of pigs. Zentralbl Veterinarmed 37: 570 574, 1990. PYE G, EVANS DF, LEDINGHAM S, AND HARDCASTLE JD. Gastrointestinal intraluminal pH in normal subjects and those with colorectal adenoma or carcinoma. Gut 31: 13551357, 1990. QUESADA CF, KIMATA H, MORI M, NISHIMURA M, TSUNEYOSHI T, AND BABA S. Piroxicam and acarbose as chemopreventive agents for spontaneous intestinal adenomas in APC gene 1309 knockout mice. Jpn J Clin Cancer Res 89: 392396, 1998. RABEN A, ANDERSEN K, KARBERG MA, HOLST JJ, AND ASTRUP A. Acetylation of or -cyclodextrin addition to potato starch: benecial effects on glucose metabolism and appetite sensations. Am J Clin Nutr 66: 304 314, 1997. RAFTER JJ, ENG VW, FURRER R, MEDLINE A, AND BRUCE WR. Effects of calcium and pH on the mucosal damage produced by deoxycholic acid in the rat colon. Gut 27: 1320 1329, 1986. RAMAKRISHNA BS, VENKATARAMAN S, SRINIVASAN S, DASH P, YOUNG GP, AND BINDER HJ. Amylase-resistant starch plus oral rehydration solution for cholera. N Engl J Med 342: 308 313, 2000. RECHKEMMER G AND ENGELHARDT WV. Concentration- and pH-dependence of short-chain fatty acid absorption in the proximal and distal colon of guinea pig (Cavia porcellus). Comp Biochem Physiol A Physiol 91: 659 663, 1988. REIMER RA AND MCBURNEY MI. Dietary ber modulates intestinal proglucagon messenger ribonucleic acid and postprandial secretion of glucagon-like peptide-1 and insulin in rats. Endocrinology 137: 3948 3956, 1996. RERAT A, FISZLEWICZ M, GIUSI A, AND VAUGELADE P. Inuence of meal frequency on postprandial variations in the production and absorption of volatile fatty acids in the digestive tract of conscious pigs. J Anim Sci 64: 448 456, 1987. ROBERFROID MB. Role of dietary factors in the modulation of cancer

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Volume 81

241.

242.

243. 244.

245.

246.

247.

248.

249.

250.

251.

252.

253.

254.

255.

256.

257.

258. 259.

260.

261.

262.

