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Review Bioscience Microflora Vol. 21 (1), 35-42, 2002 Physiological Effects of Short-Chain Produced from Prebiotics in the Colon

Hiroshi HARA*

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Presented at International Symposium on , held in Tokyo, July 3, 2001. Received for publication, September 7, 2001

Short-chain fatty acids are products of nondigestible oligo- and poly-saccharides fermented by vast numbers of colonic called microbial flora. Produced in the colon they are readily absorbed and are metabolized in the liver and muscle tissues providing energy to the body. The colonic mucosal cells consume , a kind of short-chain fatty acid, as a main and indispensable energy source. A deficiency in butyric acid leads to a malfunction of the colon. This means that nondigestible sugars, including prebiotics, are essential for human health. Short-chain fatty acids are not merely sources of energy; they provide beneficial physiological actions. These include improving the absorption of water and several minerals, especially calcium, magnesium, and iron, and suppressing the synthesis of liver cholesterol. Butyric acid is marked as a suppressor of colon cancer, a lifestyle-related disease. This product reduces cell proliferation and induces in the mutated cells. Butyric acid is very likely to be effective for the prevention of this fatal disease. How do short-chain fatty acids work in various cells? This has not been revealed. The research into short-chain fatty acids may clarify the physiological implications of the colonic fermentation and prebiotics.

Key words: short-chain fatty acids; nondigestible sugar; colonic fermentation; prebiotics

flowing into the provide some of the ni- INTRODUCTION trogen and sulfur that the microbial flora need. Non- Short-chain fatty acids are major products of fermen- digestible proteins in foods (resistant proteins) are ex- tation by microbial flora living in the large intestine of ogenous sources; some digestive are also avail- and other monogastric animals. These acids able for microbial fermentation as endogenous sources. consist mainly of acetic, propionic, and butyric acids, Table 1 (20, 28) shows the amount of each substrate all of which are volatile and have very short available for colonic fermentation for human. The to- chains. During colonic fermentation, the short-chain tal amount of these compounds flowing into the large fatty acids are derived from nondigestible substances intestine is 30 to 100 g, which is much greater than the that contain prebiotics. Not only are these acids a source recommended daily intake of , 20 to 30 g. of energy for the human body, but they are also in- Nondigestible sugars are hydrolyzed into monosac- volved in the beneficial effects of prebiotics on human charides by various bacterial glycosidases that the body health. Thus, as the products of fermentation, short- lacks as digestive enzymes. The sugar units are incor- chain fatty acids are in two aspects: as energy porated into , then metabolized and converted and as regulators of body functions. into various fermentation products. Most end products of fermentation from monosaccharides (hexose or pen- PRODUCTIONOF SHORT-CHAIN FATTY ACIDS tose) are short-chain fatty acids. Each short-chain fatty IN THE LARGE INTESTINE acid is produced with interactions among many bacte- Short-chain fatty acids are derived through micro- rial , resembling the microbial ecosystem in the bial fermentation of nondigestible substances in the . A large amount of propionate is converted large bowel. The nondigestible substances originate from succinate, which is a fermentation product of other from foods and from the intestines themselves. The bacteria, and acetate is produced by acetogenesis in part former includes mainly nonstarch polysaccharide (di- by the use of H2. Almost all lactate produced is usually etary fiber), , and nondigestible oligosac- converted to short-chain fatty acids. charide, though the latter is mainly mucin. Proteins Branched-chain fatty acids, isobutyrate and isoval- erate, are fermentation products of amino acids. These *Corresponding author . Mailing address: Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. Phone: +81-11-706-3352. fatty acids are minor end products of colonic fermenta- Fax. +81-11-706-2504. tion. We previously showed that oxidized inges-

35 36 H. HARA

Table 1. Substrates for human colonic Table 2. Production ratio (%) of short-chain fatty fermentation (nondigestible substances). acids from nondigestible substances by fermentation with human feces (5).

Fig. 2. Absorption and of SCFA by a colonic mu- cosal cell.

minor components of the fermentation products. Also, lactic and succinic acids are usually minor components in the colonic lumen. In the present study, three major short-chain fatty acids were produced in proportions Fig. 1. Colonic fermentation pathways. different from various nondigestible sugars by in vitro fermentation using human fecal bacteria (Table 2) (5). tion induced higher levels of isobutyrate and lower lev- The fermentation products of fructooligosaccharides els of butyrate (11). The physiological effects of the and are rich in acetate, but resistant starch is rich branched-chain fatty acids are still not well understood. in butyrate.

