Effect of purified cellulose, pectin, and a low-residue diet on fecal volatile fatty acids, transit time, and fecal weight in humans1 Gene A. Spiller, Miriam C. Chernoff, Rebecca A. Hill, Joan E. Gates, Jorge J. Nassar, and Elizabeth A. Shipley ABSTRACT Some relationships have been proposed between fecal volatile fatty acids, transit time, fecal weight, and dietary fiber intake. In this study, the effect of purified cellulose, purified pectin, and a natural low-residue diet on fecal acetic, propionic, butyric, isobutyric, valeric and isovaleric acids, transit time, and fecal weight was investigated. Forty-two healthy male and female adults were fed low-residue diets for 2 weeks, followed by 3 weeks on the same diet plus either 1 g/day cellulose or 6 g/day pectin or a sucrose placebo. Feces were collected for 7 days during weeks 2 and. From week 2 to, transit time decreased by 2. days with cellulose, 0.2 with placebo, and increased by 0.1 with pectin; fecal weight increased by 3 g/day with cellulose but decreased by 1 g/day with placebo and by 0.32 g/day with pectin (P < 0.0); volatile fatty acids decreased with placebo (-1.2 g/7 days), but increased with cellulose (+ 1.3 g/7 days) and pectin (+0.6 g/7 days) (P < 0.0). Volatile fatty acid changes with placebo and cellulose paralleled changes in fecal weight, thus fecal volatile fatty acid concentration did not change. Conversely, volatile fatty acid increase of the pectin group was not paralleled by fecal weight increase, signifying an increase in volatile fatty acid concentration (P < 0.0) of possible physiological significance. Some volatile fatty acids are probably absorbed, a fact worthy of further investigation. This study confirms: 1) that generalization of the effects of dietary fiber on volatile fatty acids, fecal weight and transit time should be avoided, 2) the mild antidiarrheal effect of pectin and, 3) the bulkung properties of cellulose. Am. J. Clin. Nutr. 33: 7-79, 1980. Organic anions from volatile fatty acids (VFA s) are the major solute in the feces (1) and most likely they are the product of digestion of dietary carbohydrates that reach the cecum undigested by gastric and intestinal enzymes. In health, the major carbohydrate polymers reaching the cecal region of humans are constituents of dietary fiber (2). In the cecum extensive fermentation by colonic microorganisms yields various volatile fatty acids, some of which are probably absorbed in their passage through the colon and some of which are excreted as part of the solutes of the feces. The degree of digestion of various plant fiber polymers by bacteria differs depending on the chemical and physical structure of the plant fiber and has been the object of many studies by animal nutritionists (3) but of only limited studies by human nutritionists (, ). In this study, to clarify some of the basic physiological effects of dietary fiber, it was decided to use two highly purified fiber polymers (pectin and cellulose) and to investigate their effect on fecal volatile fatty acids (FVFA s), transit time, and fecal weight in healthy human subjects fed a natural lowresidue diet. These two polymers were chosen because cellulose is known to be only partially digested by colonic bacteria while pectin is practically totally digested (6-8). In addition, the effects of a low-fiber diet were studied for comparison. Materials and methods Subjects and diets Forty-two healthy male and female adults, all residents of the San Francisco (Calif.) Bay region, aged 23 to 60 years, were selected for this study. They were chosen on the basis of slow intestinal transit time and low fecal output while they were consuming their normal From the Department of Nutritional Science, Syntex Research, Palo Alto, California 930. 7 The American Journal of Clinical Nutrition 33: APRIL 1980, pp. 7-79. Printed in U.S.A.