induction. In: Food and Cancer Prevention: Chemical and Biological Aspects, edited by Waldron KW, Johnson IT, and Fenwick GR. Cambridge, UK: Royal Society of Chemistry, 1993, p. 255. ROBERTSON JB AND HORVATH PJ. Detergent analysis of foods. In: CRC Handbook of Dietary Fiber in Human Nutrition, edited by Spiller GA. Boca Raton, FL: CRC, 1993, p. 49. ROE M, BROWN J, FAULKS R, AND LIVESEY G. Is the rat a suitable model for humans on studies of cereal digestion? Eur J Clin Nutr 50: 710 712, 1996. ROEDIGER WEW. Role of anaerobic bacteria in the metabolic welfare of the colonic mucosa in man. Gut 21: 793798, 1980. ROEDIGER WEW. The place of short-chain fatty acids in colonocyte metabolism in health and ulcerative colitis: the impaired colonocyte barrier. In: Physiological and Clinical Aspects of Short-Chain Fatty Acids, edited by Cummings JH, Rombeau JL, and Sakata T. Cambridge, UK: Cambridge Univ. Press, 1995, p. 337. ROEDIGER WEW, HEYWORTH M, WILLOUGHBY P, PIRIS J, MOORE A, AND TRUELOVE SC. Luminal ions and short chain fatty acids as markers of functional activity of the mucosa in ulcerative colitis. J Clin Pathol 35: 323326, 1982. ROEDIGER WEW AND MOORE P. Effect of short chain fatty acids on sodium absorption in isolated human colon perfused from the vascular bed. Dig Dis Sci 26: 100 106, 1981. ROEDIGER WEW AND NANCE S. Metabolic induction of experimental ulcerative colitis by inhibition of fatty acid oxidation. Br J Exp Pathol 67: 773782, 1986. ROLAND N, NUGON-BAUDON L, ANDRIEUX C, AND SZYLIT O. Comparative study of the fermentative characteristics of inulin and different types of ber in rats inoculated with a human whole fecal ora. Br J Nutr 74: 239 249, 1995. C, LE QUELLEC A, HOLST JJ, FU-CHENG X, ROPERT A, CHERBUT C, ROZE BRULEY DES VARANNES S, AND GALMICHE JP. Colonic fermentation and gastric tone in humans. Gastroenterology 111: 289 296, 1996. RUBALTELLI FF, BIADOAIOLI R, PECILE P, AND NICOLETTI P. Intestinal ora in breast- and bottle-fed infants. J Perinat Med 26: 186 191, 1998. M. Mutation of p53 tumor suppressor gene RUBIO CA AND RODENSJO in at neoplastic lesions