I. INTRODUCTION

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Early observational studies showed that native East Africans, consuming a diet high in unrefined 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 fiber 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 profile reflects 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 confined 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 influenced 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 deficit may be filled 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 fiber 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 difficult. Thus health authorities have been able to make dietary recommendations for fiber 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 microflora, 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 fiber hypothesis led to expectations of a strong protective role for it in laxation, and this is well-documented, especially for insoluble fiber. This is not so for colorectal cancer, an important malignancy in affluent countries. Experimental work (largely in rats) suggested strong protection by fiber 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 fiber supplements (usually as cereal brans), have also yielded disappointing data on progression or recurrence. Conversely, studies in rats have indicated no beneficial or adverse effects of RS on tumors in genetically or chemically induced tumors, while epidemiological 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 benefits in diarrhea may be due to interactions between RS and the microflora. 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 microflora 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 microflora appear to be of benefit to the host in the short and long term. In countries with low intakes of starch and RS, it may be of health benefit 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

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The first systematic links between dietary fiber and human health were expressed in terms of its indigestibility (45, 46). Although breakdown of some fiber components on passage through the gut was recognized, it was considered largely as a bulking agent (45) and was defined as "plant structural and exudative components not digested by human digestive enzymes" (275). This has been called the "roughage model" whereby any protection by fiber 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 fiber components (241). Application of this, rather limited, technology to humans showed that the breakdown of fiber 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 fiber survived (282). The neutral detergent fiber (NDF) procedure used in that study is a substantial underestimate as soluble material, including important fiber components, are extracted and so not included in the fiber value (241). Comprehensive analyses have been developed for the major fiber components that obviate such losses (e.g., Ref. 293), and their application shows that fiber 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 Official 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 fiber (TDF). There are other components such as oligosaccharides that are also not measured routinely. These can be regarded as components of dietary fiber 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 15-20+ g/day for NSP (22). Thus NSP could be taken as the principal contributors to dietary fiber 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

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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 identified in human feces (100, 120, 138, 258). The dominant organisms in terms of numbers are anaerobes including bacteroides, bifidobacteria, eubacteria, streptococci, and lactobacilli, while others, such as enterobacteria, also may be found, usually in 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 microflora 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 reflects 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 reflected by a decline in SCFA production (177, 178). In vitro endogenous production was ~250 mmol SCFA·kg¹·48 h¹ during incubation with proximal colonic inocula falling to ~50 mmol SCFA·kg¹·48 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 first 1-3 days after birth when fecal concentrations of these organisms peak at ~10¹¹ cfu/g feces before declining (201). Bifidobacteria appear in feces after 2 or more days after birth and become the dominant species at ~4-5 days. Colonization by bifidobacteria is significantly 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, bifidobacteria decrease and a more "adult" profile develops, presumably reflecting dietary change (201). The relationship of dietary NSP and RS to this process is unknown.

The colonic microflora 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 bifidobacteria 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 fiber on large bowel microflora and SCFA (186). Information in these areas is limited because conventional microbiological 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 bifidobacteria and lactobacilli, whereas with genus-specific 16S rRNA oligonucleotide probes, bifidobacteria 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 specific 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.

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  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 of feces was found in breast-fed babies. Formate and ethanol have been found in quantity in feces from breast-fed babies (333). The SCFA profile 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 profile has emerged (198). Presumably, the product profile during milk feeding contributes to the specific metabolic needs at this period of development, but this remains to be established as do the changes in individual SCFA during weaning and maturation.

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 fiber (110). They have been used clinically in ulcerative colitis (245) where the severity of inflammation 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 effluent 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.

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 profile has been confirmed in pigs where the fall can be substantial (20, 32, 78, 124, 183-185, 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 individual 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-fiber 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 µmol/g wet wt (wheat bran), 202 and 59 µmol/g wet wt (senna), and 82 and 6 µmol/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 significantly 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 fluid 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 large bowel disease, especially cancer (54), which is an important malignancy in terms of numbers affected, particularly in affluent 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?

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) fiber test meals and found that evolution was 158 and 167 ppm H₂/h on days 3 and 5 of consumption of the low-fiber meals and 492 and 554 ppm H₂/h with the high-fiber 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 µM against a baseline of 54 µM (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 reflect 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 difficult without considerable sample concentration (209, 332). Wolever et al. (332) recorded values of 4.5-6.6 and 2.0-3.9 µM for propionate and butyrate, respectively. Corresponding values reported by Muir et al. (209) were higher at 17.3-32.8 and 36.3-65.5 µM, 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) 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 difficult. 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 influence SCFA production. Protein may have an influence 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 µmol/cecum, respectively. Partial replacement of casein with an RP (autoclaved egg white) lowered succinate to 381 µmol/cecum and raised butyrate to 111 µmol/cecum. Substrate concentration is important and varies with fiber 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 five 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 influences 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 significantly higher with the former (183 vs. 116 mmol/kg dry fecal wt). These limited data confirm that human colonic SCFA seem to respond to change in fermentable substrate as they do in model animal species.