FECAL WEIGHT AND FIBER INTAKE 7 diet. These parameters were determined by daily fecal collection during a screening period of days. Anyone with a history of major gastrointestinal disease, diabetes, or under treatment for such diseases was excluded. All subjects were within ±20% of their ideal body weight. Subjects were not allowed to take any medication that could affect gastrointestinal function for at least 2 weeks before and during the study and were placed on a lowresidue diet for the entire study period. This diet allowed only refmed breads and cereals, meats, milk products, and a limited selection of cooked vegetables and canned fruits (two servings per day). All whole grain products, legumes, raw vegetables, and raw fruits were excluded from the diet. Study design The subjects adhered to the restricted diet without taking any treatment for the base-line period (days I to 1) and collected feces daily on days 8 to 1 (week 2). Subjects were then assigned to treatment groups so that mean fecal weight and transit time were similar for all groups. The treatment period consisted of days 17 to 1, with daily fecal collections occurring on days 2 to 31 (week ) and on days 3 to 1 (week ). Treatments During the treatment period (days 17 to 1), subjects ingested either 1 g/day of cellulose (Solka-floc, manufactured by Brown and Co., Berlin, N.H., 99% cellulose on a dry basis), 6 g/day of pectin (Pectin, N.F., a highmethoxyl undiluted citrus pectin, manufactured by Sunkist Growers, Ontario, Calif.), or a sucrose nonfiber placebo. All the treatments were in powder form and were flavored with sucrose and natural lemon flavor. It appeared reasonable to feed much less pectin than cellulose to somewhat simulate the ratio of these polymers in a mixture of unrefmed carbohydrate foods, even though the amounts and ratios in actual diets are not readily quantifiable and certainly extremely variable. The daily dose of the appropriate treatment was mixed with 10 oz of water before ingestion and taken after the evening meal. Physiological measurements and assays Intestinal transit time was measured according to the method of Hinton Ct al. (9). Transit markers, in the form of radioopaque, barium impregnated pellets (Portex Ltd., Kent, England), were administered to the subjects on day 8 of the base-line period and days 2 and 3 of the treatment period. All fecal samples were collected and x-rayed for the next 7 days to count the numbers of markers excreted: 80% appearance of the markers in the feces was taken as the mean transit time. After x-ray, the fecal samples were frozen. Before preparation of the homogenate, the fecal material was allowed to thaw and deionized water was added. Using a Polytron Model PT-lO-3 (Brinkman Instruments, New York, N.Y.) with a generator mixing head Model PT-3 at high speed for 2 min, the fecah material was dispersed until completely homogenized and 1% of formaldehyde solution (37%) was added. Aliquots of the homogenate were stored frozen and were used for subsequent analyses. All the fecal analyses were carried out on this 7-day fecal composite. FVFA assays were performed by gas liquid chromatograph (830A Reporting Gas Chromatograph, Hewlett Packard, Palo, Alto, Calil) equipped with flame ionization detector using a 6 ft by V in id glass column packed with 10% SP-l200/l% H3PO on 80/100 chromosorb WAW. Column temperature was programed from 80 to 10 C at 20 C/min. To 1 g of fecal homogenate. 0.1 ml of 8% H3PO and a known amount of caprylic acid (internal standard) were added and then extracted three times with anhydrous ethyl ether, centrifuged, and the ether layer transferred to a 10-nil volumetric flask and diluted to the mark with ether. Three microhiters of the ether extract was injected directly onto the column. Calibration and calculation were made by the internal standard method. Calibration curves were obtained for acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids by chromatographing standard solutions at five different concentration levels. The calibration curves were prepared by plotting the area ratios (weight of active per weight of internal standard). Results Total fecal wet weight Total fecal wet weight increased from baseline to treatment for the purified cellulose group (Tables 1 and 2). Total fecal wet weight decreased from base-line to treatment for the pectin group and for the placebo group. The decreases of the pectin and placebo groups were significantly different from the increases of the cellulose group (F> 0.0). Mean fecal output changes from base-line to week were similar to changes from baseline to week for all treatment groups (P = 0.1 for analysis of variance test of period effect). Intestinal transit time Only subjects with base-line transit times greater than or equal to 3 days were included in the analysis of transit time. Thus, each treatment group when analyzed for transit times contained only 10 subjects. Base-line mean transit times were comparable among treatment groups (P = 0.069). They ranged from.9 to.7 days. The mean decrease in transit time for the cellulose treatment group was significantly different from the changes of both the placebo and the pectin groups (F> 0.0). The pectin and the placebo treatment groups had comparable transit time changes. The transit time changes from base-line to
76 SPILLER ET AL. TABLE 1 Treatment group mean values for transit times, fecal weight, and VFA s in feces for the 3 collection weeks Variable Period Cellulose Pectin Placebo Transit time (days) Wk 2 (base-line).1 ± 16b 2.7 ± 1.1 2.6 ± 1.0.9 ± 1.7.2 ± 2.6.0 ± 2.2.9 ± 1.6.0 ± 1.2.8 ± 2. Total fecal wet weightc (g/7 days) Wk 2 (base-line) Total VFAC (g/7 days) Wk 2 (base-line) 7.1 ± 17.3 719. ± 213.8 682.0 ± 276. 3.0 ± 2.. ± 2.7.3 ± 3. 38.8 ± 118.9 382.3 ± 26.8 378.7 ± 187.9 2.7 ± 2.6 3.8 ±.3 3.3 ± 3.7 86.1 ± 12.8 08. ± 120.6 37.9 ± 180.0 3.6 ± 1.6 3.0 ± 1. 2. ± 1. Percentage VFAC Wk 2 (base-line) 0.61 ± 0.29 0.66 ± 0.2 0.72 ± 0.2 0.61 ± 0.23 0.83 ± 0.1 0.72 ± 0.21 0.9 ± 0.2 0.7 ± 0.9 0.6 ± 0.23 Sample sizes for transit time were: 10-cellulose, lo-pect, and 10-placebo. 6Mean ± SD. csample sizes were: 13-cellulose, 12-pectin, and 1-placebo (one and two subjects were lost on cellulose and pectin, respectively, for reasons unrelated to the study.) TABLE 2 Treatment group mean values for 7 days for total amount of individual VFA s in feces for the 3 collection weeks Variable Period Cellulose Pectin Placebo Acetic (g) Wk 2 (base-line) Wk Propionic (g) Wk 2 (base-line) lsobutyric (g) Wk 2 (base-line) Butyric (g) Wk 2 (base-line) Isovaleric (g) Wk 2 (base-line) Valeric (g) Wk 2 (base-line) 1. ± 1.1 2.1 ± 1.2 2.1 ± 1.6 0. ± 0. 0.9 ± 0. 0.9 ± 0.7 0. ± 0.6 0.8 ± 0.7 0.7 ± 0.8 Sample sizes were: 13-cellulose, 12-pectin, and 1-placebo. bmean ± SD. 1.3 ± 1.1 2.0± 2.3 1.7 ± 1.9 0. ± 0. 0.7 ± 0.8 0.6 ± 0.7 0. ± 0.6 0.7 ± 1.0 0.6 ± 0.7 1.8 ± 0.7 1.± 0.6 1.2 ± 0.7 0.7 ± 0.3 0. ± 0.3 0. ± 0.3 0.6 ± 0.3 0. ± 0.3 0. ± 0.2 0.2 ± 0.2 study week were not significantly different from those to study week (F = 0.31). Total FVFA s The mean changes from base-line to treatment in the total amount of FVFA s (gil days) differed significantly among treatment groups (P < 0.0). Total FVFA s for the placebo treatment group decreased steadily to weeks and from the base-line. Total FVFA s increased from base-line to both week and for the
FECAL WEIGHT AND FIBER INTAKE 77 cellulose and the pectin groups. The mean decrease in total FVFA s from base-line to weeks and for the placebo group was significantly different from the mean increases during the corresponding time period for the other two groups. These mean increases were comparable for the latter two groups. Concentration of FVFA s Base-line mean percent FVFA s ranged from 0.61% for the cellulose group to 0.7% for the placebo group. These values were not significantly different (P = 0.7606). The mean changes in percentage FVFA s from base-line to treatment were found to be different among the three treatment groups (P = 0.0). Percentage FVFA s for the placebo group decreased from base-line to week and to. Whereas the total FVFA s for the cellulose treatment group increased from base-line, the percentage FVFA s decreased from base-line to weeks and. The mean increase in percentage FVFA s for the pectin treatment was statistically significant (P < 0.0). Effect on individual fatty acids Individual FVFA s for all treatments and periods are given in Table 2. The major FVFA was acetic acid (approximately 0% of measured FVFA s) for all diet periods and