ROBERT W. CULLEN ANDSUSAN M. OACE 2 Department of Nutritional Sciences, University of California, Berkeley, California 94720 ABSTRACT The purpose of this study was to compare the effects of various levels of two components of dietary fiber, cellulose and pectin, on the utilization of vitamin 612 by the rat. Compared with the fiber-free group, the rats fed purified diets of 40 or 50% of cellulose excreted about twice as much methylmalonic acid (MMA) after 10 weeks of Bi2 deple tion. In contrast, rats consuming purified diets containing 5% to 15% of pectin experienced a 9- to 21-fold elevation of group average MMA excre tion, respectively. Compared with the fiber-free group, rats offered celluloseor pectin-containing diets exhibited significantly greater fecal excretion of the radioactive Bia dose that was injected after several weeks of Bi2 deple tion. However, despite the large difference in MMA excretion between the cellulose and pectin groups, the two fibers supported surprisingly similar rates of fecal excretion of "Co. Urinary MMA excretion decreased significantly in all groups following the injection of 0.5 ^g of vitamin BIO, thus indicating that the observed defect in MMA metabolism was Bi2responsive. Mechanisms consistent with these results are discussed. These data indicate that two dietary fibers, cellulose and pectin, have a deleterious effect on vitamin Bi2 status which is due in part to increased fecal excretion of the vitamin. The dramatically greater influence of pectin than of cellu lose on MMA excretion emphasizes the importance of investigating sepa rately the metabolic effects of the various components of dietary fiber. J. Nutr. 108: 640-647, 1978. INDEXING KEY WORDS vitamin Bi2 deficiency •methylmalonic acid • dietary fiber •cellulose • pectin • intestinal flora fiber-Bi2 interaction Epidemiological evidence suggests that the diet of modern Western civilizations is correlated with the incidence of a number of diseases such as gastrointestinal disorders, heart disease and others. Burkitt et al. (1) have pointed to the relative lack of dietary fiber in the Western diet as a likely etiological factor in the devplnnmpnf-
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nf
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fiber supplements currently are very popular. However, little is known about potential
interactions
of dietary
vitamin status or about comparative effects of fibers of differing composition, Fiber, by virtue of its indigestibility by mammalian enzymes, is available in the lower intestine to support growth of the microbial population. Previous work in our Receivedfor publicationAugust25, 1977.
* Portions of these data were presented at the annual meetlng of the Federation of American so-
dettes for Experimental Biology, April 1977, Chicago,
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Methylmalonic Acid and Vitamin B,, Excretion of Rats Consuming Diets Varying in Cellulose and Pectin1
DIETARY FIBER AND B^ METABOLISM
MATERIALS
AND METHODS
Male, 28-day old Fischer rats4 were housed individually in mesh-bottomed me tabolism cages. After consuming a closed formula, cereal based stock diet5 for 3 days, groups of six rats were assigned to dietary treatments such that group average weights were not significantly different (ca. 70 g). Food and tap water were al lowed ad libitum throughout the study. Fresh food was given every 2 to 3 days; water bottles were changed two times a week. A vitamin Bi2-deficient purified basal diet ( table 1 ) served as our fiber-free con trol (FF). It was made in batches of less than 15 kg as needed throughout the study. Cellulose8 (C) was added to the FF diet to final concentrations of 10%, 20%, 30%,
TABLE l Composition of basai fiber-free diet diet20.070.05.03.51.00.5 g Soy-bean protein1Glucose monohydrate2Corn oil3Salt mix4Vitamin mix6D.L-Methionine6g/100 1Soya Assay Protein ; Teklad Test Diets, Madi son, Wisconsin. 2Cerelose; Corn Products, CPC International, Inc., Englewood Cliffs, New Jersey. 3Mazóla; Best Foods, CPC International, Inc., Englewood Cliffs, New Jersey. 4Provides : (in mg/100 g diet) CaCO3) 725; CaHPO4. 1130; Na2HPO4, 651; KC1, 730; MgS04, 230; MnS04 •H20,15.4; CuS04, 1.3; ferric citrate, 15.1; ZnC03, 2.1 ; KIOS, 0.1. 'In glucose, provides : (in mg/100 g diet) D-biotin, 0.2 ; folie acid, 1 ; thiamin • HC1, 1.5; riboflavin, 1.5; pyridoxine • HC1, 1.5; menadione, 1.5; nicotinic acid, 5; o-calcium pantothenate, 5.0; choline bitartrate, 100; (in IU/100 g diet) D,L-Otocopherol, 5; ergocalciferol, 125; retinyl acetate, 1,000. 6Nutrional Biochemical«Corp., Cleveland, Ohio.