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 modified 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 fiber (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 effluent (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 modified 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 specifically. This has very important implications for the digestion of RS but not necessarily of fiber. The fermentation of insoluble fiber 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 flaked 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 fiber 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.

View larger version (83K): [in this window] [in a new window]   Fig. 1. Scanning electron micrographs of native high-amylose starch granules (A) and granules isolated from human ileostomy effluent (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 high-amylose (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 microflora. [Modified from Brown et al. (43).]

	IV. METABOLIC EFFECTS OF SHORT-CHAIN FATTY ACIDS IN THE LARGE BOWEL

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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 fiber hypothesis (302) and is a recognized attribute of foods such as cereal brans (23, 116, 152, 164, 165). Fiber analogs such as plastic "bran" flakes also speed transit and promote laxation (171), indicating the importance of the roughage effect of fiber. The actions of SCFA are wider in scope and more significant 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.

SCFA are the principal luminal anions in humans and other omnivores. They are relatively weak acids with pK_a values ~4.8, and raising their concentrations through fermentation lowers digesta pH. Food supply is an important variable. In rats fed a nonpurified 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 nonpurified 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 fiber, 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 significantly 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 flour, cecal pH is lowered by 1-2 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 reflect 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-fiber 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 fiber (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 find any effect of an RS on SCFA or pH, but that may reflect 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-fiber diets (196), pH values are lowered significantly. 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 influences 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 findings, 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 fluid loss substantially and speeds remission by up to 50% (236). Diarrhea has been shown in rats when they are fed purified 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 microflora. Normally it seems that SCFA production and absorption from RS and NSP is associated with diminished fluid 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 specific beneficial lactic acid bacteria, i.e., probiotics (usually bifidobacteria or lactobacilli) rather than the flora 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 defined as "nondigestible food ingredients that beneficially 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 bifidobacteria in human volunteers. Kleessen et al. (157) found that at a dose of 40 g/day, inulin consumption increased bifidobacteria 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 bifidobacteria numbers were higher than in controls (51). Feeding trials in pigs with RS have shown greater fecal numbers of bifidobacteria 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 modified or unmodified starch granules (31). This adhesion has been studied relatively little as has the interaction between RS, SCFA, and the large bowel microflora. New technologies for bacterial enumeration will facilitate development of a fuller understanding of these relationships.

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 fluid and electrolyte transport (66), although an inhibition of NSP breakdown (with attendant laxation) is possible (204). Enhanced fluid 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 fiber (72). Apparent colonic absorption was increased significantly 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 (5.5-7.5) 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 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 inflammation of the defunctioned segment in humans (3, 130), although this has not been confirmed (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 benefit, especially in the distal colon, which is the region most at risk of pathology.

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 flow (203). Greater colonic blood flow 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 flow 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 flow 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 influence 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 volunteer's 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 flow enhances tissue oxygenation and transport of absorbed nutrients.

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/µg 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 effects of SCFA may be due to low intra-colorectal pH rather than any specific 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 fivefold 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 floating 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 significant 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 first 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 floating 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 significantly 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

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The influence 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 fiber (measured as NDF) in wheat bran was 43% in women and 37% in men when they consumed 30 g of wheat bran fiber/day. Fecal bulking was lower and mouth to anus transit was longer in women than in men and varied with fiber type (vegetable vs. wheat) and level of intake (10 or 30 g/day). In women consuming a low-fiber 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.

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 reflect 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).

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 fiber-rich foods including bran fractions from wheat, oats, barley, corn and rice, soybean fiber, vegetable extracts, and pea fiber (25, 73, 189, 207). Purified 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 purified 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 fluid (203). Additionally, greater fermentability may be associated with a more rapid fermentation (25). The large European interlaboratory study that examined a number of fiber 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 fiber study was of sufficient size to allow for the variables that can impact on fermentation and showed clearly the wide range of values obtained in five 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 nonpurified 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 confined to the cecum. These experiments confirm that enrichment of the diet with NSP, RS, and OS leads to elevation of large bowel SCFA. Feeding diets containing complex fiber mixtures (187) or fiber-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 pericarp-seed 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 fiber. When intakes of dietary water-soluble carbohydrates exceed the fermentative capacity of the microflora, SCFA fall due to osmotic diarrhea secondary to their presence in the digesta.

In pigs, feeding fiber or RS elevates large bowel SCFA concentrations and pools. When pigs were fed a western-type diet (i.e., high in fat and protein and low in fiber), increasing fiber 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 fiber) 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 reflect 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.

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 breast-fed 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,