all groups. Propionic and butyric acid were present in approximately equivalent amounts and appeared to be the most important components after acetic acid, each one of them present at approximately the 20% level. Insovaleric and valeric acids were present in small amounts (about % of each) and only occasionally traces of isobutyric were found. It is noteworthy that there was a slight change in the ratio of acetic to propionic acid as the total FVFA increased in the cellulose group. This ratio dropped from 3.0 to 2.3 from base-line to treatment periods. Discussion It becomes apparent from this study that we must carefully distinguish between the effect of a dietary fiber polymer on the total VFA s excreted in the feces and the effect on their concentration in the feces. Physiologically and clinically, these changes might have different significance in their effect on the microecology of the colonic lumen, colonic ph, and in general on colonic function. No information clearly emerged from this study on the possible interrelationship of FVFA and transit times. The fact that pectin increased concentration and total FVFA s but not fecal bulk is in agreement with the almost complete digestion of pectin by colonic bacteria in humans and other mammals (6, 7). Kay and Truswell (8) fed 1 g/day of pectin (over twice the amount fed in this study) and observed only a slight increase in fecal wet weight in agreement with our fmdings. Pectin, even though not statistically significantly, slows down transit time. This effect could be the reason for the efficacy of pectin as a mild antidiarrheal product. Cellulose induced a significant decrease in transit time, but did not change significantly the concentration of FVFA s. Because of the increase in fecal bulk induced by this polymer, there was a greater amount of FVFA s excreted than in the subjects on either pectin or placebo. Differently from the pectin group, there was no concomitant increase in percentage of FVFA s. Subjects who continued to eat a low-residue diet after the base-line showed a continuous and steady decrease in both total FVFA s and in their concentration. This agrees with the decrease in fecal weight and with no significant changes in transit time. In view of these results with the placebo group, it may be further hypothesized that the increase in FVFA s for the two fiber treatments is actually greater than it appears as, had these groups been on the low-residue diet without the addition of fiber, it is logical to suppose that their FVFA s would have actually decreased. The physiological significance of the amount of FVFA s excreted is not clear as we do not have a good understanding of the amount absorbed through the colonic mucosa and the relation of production/absorption! excretion as affected by speed of transit through various regions of the colon of humans. The percentage of acetic acid in the feces
78 SPILLER ET AL. agrees with values reported in the literature: Cummings (1) found that about 60% of the total FVFA is acetic acid and Grove et al. (11) found a range of acetic acid of to 60%. It is important to always consider that the amount of VFA s excreted in the feces is the difference between the amount produced by bacterial action and the amount absorbed through the colonic mucosa. Any factor affecting production (e.g., amount and type of fiber polymer present in the diet) and/or absorption (e.g., rate of transit from cecum to rectum) will affect VFA s output in the feces. Recently, McNeil et a!. (10) showed that acetate is absorbed from the human rectum. This study involved the insertion of dialysis tubing and there is now a need to combine the study of VFA excretion with the study of their absorption in vivo in humans. Animal studies have shown (3) the complex interactions among diet, absorption, and excretion and the clinical and physiological importance to develop methodologies applicable to the human appear important. Stevens (3) points out the effect on VFA production of the ratio of dietary fiber to starch in the diet in animals. This confirms that, if other dietary components reach the human colon, their effect cannot be overlooked and that their interaction should always be taken into account in studies of the effects of dietary fiber (12) especially in view of the power of fibrous polymers to adsorb or bind other dietary components. Despite the limited knowledge of the amount of VFA s absorbed, the variation of YFA s that remain as part of the fecal mass per se appears an imporant physiological and clinical fact for the possible effect of these volatile fatty acids on fecal elimination and on the mucosas of the rectum and of the sigmoid colon, which are the site of many diseases in humans. Whether the change from base-line to treatment in the cellulose group in the ratio of acetic propionic acid (two of the three major fecal organic acids analyzed in this study) from 3.0 to 2.3 has physiological or clinical significance is not known. It could be due to alteration in production or absorption or both. It is noteworthy that, in this study, as the total FVFA s increased or decreased, only cellulose induced a change of the ratio of acetic to propionic acid. Conclusion There is no doubt that dietary fiber polymers extensively affect colonic function. The measurement of changes in FVFA s, transit time, and fecal bulk are ways to study the effect of diet on the colonic environment and its possible relations to health and disease. These fecal measurements are important for practical reasons since it is difficult to study ceca! and colonic contents or the absorption of VFA s in vivo. The fact that the FVFA s are affected differently by different forms of dietary fiber (cellulose, pectin, and low fiber in this study), confirms that the effects of dietary fiber cannot be generalized (13). The individual fiber components must be examined for their effects on such parameters as feca! weight, transit time, and VFA s. Even though the effect of purification and isolation of fiber polymers on their physiological function is not well known, it appears that one must study isolated polymers as well as natural foods for both their importance in the knowledge of fundamental nutrition as well as for the possible dietary use of both purified and natural dietary fiber with their clinical implications. U The authors thank William C. Cooper, M.D., Ms. Carol Flatman, RN., Linda Kuly, RN., and Constance Northway, RN. of the Institute of Clinical Medicine, Syntex Research, for their assistance in screening subjects and for clinical support. References I. CUMMINGS, J. H. Absorption and secretion by the colon. Gut 16: 323, 197. 2. CuMMINGs, J. H. What is fiber? In: Fiber in Human Nutrition, edited by 0. A. Spiller and R. J. Amen. New York: Plenum, 1976, p. 1. 3. Smvzs, C. E. Physiological implications of microbial digestion in the large intestine of mammals: relation to dietary factors. Am. J. Cliii. Nutr. 31: 161, 1978.. SPILLER, G. A., J. A. Bix..a AND E. A. SHIPLEY. Recent progress in dietary fiber (plantix) in human nutrition. CRC Crit. Rev. Food Sci. Nutr. 10: 31, 1978.. KELSAY, J. L. A review of research on effects of fiber intake in man. Am. J. Clin. Nutr. 31: 2, 1978. 6. CAMPBELL, L. A., AND G. H. PALMER. Pectin. In: Topics in Dietary Fiber Research, edited by 0. A. Spiller and R. J. Amen. New York: Plenum, 1978, p. 10. 7. WERCH, S. C., R. W. YOUNG, A. A. DAY, T. E. FRIEDEMANN AND A. C. IVY. The decomposition of
FECAL WEIGHT AND FIBER INTAKE 79 pectin and galacturomc acid by intestinal bacteria. J. Infect. Diseases. 70: 231, 192. 8. KAY, R. M., AND A. S. TRUSWELL. Effect of citrus pectin on blood lipids and fecal steroid excretion in man. Am. J. Clin. Nutr. 30: 171, 1977. 9. HINTON, J. M., J. E. LnAIw-Jot &ns AND A. C. YOUNG. A new method for studying gut transit time using radioopaque markers. Gut 10: 82, 1969. 10. MCNEil., N. I., J. H. CUMMINGS AND W. P. T. Jms. Short chain fatty acid absorption in the human large bowel. Gut 18: A2, 1977. 11. GROVE, F. W., W. H. Ou.smD AND K. KOENING. The effect of diet and catharsis on the lower volatile fatty acids in the stools of normal man. J. Biol. Chem. 8: 127, 1929. 12. SPILLER, G. A. Interaction of dietary fiber with other dietary components a possible factor in certain cancer etiologies. Am. J. Clin. Nutr. 31: 231, 1978. 13. SPILLER, 0. A., AND J. E. OAThs. Defining dietary plant fibers in human nutrition. In: Nutritional Improvement of Food and Feed Proteins, edited by M. Friedman. New York: Plenum, 1978, p. 16.