of the colorectal mucosa. Dis Colon Rectum 39: 143147, 1996. RUPPIN H, BAR-MEIR S, SOERGEL KH, WOOD CM, AND SCHMITT MG JR. Absorption of short-chain fatty acids by the colon. Gastroenterology 78: 1500 1507, 1980. SAKAMOTO J, NAKAJI S, SUGAWARA K, IWANE S, AND MUNAKATA A. Comparison of resistant starch with cellulose on 1,2 dimethylhydrazine-induced colonic carcinogenesis in rats. Gastroenterology 110: 116 120, 1996. SAKATA T AND YAJIMA T. Inuence of short chain fatty acids on the epithelial cell division of digestive tract. Q J Exp Physiol 69: 639 648, 1984. SALVADOR V, CHERBUT C, BARRY JL, BERTRAND D, BONNET C, AND DELORT-LAVAL J. Sugar composition of dietary bre and short-chain fatty acid production during in vitro fermentation by human bacteria. Br J Nutr 70: 189 197, 1993. SAMESHIMA S, KUBOTA Y, SAWADA T, WATANABE T, KURODA T, TSUNO N, HIGUCHI Y, SHINOZAKI M, SUNOUCHI K, MASAKI T, SAITO Y, AND MUTO T. Overexpression of p53 protein and histologic grades of dysplasia in colorectal adenomas. Dis Colon Rectum 39: 562567, 1996. SANDBERG AS, ANDERSSON H, BOSAEUS I, CARLSSON NG, HASSELBLAD K, AND HARROD M. Alginate, small bowel excretion, and absorption of nutrients in ileostomy subjects. Am J Clin Nutr 60: 751756, 1994. SAVAGE DC. Gastrointestinal microora in mammalian nutrition. Annu Rev Nutr 6: 155178, 1986. SCHEPPACH W, FABIAN C, AHRENS F, SPENGLER M, AND KASPER H. The effect of starch malabsorption on colonic function and metabolism in humans. Gastroenterology 95: 1549 1555, 1988. SCHEPPACH W, FABIAN C, SACHS M, AND KASPER H. Effect of starch malabsorption on fecal short chain fatty acid excretion in man. Scand J Gastroenterol 23: 755759, 1988. SCHEPPACH W, MULLER JG, BOXBERGER F, DUSEL G, RICHTER F, BARTRAM P, CHRISTL SU, DEMPFLE CE, AND KASPER H. Histological changes in the colonic mucosa following irrigation with short-chain fatty acids. Eur J Gastroenterol Hepatol 9: 163168, 1997. SCHEPPACH W, POMARE EW, ELIA M, AND CUMMINGS JH. The contri-

263.

264.

265.

266.

267. 268.

269.

270. 271.

272.

273.

274. 275.

276.

277.

278.

279.

280. 281.

282. 283.

284.