40%, and 50%, and unhydrated pectin7 (P) was added to final concentrations of 5%, 10%, 15%, 20%, and 30%. All diets were stored in a freezer. Body weight and food intake were re corded three times a week. Twenty-fouror 48-hour fecal samples were collected periodically during the study for all rats. Twenty-four-hour urine samples were collected in vials containing 3 ml of 0.72 N H2SO4 (approximately 2 meq) in order to maintain an acid pH for stability of MMA and to inhibit bacterial growth. Funnels with a screen baffle and a small screen over the collection vial separated urine and feces. Urine samples were fil tered through Whatman number 1 filter paper into graduated cylinders and di luted to a standard volume, usually 30 ml. Urinary methylmalonic acid was analyzed colorimetrically 8 by the method developed 3 Chen, S. C. H. & Oaee, S. M. (1974) Decreased depletion rate and prolonged biogloglcal half-life of vitamin B12 in germfree rats. Federation Proc. 33, 605 (abstract). 4 Charles River Breeding Laboratories, Inc., Wil mington, Massachusetts. 6 Purina Rat Chow, Ralston Purina Company, St. Louis, Missouri. • Alpini rol. ICN Pharmaceuticals, Inc., Cleveland, Ohio. ' Citrus Pectin, ICN Pharmaceuticals, Inc., Cleve land. Ohio. 8 Spectronic 20 spectrophotometer ; Bausch and Lomb, Rochester, New York.
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laboratory 3 demonstrated that the vitamin Bi2 depletion rate is significantly greater in the presence than in the absence of in testinal microorganisms. It has also been shown that intestinal bacterial overgrowth, such as in blind loop syndrome, is accom panied by Bi2 depletion, presumably due to competition for available B12 between the gut flora and the host (2). It therefore seemed possible that fiber, by stimulating intestinal bacterial growth, might increase the rate of vitamin Bi2 utilization of the host animal. To test this, groups of rats were assigned to Bi2-deficient diets differ ing in level and type of two dietary fiber constituents, cellulose and pectin. Two approaches were used to study the interaction of dietary fiber with B12 metab olism. First, urinary methylmalonic acid (MMA) was measured at intervals during the first 10 weeks of B12 depletion. MMA accumulates when there is a block in the propionate metabolic pathway at the BJ2containing enzyme methylmalonyl CoA (MMCoA) mutase (3). Urinary MMA ex cretion is elevated in pernicious anemia in humans ( 4, 5 ) and in experimental vitamin B12 deficiency in rats (6-8). Relatively higher urinary MMA of a particular fiber level would be taken as an indicator of greater vitamin Bi2 depletion. Secondly, a small tracer dose of 57Co-cyanocobalamin was injected. Urinary and fecal excretion of radioactivity would reflect any alteration in the rate of vitamin elimination.
641
642
ROBERT W. CULLEN
AND SUSAN M. OACE
TABLE 2 Responses to cellulose and pectin diets Diet1
Weight gain
Food efficiency consumed0.335 total food consumed
weeks128 FF (12)!IOC
(6)10P (6)15P (6)20P (6)30P (6)g/4
±0.0040.352 ±0.00840.238±0.00740.202±0.00440.177±0.00340.322 ±0.0100.340±0.0100.336
±5108 ±64129±791
±0.0060.354±0.0060.340±0.0040.318±0.0090
±0.0040.286±0.00840.197±0.01340.129±0.0234rats
±7*50 ±4431 ±64(allgam:
±0.015»0.161 ±0.0294and lost weight during first0.335 second weeks)
1FF = fiber-free control diet (described in table 1) ; C = cellulose; P = pectin; number preceding letter indicates percent of fiber in diet (see text). 2 Number of animals per group. *Mean±SEM. 4 Group means which differ significantly from the control mean, P < 0.05. Nested analysis of variance (réf.23, pp. 144-147) and Dunnet's multiple range test (24).