What Quantities of Short-Chain Fatty Acids Are Pro- Where Do Short-Chain Fatty Acids Go? duced Short-chain fatty acids produced in the colon are Short-chain fatty acids are derived from nondigestible readily absorbed via the colonic mucosa (Fig. 2). Pro- substances that have reached the large intestine. The tonated nonionic short-chain fatty acids are diffusionally energy reserved as short-chain fatty acids is maximally absorbed into the colonic mucosal cells, and ionized 40 to 50% of gross energy in the nondigestible sub- short-chain fatty acids are absorbed mainly across the stance, which is much higher than the energy released colonic epithelial cell membrane with exchange to bi- as gases or heat during fermentation. From the quan- carbonate (21). However, absorptive mechanisms tity of nondigestible substances reaching the colon, we of short-chain fatty acids are still not fully understood. can estimate that the daily production of short-chain Acetic and propionic acids across the membrane are fatty acids in human is 20 to 30 g. The concentration of transported through the epithelial cells into the portal total short-chain fatty acids in the colonic contents is blood. The absorbed fermentation products are metabo- more than 100 mmol/l, which is extremely high corn- lized mostly in the liver and muscle. In contrast, bu- pared with concentrations of other metabolites in the tyrate is consumed within the colonic epithelial cells. body. is usually the most abundant, fol- The mucosal cells prefer butyrate as an energy source. lowed by propionic and butyric acids. Other short-chain The energy provided by colonic fermentation contrib- fatty acids isobutyric, valeric and isovaleric-are utes 2 to 10% of the body's daily energy consumption. PHYSIOLOGICAL EFFECTS OF SHORT-CHAIN FATTY ACID PRODUCED FROM PREBIOTICS IN THE COLON 37

Polydextrose, which is also a soluble and low-viscos- ity fiber, has much lower available energy than guar gum or gum arabic. This agrees with the results that short-chain fatty acid contents in the cecum of rats fed polydextrose is lower than in rats fed guar gum (13). The available energy of is very low because this dietary fiber usually has very low fermentability. This figure also demonstrates that available energy in nondigestible sugars in humans (vertical axis) is very similar to those in rats (horizontal axis). We also should note that fermentability, nearly equal to energy avail- ability, is not constant and may vary with host condi- tions, such as illnesses, mental stress, physical activity, and drug administration.

Butyrate Is an Essential for the Colonic Mu- cosal Cells As described above, butyrate is consumed by the absorptive (epithelial) cells in the large intestine as en- ergy. Actually, butyrate is important more than as just an energy source for the colonic epithelial cells. It is an indispensable nutrient for the colonic cells. Defective butyrate metabolism in the colonic mucosal cells is re- ported to be a cause of (27), the inci- dence of which has been increasing worldwide. We know the importance of butyrate produced by colonic Fig. 3. Availableenergy in nondigestiblecarbohydrates (20). fermentation because the colonic mucosal cells con- sume only butyrate when and glutamine coex- ist (26). Colonic fermentation is the sole source of bu- Table 3 shows the metabolizable energy (the amount tyrate for the colonic mucosa because ingested butyrate of energy the body gets from each nutrient) and the is totally absorbed in the stomach and the small intes- respiratory quotient (RQ) of short-chain fatty acids. The tine. Moreover, fermentation products are the only avail- metabolizable energy of short-chain fatty acids is higher able energy source for mucosal cells in the colonic lu- than that of sugar (4 kcal/g) and lower than that of men. It is interesting that only butyrate within the prod- (9 kcal/g). The energy value of butyrate, the longer car- ucts is indispensable for the colonic mucosal cells. bon-chain acid, is higher than in the shorter carbon- chain acids, propionate and acetate. The RQ of acetate SHORT-CHAINFATTY ACIDS ARE HEALTH- and butyrate are 1.0 and 0.8, respectively, which indi- PROMOTING AGENTS cates that acetate is metabolized like sugar and butyrate Short-chain fatty acids have various physiological like fat. effects not only on the colon, but also on the metabo- Different nondigestible sugars have different quan- lism of the whole body. Once they are absorbed, these tities of metabolizable energy (Fig. 3). The available acids directly affect the body's metabolism. Short-chain energy in these sugars depends on their fermentability fatty acids may also affect some functions of tissue with colonic microbial flora. Guar gum and guar Ara- besides the colon through the enteric endocrine and ner- bic, two water-soluble dietary fibers, have the highest vous systems or the gut . metabolizable energy. Intestinal microorganisms almost The physiological effects of short-chain fatty acids entirely degrade and ferment these fibers. The value differ depending on certain factors. Their amount and shown with these soluble fibers, just below 2 kcal/g, the proportion of each of the three major ones are chief may be the maximum available energy of nondigestible determinants of the physiological effects of colonic fer- sugars. The rate of metabolizable energy of fructooli- mentation. The effects of short-chain fatty acids are gosaccharides is similar to that of these soluble fibers. usually dose-dependent, and each of the three major 38 H. HARA.