285.

bution of the large intestine to blood acetate in man. Clin Sci 80: 177182, 1991. SCHEPPACH W, SOMMER H, KIRCHNER T, PAGANELLI GM, BARTRAM P, CHRISTL SU, RICHTER F, DUSEL G, AND KASPER H. Effect of butyrate enemas on the colonic mucosa in distal ulcerative colitis. Gastroenterology 103: 5156, 1992. SCHWARTZ B, LAMPRECHT SA, POLAK-CHARCON S, NIV Y, AND KIM YS. Induction of differentiated phenotype in human colon cancer cell is associated with the attenuation of subcellular tyrosine phosphorylation. Oncol Res 7: 277287, 1995. SEGAL I, HASSAN H, WALKER ARP, BECKER P, AND BRAGANZA J. Fecal short chain fatty acids in South African urban Africans and whites. Dis Col Rectum 38: 732734, 1995. SGHIR A, CHOW JM, AND MACKIE RI. Continuous culture selection of bidobacteria and lactobacilli from human fecal samples using fructooligosaccharide as substrate. J Appl Microbiol 85: 769 777, 1998. SHETTY PS AND KURPAD AV. Increasing starch intake in the human diet increases fecal bulking. Am J Clin Nutr 43: 210 212, 1986. SIAVOSHIAN S, BLOTTIERE HM, CHERBUT C, AND GALMICHE JP. Butyrate stimulates cyclin D and p21 and inhibits cyclin-dependent kinase 2 expression in HT-29 colonic epithelial cells. Biochem Biophys Res Commun 232: 169 172, 1997. SIEVERT D AND POMERANTZ Y. Enzyme-resistant starch. I. Characterization and evaluation by enzymatic, thermoanalytical, and microscopic methods. Cereal Chem 66: 342347, 1989. SIIGUR U, ORMISSON A, AND TAMM A. Faecal short-chain fatty acids in breast-fed and bottle-fed infants. Acta Paediatr 82: 536 568, 1993. SILVESTER KR, ENGLYST HN, AND CUMMINGS JH. Ileal recovery of starch from whole diets containing resistant starch measured in vitro and fermentation of ileal efuent. Am J Clin Nutr 62: 403 411, 1995. SKUTCHES CL, HOLROYDE CP, MYERS RN, PAUL P, AND REICHARD GA. Plasma acetate turnover and oxidation. J Clin Invest 64: 708 713, 1979. SNOSWELL AM, TRIMBLE RP, FISHLOCK RC, STORER GB, AND TOPPING DL. Metabolic effects of acetate in perfused liver: studies on ketogenesis, glucose output, lactate uptake and lipogenesis. Biochim Biohys Acta 716: 290 297, 1982. SOUTHGATE DAT. How much and what classes of carbohydrates reach the colon? Eur J Cancer Prev 7, Suppl 2: S81S82, 1998. SPILLER GA. Denition of dietary ber. In: CRC Handbook of Dietary Fiber in Human Nutrition, edited by Spiller GA. Boca Raton, FL: CRC, 1993, p. 15. SQUIRES PE, RUMSEY RDE, EDWARDS CA, AND READ NW. Effect of short-chain fatty acids on contractile activity and uid ow in rat colon in vitro. Am J Physiol Gastrointest Liver Physiol 262: G813 G817, 1992. STANOGIAS G AND PEARCE GR. The digestion of bre by pigs. 2. Volatile fatty acid concentrations in large intestine digesta. Br J Nutr 53: 531536, 1985. STEINHART AH, HIRUKI T, BRZEZINSKI A, AND BAKER JP. Treatment of left-sided ulcerative colitis with butyrate enemas: a controlled trial. Aliment Pharmacol Ther 10: 729 736, 1996. STEINHART AH, JENKINS DJ, MITCHELL S, CUFF D, AND PROKIPCHUK EJ. Effect of dietary ber on total carbohydrate losses in ileostomy efuent. Am J Gastronterol 87: 48 54, 1992. STEINMETZ K AND POTTER JD. Vegetables, fruit and cancer. I. Epidemiology. Cancer Causes Control 2: 325357, 1991. STEPHEN AM. Starch and dietary bre: their physiological and epidemiological interrelationship. Can J Physiol Pharmacol 69: 116 120, 1991. STEPHEN AM AND CUMMINGS JH. Mechanism of action of dietary bre in the human colon. Nature 284: 283284, 1980. STEPHEN AM, HADDAD AC, AND PHILLIPS SF. Passage of carbohydrate into the colon. Direct measurement in humans. Gastroenterology 85: 589 595, 1983. STEVENS CE AND HUME ID. Contributions of microbes in vertebrate gastrointestinal tract to production and conservation of nutrients. Physiol Rev 78: 393 427, 1998. STEVENS JD, LEVITSKY DA, VAN SOEST PJ, ROBERTS JB, KALKWARF HJ, AND ROE DA. Effect of psyllium gum and wheat bran on spontaneous energy intake. Am J Clin Nutr 46: 812 817, 1987.