by Oace and Chen (9) using automated sampling and pumping equipment.9 It is an adaptation of the original colorimetrie method of Giorgio and Flaut (10). Onetenth of each urine sample was applied to an ion exchange resin column 10 after ad justment to pH 6.5 with NaOH. Columns were rinsed twice with 50 ml volumes of deionized water. MMA was eluted with 30 ml of 0.1 N HC1. The eluant was ana lyzed directly or diluted appropriately with 0.1 N HC1 so that it fell on the stan dard curve. Creatinine was used as a basis to ex press MMA excretion in order to correct for occasional incomplete or spilled col lections. An automated modification of the Jaffe reaction utilizing picric acid and NaOH (11) was used to assay creatinine. An autoanalyzer was used for this analy sis.8'9 After 10 weeks of B12 deprivation, repre sentative groups (FF, 20C, 40C, 5P, and 10P) were injected intraperitoneally with 0.1 jug of B12 containing 0.87 /«Ciof 57Cocyanocobalamin (179 ^.Ci/^g in 0.9% benzyl alcohol).11 All other groups were injected with an equivalent amount of nonradioactive vitamin Bia.12 Urine and feces were collected quantitatively from "Co-Bi2-injected rats for 5 days. Urine samples were filtered and diluted to 30 ml as for MMA analyses. Three-milliliter
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(6)20C (6)30C (6)40C (6)50C (6)5P
gain: fiber-free diet
±53137±7128±6118±2112 ±0.0040.308±0.00440.282 ±0.0040.342
samples were counted in 16 X 25 mm poly styrene counting vials.11 Feces from rats consuming FF, 5P, and 10P diets were counted directly in the counting vials since volumes were less than or equal to 3 ml. Counting efficiency varied little with this range of volumes. Feces from rats fed 20C and 40C diets were weighed, thoroughly ground in a mortar and pestle, and a weighed portion was counted. Samples were counted in a well-type gamma counter.13 All values were expressed as a percentage of the injected dose by com paring samples to standards made from the injection solution. RESULTS The growth and food efficiency of the experimental groups during the first 4 weeks of the study varied with type and amount of dietary fiber. Up to 40% cellu lose did not affect body weight gain sig nificantly during this period (table 2). Weight gain averaged 4 to 5 g/day. Fifty percent cellulose slightly depressed body • Proportioning Pump and Sampler ; Technicon Instruments Corporation, Chauncey, New York. 10AG-3, 200-400 mesh chloride form ; Bio-Rad. Lab., Richmond, California. "Amersham Corporation, Arlington Heights, Illinois. " Merck and Company, Inc., Rahway, New Jersey. u Nuclear-Chicago, Model 1085 ; G. D. Searle and Company, Des Plaines, Illinois.
DIETARY FIBER AND B* METABOLISM
TABLE 3 Effect of dietary fiber consumption on fecal mass Diet1FF
weightg/48
consumedg/48
hr1.54±0.1335.03±0.424 hr—3.72±0.254 (12)2IOC (6) 20C (6) 300 (6) 40C (6) 50C (6)5P 10P 15P 20P 30P
(6) (6) (6) (6) (3)Fecal
8.88±0.344 7.67±0.224 15.2 ±0.70* 13.5 ±0.594 20.9 ±0.554 19.4 ±0.544 27.8 ±1.442.17±0.28 25.8 ±1.7«1.88±0.08< 1.60±0.11 1.22 ±0.18 1.88±0.37 0.53±0.18Fiber
2.9C±0.144 3.22 .^0.154 4.23±0.244 4.30±0.204
1See footnote 1, table 2. 2 Number of animals per group. 3MeanisEM. 4Group means which differ significantly from the control mean, P < 0.05. Nested analysis of variance (réf.23, pp. 144-147) and Dunnet's multiple range test (24).
Rats offered the 5% pectin diet experi enced the same weight gain and food ef ficiency as did the fiber-free controls dur ing the first 4 weeks (table 2). Subse quently, their weight gain was depressed significantly compared to the FF controls, perhaps due to intermittent diarrhea. Higher levels of pectin progressively and significantly depressed weight gain and food efficiency as well as food intake. The fiber-free food efficiency of these groups indicates a decreased utilization of the food consumed as pectin levels increase. In contrast to cellulose, pectin disappeared from the intestinal tract. Pectin did not increase fecal weight (table 3), indicating that it was metabolized by intestinal bac teria. Rats fed the highest level of pectin failed to gain weight. After 1 week, one rat died and survivors were transferred to a fiber-free diet during the second and third weeks. Methylmalonic acid excretion was mea sured after 6 and 10 weeks of vitamin Bt2 deprivation. Among groups assigned cellu lose diets, very little difference in MMA excretion occurred during the first 5 weeks (table 4). Even at 10 weeks, only the highest level of cellulose, 50%, was asso ciated with a significant increase in MMA excretion. In contrast, each level of pectin was ac companied by a significantly greater in-
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weight compared with the fiber-free and other cellulose groups. When calculated using the weight of diet consumed, food efficiency of the cellu lose groups decreased proportionally with increasing fiber; food efficiency based on the amount of fiber-free diet consumed was equivalent for all cellulose groups (table 2). Since the fiber-free food effi ciency ratios were the same, the observed differences in body weight appeared to be due to a lower intake of energy and not to a deficiency of other essential nutrients. The high-cellulose groups did not increase food intake sufficiently to overcome the energy dilution of their diet. These rats appeared to be less fat but not smaller than the rats fed lower levels of cellulose. However, body composition data were not collected. Another implication of the fiberfree food efficiency data is that our source of cellulose was not appreciably metab olized by intestinal bacteria. This conclu sion is further supported by an analysis of the fecal weights of rats fed cellulose. Compared with the group average 48-hour fecal weight of rats consuming FF diet, fecal weights of cellulose-fed rats increased equivalent to the amount of cellulose con sumed (table 3). When fecal weight is plotted versus cellulose consumed, the simple linear regression equation is: Y = 1.01 X + 1.44, where Y = fecal weight in g, and X = g of cellulose consumed The slope, which is near unity, predicts com plete excretion of cellulose; and the Y-intercept (1.44), which predicts fecal weight when cellulose consumption is zero, is rea sonably close to the observed data from the FF group (1.54 g). The correlation coefficient ( r = 0.996 ) indicates that linear ity was significant. Since cellulose content of feces was not measured directly, it is possible that some cellulose was digested by bacteria and that the increased fecal weight and decreased gross food efficiency of cellulose-fed rats resulted from increased excretion of other carbohydrate, protein and lipid. However, the appearance of the fecal material, which was progressively whiter and more powdery as the cellulose content of the diet increased, suggests that at least a substantial proportion of the cellulose was not digested.