Fig. 4. Calcium absorption across the striped cecal mucosa by using Ussing chamber system (24).

Mucosal fluid, Ca 10 mmol//, serosal fluid, Ca 1.25 mmol/l, 37•Ž for 30 min.

ones has a different action on cells. Moreover, the rate pionate (1 to 30 mmol/l), the absorptive rate was twice and site of short-chain fatty acid production also influ- that with no addition. In the colonic striped mucosa, ence the actions. A faster production rate seems to en- the rates of transepithelial calcium transport were lower hance mineral absorption in the large intestine. To work than those in the cecal mucosa (8-25 nmol/min •E cm2). as a cancer-preventing agent, butyrate must be produced The stimulative effect of butyrate was higher than that in the distal site of the large intestine because the gen- of acetate in the colon, which is the opposite of the eration of colon cancer in the distal parts of the colon is order in the cecal mucosa. The addition of glycerol, limited (22). polyethylene glycol, or HCl had no effect on calcium absorption, which shows that only short-chain fatty Promotion of Intestinal Calcium Absorption acids (at least organic acids with the carboxy group)

Recently, fermentable nondigestible saccharides have stimulate calcium absorption via a paracellular path- become known to enhance the absorption of calcium, way. magnesium, and iron in the large intestine (4, 12, 25). Most reports have proposed that acidification with in- Effects of Short-Chain Fatty Acids on Cholesterol Syn- creasing fermentation dissolves these minerals flowing thesis into the large intestine and increases their absorption It is well known that soluble dietary fibers lower

(32). However, we suggest that the products of fermen- plasma cholesterol concentration (3, 19). Effective fi- tation, short-chain fatty acids, are more important fac- bers are mostly highly fermentable. Decreases in the tors for the promotion of mineral absorption than lumi- intestinal absorption of cholesterol and bile acids with nal acidification is (14, 15). Trinidad et al. (30) showed fiber itself are known to contribute to the reduction of that propionate and acetate increased calcium absorp- plasma cholesterol. It is proposed that short-chain fatty tion with human colonic perfusion experiments. In a acids produced from these fibers contribute to the cho- previous study, we made it evident that all short-chain lesterol reduction via another mechanism. Propionate fatty acids directly stimulate calcium absorption across suppresses cholesterol synthesis in the hepatic cell cul- the rat-stripped cecal mucosa via a paracellular path- ture (31). Short-chain fatty acids produced by colonic

way by using the Ussing chamber system (Fig. 4) (24). fermentation are absorbed into the portal blood and The addition of each short-chain fatty acid increased reach the liver; however, the effective concentration of calcium transport dose-dependently from the mucosal propionate for the cultured cells is high. It has not been side to the serosal side of the cecum, and the absorp- clarified whether the physiological concentration of

tive rate with 100 mmol/l acetate was four times higher short-chain fatty acids suppresses in vivo cholesterol than that with no addition. Within the physiological synthesis.

concentrations of acetate (10 to 100 mmol//) and pro- We showed in rats that in vitro fermentation prod- PHYSIOLOGICAL EFFECTS OF SHORT-CHAIN FATTY ACID PRODUCED FROM PREBIOTICS IN THE COLON 39