July 2001

SHORT-CHAIN FATTY ACIDS AND HUMAN COLONIC FUNCTION

1063

286. STORER GB, ILLMAN RJ, TRIMBLE RP, SNOSWELL AM, AND TOPPING DL. Plasma and caecal volatile fatty acids in male and female rats: effects of dietary gum arabic and cellulose. Nutr Res 4: 701707, 1984. 287. SUNVOLD GD, FAHEY GC JR, MERCHEN NR, TITGEMEYER EC, BOURQUIN LD, BAUER LL, AND REINHART GA. Dietary ber for dogs. IV. In vitro fermentation of selected ber sources by dog fecal inoculum and in vivo digestion and metabolism of ber-supplemented diets. J Anim Sci 73: 1099 1109, 1995. 288. SZYLIT O, MAURAGE C, GASQUI P, FAVRE S, GOLD F, AND BORDERON JC. Fecal short-chain fatty acids predict digestive disorders in premature infants. J Parenter Enteral Nutr 22: 136 141, 1998. 289. TAKAHASHI H, YANG SI, HAYASHI C, KIM M, YAMANAKA J, AND YAMAMOTO T. Effect of partially hydrolysed guar gum on fecal output in human volunteers. Nutr Res 13: 649 657, 1993. 290. TANNOCK GW. A fresh look at the intestinal microora. In: Probiotics: A Critical Review, edited by Tannock GW. Wymondham: Horizon Scientic Press, 1999, p. 5. 291. TAYLOR CW, KIM YS, CHILDRESS-FIELDS KE, AND YEOMAN LC. Sensitivity of nuclear c-myc levels and induction to differentiation-inducing agents in human colon tumor cell lines. Cancer Lett 62: 95105, 1992. 292. TERPSTRA OT, VANBLANKENSTEIN M, DEES J, AND EILERS GA. Abnormal pattern of cell proliferation in the entire colonic mucosa of patients with colon adenoma or cancer. Gastroenterology 92: 704 708, 1987. 293. THEANDER O, MAN P, WESTERLUND E, ANDERSSON R, AND PETTERSSON D. Total dietary ber determined as neutral sugar residues, uronic acid residues, and Klason lignin (the Uppsala method): collaborative study. J AOAC Int 78: 1030 1044, 1995. 294. THORNTON JR, DRYDEN A, KELLEHER J, AND LOSOWSKY MS. Superefcient starch absorption. A risk factor for colonic neoplasia? Dig Dis Sci 32: 1088 1091, 1987. 295. THORUP IO, MEYER O, AND KRISTIANSEN E. Effect of potato starch, cornstarch and sucrose on aberrant crypt foci in rats exposed to azoxymethane. Anticancer Res 15: 21012105, 1995. 296. THUN MJ, CALLE EE, NAMBOODIRI MM, FLANDERS WD, COATES RJ, BYERS T, BOFFETTA P, GARFINKEL L, AND HEATH CW JR. Risk factors for fatal colon cancer in a large prospective study. J Nat Cancer Inst 84: 14911500, 1992. 297. TIL HP, FERON VJ, IMMEL HR, AND VOGEL WF. Chronic (89-week) feeding study with hydroxypropyl distarch phosphate, starch acetate, lactose and sodium alginate in mice. Food Chem Toxicol 24: 825 834, 1986. 298. TOMLIN J AND READ NW. The effect of resistant starch on colon function in humans. Br J Nutr 64: 589 595, 1990. 299. TOPPING DL. Soluble ber polysaccharides: effects on plasma cholesterol and colonic fermentation. Nutr Rev 49: 195203, 1991. 300. TOPPING DL. Physiological effects of dietary carbohydrates in the large bowel: is there a need to recognize bre equivalents? Asia Pacic J Clin Nutr 8 Suppl: S22S26, 1999. 301. TOPPING DL, GOODEN JM, BROWN IL, BIEBRICK DA, MCGRATH L, TRIMBLE RP, CHOCT M, AND ILLMAN RJ. A high amylose (amylomaize) starch raises proximal large bowel starch and increases colon length in pigs. J Nutr 127: 615 622, 1997. 302. TOPPING DL AND ILLMAN RJ. Bacterial fermentation in the human large bowel. Time to change from the roughage model of dietary bre? Med J Aust 144: 307309, 1986. 303. TOPPING DL, ILLMAN RJ, CLARKE JM, TRIMBLE RP, JACKSON KA, AND MARSONO Y. Dietary fat and ber alter large bowel and portal venous volatile fatty acids and plasma cholesterol but not biliary steroids in pigs. J Nutr 123: 133143, 1993. 304. TOPPING DL, ILLMAN RJ, TAYLOR MN, AND MCINTOSH GH. Effects of wheat bran and porridge oats on hepatic portal venous volatile fatty acids in the pig. Annu Nutr Metab 29: 325331, 1985. 305. TOPPING DL, ILLMAN RJ, AND TRIMBLE RP. Volatile fatty acid concentrations in rats fed diets containing gum arabic and cellulose separately and as a mixture. Nutr Rep Int 32: 809 814, 1985. 306. TOPPING DL AND TRIMBLE RP. Effects of insulin on the metabolism of the isolated working rat heart perfused with undiluted rat blood. Biochim Biophys Acta 844: 113118, 1985. 307. TOPPING DL, MOCK S, TRIMBLE RP, AND ILLMAN RJ. Effects of varying

308.

309.

310. 311.

312.

313.

314.

315.

316.

317.

318.

319.

320.

321. 322.

323.

324.

325.

326.

327.

328.

329.

the content and proportions of gum arabic and cellulose on cecal volatile fatty acids in the rat. Nutr Res 8: 10131020, 1988. TRINIDAD TP, WOLEVER TMS, AND THOMPSON LU. Effect of acetate and propionate on calcium absorption from the rectum and distal colon of humans. Am J Clin Nutr 63: 574 578, 1996. TROCK B, LANZA E, AND GREENWALD P. Dietary ber, vegetables, and colon cancer: critical review and meta-analyses of the epidemiological evidence. J Natl Cancer Inst 82: 650 661, 1990. TRUSWELL AS. Glycaemic index of foods. Eur J Clin Nutr 46, Suppl 2: S91S101, 1992. UMESAKI Y, YAJIMA T, YOKOKURA T, AND MUTAI M. Effect of organic acid absorption on bicarbonate transport in rat colon. Pu gers Arch 379: 43 47, 1979. VAN DOKKUM W, WEZENDONK B, SRIKUMAR TS, AND VAN DEN HEUVEL EG. Effect of non-digestible oligosaccharide on large-bowel functions, blood lipid concentrations and glucose absorption in young, healthy male subjects. Eur J Clin Nutr 53: 17, 1999. VAN MUNSTER IP, DE BOER HM, JANSEN MC, DE HAAN AF, KATAN MB, VAN AMELSVOORT JM, AND NAGENGAST FM. Effect of resistant starch on breath-hydrogen and methane excretion in healthy volunteers. Am J Clin Nutr 59: 626 630, 1994. VAN MUNSTER IP, TANGERMAN A, AND NAGENGAST FM. Effect of resistant starch on colonic fermentation, bile acid metabolism and mucosal proliferation. Dig Dis Sci 39: 834 842, 1994. VAN SOEST PJ. Comparative aspects of animal models. In: Dietary Fiber in Health and Disease, edited by Kritchvesky D and Boneld C. St. Paul, MN: Eagan, 1995, p. 321. VELAZQUEZ OC, SETO RW, BAIN AM, FISHER J, AND ROMBEAU JL. Deoxycholate inhibits in vivo butyrate-mediated BrDU labeling of the colonic crypt. J Surg Res 69: 344 348, 1997. VELAZQUEZ OC, SETO RW, CHOI J, ZHOU D, BREEN F, FISHER JD, AND ROMBEAU JL. Butyrate inhibits deoxycholate-induced increase in colonic mucosal DNA and protein synthesis in vivo. Dis Colon Rectum 40: 1368 1375, 1997. VERNIA P, CAPRILLI R, LATELLA G, BARBETTI F, MAGLIOCCA M, AND CITTADINI M. Fecal lactate and ulcerative colitis. Gastroenterology 95: 1564 1568, 1988. VERNIA P, CIARNIELLO P, CITTADINI M, LORENZOTTI A, ALESSANDRINI A, AND CAPRILLI R. Stool pH and SCFA in colorectal cancer and polyps. (Abstract). Annu Meet Am Gastoenterologic Assoc 89th Washington DC 1989, p. A528. WALKER ARP, WALKER BF, AND WALKER AJ. Fecal pH, dietary bre intake and proneness to colon cancer in four South African populations. Br J Cancer 53: 489 495, 1986. WASAN HS AND GOODLAD RA. Fiber-supplemented foods may damage your health. Lancet 348: 319 320, 1996. WEAVER GA, KRAUSE JA, MILLER TL, AND WOLIN MJ. SCFA distribution of enema samples from a sigmoidoscopy population: an association of high acetate and low butyrate ratios with adenomatous polyps and colon cancer. Gut 29: 1539 1543, 1988. WEAVER GA, KRAUSE JA, MILLER TL, AND WOLIN MJ. Constancy of glucose and starch fermentations by two different human faecal microbial communities. Gut 30: 19 25, 1989. WEAVER GA, KRAUSE JA, MILLER TL, AND WOLIN MJ. Cornstarch fermentation by the colonic microbial community yields more butyrate than does cabbage fermentation; cornstarch fermentation rates correlate negatively with methanogenesis. Am J Clin Nutr 55: 70 77, 1992. WEAVER GA, TANGEL C, KRAUSE JA, ALPERN HD, JENKINS PL, PARFITT MM, AND STRAGAND JJ. Dietary guar gum alters colonic microbial fermentation in azoxymethane-treated rats. J Nutr 126: 1979 1991, 1996. WEAVER GA, TANGEL CT, KRAUSE JA, PARFITT MM, JENKINS PL, RADER JM, LEWIS BA, MILLER TL, AND WOLIN MJ. Acarbose enhances human colonic butyrate production. J Nutr 127: 717723, 1997. WELLER RA AND PILGRIM AF. Passage or protozoa and volatile fatty acids from the rumen of the sheep and from a continuous in vitro fermentation system. Br J Nutr 32: 41351, 1974. WHITEHEAD RH, YOUNG GP, AND BHATHAL PS. Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM1215). Gut 27: 14571463, 1986. WILLIAMS AC, HARPER SJ, AND PARASKEVA C. Neoplastic transforma-

1064

DAVID L. TOPPING AND PETER M. CLIFTON

Volume 81

330. 331.

332.

333.

334.