Fig. 5. Diurnal changes in plasma short-chain fatty acid (sum of acetic, propionic and butyric acids) and cholesterol concentrations on the 14th day in rats fed fiber-free, sugar-beet fiber (100 g/kg diet) and short- chain fatty acid mixture diets (9). Mean values at a time not sharing a common letter are significantly different between diet groups (p < 0.05). ucts of sugar-beet fiber by cecal bacteria reduced plasma (Fig. 6) (9). In an in vitro measurement using liver slices cholesterol concentration to a degree similar to that of of rats fed the SCFA diet for 2 weeks, the value of the sugar-beet fiber feeding (10). Sugar-beet fiber is a highly synthesis rate represents cholesterol synthetic fermentable dietary fiber. We also demonstrated that activities, not the actual synthesis rate in vivo. The re- short-chain fatty acids in the fermentation products sult of the in vitro experiment is reasonable because a contribute fully to cholesterol reduction (10). Figure 5 decrease in body cholesterol induces enzymes for cho- shows diurnal changes in the sum of the three major lesterol synthesis by a feedback system. Short-chain short-chain fatty acids and cholesterol concentrations fatty acids did not exist in the cells synthesizing cho- in the portal blood of rats fed diets with or without a lesterol in liver slices used in the in vitro study, but short-chain fatty acid mixture (9). The level of each they did exist in in vivo measurement. This is the rea- short-chain fatty acid in the diet is that produced by the son for the difference between the results of the in vitro fermentation from 10% sugar-beet fiber in the diet. and in vivo experiments and reveals that short-chain Concentrations of short-chain fatty acid and cholesterol fatty acids actually suppress the cholesterol synthesis in the blood were reciprocally changed after a feeding without decreasing the enzyme levels of cholesterol syn- of the SCFA diet, as shown in Fig. 5. Plasma short- thetic pathways. chain fatty acid levels in the rats fed a sugar-beet fiber diet are also much higher than those in the SCFA- and Suppressive Effects of Butyrate on Colon Cancer fiber-free groups. These findings reveal that short-chain Dietary fiber is well known to lower the incidence of fatty acids are absorbed from the intestine lower plasma colon cancer (2), and many studies using experimental cholesterol in vivo and are at least partly responsible animals showed that nondigestible polysaccharides sup- for the cholesterol reduction as a result of sugar-beet press the generation of colon cancer (6, 18). Several fiber feeding. mechanisms have been proposed for this suppressive We have obtained conflicting results on cholesterol effect of dietary fibers. The possible mechanisms are synthesis between in vitro and in vivo studies: Short- listed in Table 4. Among them, the effect of butyrate chain fatty acid feeding increases hepatic cholesterol produced by colonic fermentation may be important synthesis in vitro and decreases the synthesis in vivo because butyrate affects the various stages of colon can- 40 H. HARA

Fig. 6. Liver cholesterol synthesis rate in vitro in liver slices and in vivo in rats fed short-chain fatty acid (SCFA) mixture diet or fiber and SCFA-free diet, for 14 day (9). The synthesis rate was determined by measuring the incorporation of 3H20 into digitonin-precipitable sterol. Each value is the mean for six rats, respectively. Mean values not sharing a common letter are significantly different between diet groups (p < 0.05).

Table 4. Possible mechanisms by which nondigestible Table 5. Number of aberrant crypt foci (ACF), substances could suppress colon cancer. total aberrant crypts (AC) induced by 1,2- dimethylhydrazine in rats intracolonically injected with butyrate.

Values are means •} SEM, n = 8-10. * Significant difference from 0 mm (control) group

(p<0.05). Ishizuka, S. et al. (unpublished data) cer development. Butyrate inhibits colonic epithelial Table 6. The gut functions affected by short-chain fatty acid. cell proliferation, induces apoptosis in genetically dam- aged cells, and induces both the differentiation of mu- tated cells and changes in (1, 8, 29). Isobutyrate, a fermentation product of butyrate , has no such effects (16). Aberrant crypt foci are precancerous lesions on the colonic mucosa with hyperproliferation of the colonic epithelial cells. Challenge by chemical carcinogens in- duces the aberrant crypt foci (23). We previously dem- unpublished data). onstrated that repeated low doses of ionizing radiation Short-chain fatty acids have many stimulative or sup- also generate the aberrant crypt foci on the colonic pressive effects on the gastrointestinal functions. The mucosa (17). Butyrate instillation on the distal colonic effects are listed in Table 6. Almost all seem to pro- lumen dose-dependently reduced aberrant crypt foci mote digestive, absorptive, and protective functions of produced by dimethylhydrazine (Table 5, Ishizuka, S., the alimentary tracts. PHYSIOLOGICAL EFFECTS OF SHORT-CHAIN FATTY ACID PRODUCED FROM PREBIOTICS IN THE COLON 41

products of sugar-beet fiber by cecal bacteria lower plasma SHORT-CHAIN FATTY ACIDS AND PREBIOTICS cholesterol concentration in rats. J Nutr 128: 688-693. Gibson and Roberfroid (7) define a as a (11) Hara H, Miyashita K, Ito S, Kasai T. 1996. Oxidized ethyl "nondigestible food ingredient that beneficially affects linoleate induces mucosal hypertrophy of the large intes- tine and affects cecal fermentation of dietary fiber in rats. J the host by selectively stimulating the growth and/or Nutr 126: 800-806. activity of one or a limited number of bacteria in the (12) Hara H, Nagata M, Ohta A, Kasai T. 1996. Increases in colon." In the present study, almost all thusly defined calcium absorption with ingestion of soluble dietary fibre, prebiotics promote bifidobacteria and Lactobacillus guar-gum hydrolysate, depend on the caecum in partially growth. These bacteria themselves produce only acetate nephrectomized and normal rats. 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