335.

336. 337. 338.

tion of a human colonic epithelial cell line: in vitro evidence for the adenoma to carcinoma sequence. Cancer Res 50: 4724 4730, 1990. WILSON RB. Species variation in response to diemthylhydrazine. Toxicol Appl Pharmacol 38: 647 650, 1976. WINDMUELLER HG AND SPAETH AE. Identication of ketone bodies and glutamine as the major respiratory fuels in vivo for postabsorptive rat small intestine. J Biol Chem 253: 69 76, 1978. WOLEVER TMS, JOSSE RG, LEITER LA, AND CHIASSON JL. Time of day and glucose tolerance status affect serum short-chain fatty acid concentrations in humans. Metabolism 46: 805 811, 1997. WOLIN MJ, YERRY S, MILLER TL, ZHANG Y, AND BANK S. Changes in production of ethanol, acids and H2 from glucose by the fecal ora of a 16- to 158-d-old breast-fed infant. J Nutr 128: 8590, 1998. WORLD CANCER RESEARCH FUND AND AMERICAN INSTITUTE FOR CANCER RESEARCH. Food, Nutrition and the Prevention of Cancer: a Global Perspective. Washington, DC: Am Inst Cancer Res, 1997 (ISBN: 1 899533 05 2). WRONG O, METCALFE-GIBSON A, MORRISON RBI, NG ST, AND HOWARD AV. In vivo dialysis of faeces as a method of stool analysis. I. Technique and results in normal humans. Clin Sci 28: 357375, 1965. WURZBURG OB. Nutritional aspects and safety of modied food starches. Nutr Rev 44: 74 79, 1986. YAJIMA T. Contractile effect of short-chain fatty acids on the isolated colon of the rat. J Physiol (Lond) 368: 667 678, 1985. YANAHIRA S, MORITA M, AOE S, SUGURI T, TAKADA Y, MIURA S, AND NAKAJIMA I. Effects of lactitol-oligosaccharides on calcium and magnesium absorption in rats. J Nutr Sci Vitaminol Tokyo 43: 123132, 1997.

339. YANG MG, MANOHARAN K, AND MICKELSEN O. Nutritional contribution of volatile fatty acids from the cecum of rats. J Nutr 100: 545550, 1970. ME SY C, BEHR S, AND DEMIGNE C. Fermentable carbo340. YOUNES H, RE hydrate exerts a urea-lowering effect in normal and nephrectomized rats. Am J Physiol Gastrointest Liver Physiol 273: G515 G521, 1997. ME SY C, AND DEMIGNE C. Acidic fermentation in the 341. YOUNES H, RE caecum increases absorption of calcium and magnesium in the large intestine of the rat. Br J Nutr 75: 301314, 1996. 342. YOUNG GP AND GIBSON PR. Butyrate and the human cancer cell. In: Physiological and Clinical Aspects of Short-Chain Fatty Acids, edited by Cummings JH, Rombeau JL, and Sakata T. Cambridge, UK: Cambridge Univ. Press, 1995, p. 319. 343. YOUNG GP, MCINTYRE V, ALBERT M, FOLINO M, MUIR JG, AND GIBSON PR. Wheat bran suppresses potato starch-potentiated colorectal tumorigenesis at the aberrant crypt stage in a rat model. Gastroenterology 110: 508 514, 1996. 344. ZHANG J AND LUPTON JR. Dietary bers stimulate colonic cell proliferation by different mechanisms at different sites. Nutr Cancer 22: 267276, 1994. 345. ZHU Y, RICHARDSON JA, PARADA LF, AND GRAFF JM. Smad3 mutant mice develop metastatic colorectal cancer. Cell 94: 703714, 1998. 346. ZORAN DL, TURNER ND, TADDEO SS, CHAPKIN RS, AND LUPTON JR. Wheat bran reduces tumor incidence in a rat model of colon cancer independent of effects on distal luminal butyrate concentrations. J Nutr 127: 22172225, 1997.