643
644
ROBERT W. CULLEN AND SUSAN M. OACE TABLE 4 Urinary methylmalonic acid excretion Length of vitamin BJ2 depletion 5 weeks
(12)2
timóleMM A/mg creatinine 7.6± 1.0s 14± 2.0 1.26.6± 0.56.6± 0.86.1± 0.76.6± 0.655
(6)20C IOC (6)30C (6)40C (6)50C (6)5P (6)10P (6)15P (6)20P (6)30P/FF (5)8.6±
±11*131 8.44221 ± ±18"293 ±34'84 ±24413±
10 weeks
Response to 0.5 f vitamin Bn % depression at 5 days 67.3
2.517± 2.617± 3.221± 4.831 3.5«122± ± 5.1«165±264300±554 (4)73.8 (4)777» (1)62.9 (1)117±32462.850.665.456.851.551.774.8
1See footnote 1, table 2. 2Number of animals per group at 5 weeks; number of survivors at other collection periods in parentheses if different. 3Mean±sEM. 4Group means which differ significantly from the control mean, P < 0.01. Single-factor analysis of variance (réf.23, pp. 133-137) and Dunnet's multiple range test (24).
crease in MM A excretion (table 4). The former 30P group, which consumed pectin for less than 3 weeks and was thereafter fed a diet lacking fiber, excreted MM A at a level significantly greater than the fiberfree controls. The fecal and urinary "Co excretion data of the representative groups (FF, 20C, 40C, 5P, 10P) injected with radio active BIOare presented in table 5. Urinary "Co excretion during the 5-day collection period was only about 2% to 3% of the dose for all groups. However, variability TABLE 5 Cumulative excretion of "Co for five days following injection
Diet1
Urine
Feces
FF (5)2
% of injected dose 2.32±0.04'.« 9.75±0.85°
20C (6) 40C (6)5P
±1.0' 20±0.02' 2.02±0.04b2.39±0.09° ±0.78C22.5 22.9
(6) 10P (6)2.
±1.5« 2.91±0.10£15.021.3 ±2.9«
1See footnote 1, table 2. 2Number of animals per group. 3Mean±sEM.Means in same vertical column not sharing a common superscript letter differ significantly, P < 0.05. Nested analysis of variance (réf. 23, pp. 144-147) and Newman-Keuls' multiple range test (.réf. 23, pp. 151-155).
within groups was small and compared with the FF group, rats fed 10% of pectin excreted a significantly greater and those consuming 40% of cellulose excreted a significantly smaller proportion of the dose. In contrast, both fibers signifi cantly increased fecal excretion of "Co. Over 20% of the "Co was recovered in the 5-day fecal collection of the 40C, 5P, and 10P groups, whereas control FF rats ex creted only about 10% of the dose by this route. The final phase of this experiment is pre sented in table 4. All rats were injected with 0.5 /xg of vitamin B1212 and again urinary MMA was measured. A significant decrease in urinary MMA occurred in all groups. DISCUSSION Fecal "Co. Normal Bi2 metabolism in the rat involves secretion of the vitamin into the intestine with bile (12) and fairly efficient reabsorption by an intrinsic factormediated mechanism in the ileum (13). Body stores of Bi2 could become depleted if this enterohepatic recycling were pre vented. In our study, both cellulose and pectin increased fecal excretion of Bi2, presum ably by interfering with reabsorption of the vitamin. Although we have no direct in-
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FF
Diet1
DIETARY FIBER AND
645
pionate in the cecum. Furthermore, in situ ations where bacterial fermentation and production of propionate would be ex pected to be minimal, such as germfree rats (8) or conventional rats that have been fasted (7) or treated with antibiotics (20), excretion of MMA is very low, de spite B12 deprivation. In our study, vitamin B12-responsive urinary MMA excretion was increased by both cellulose- and by pectin-containing diets but this occurred much earlier and to a greater extent with pectin than with rats (14). cellulose. We have ruled out a trivial ex planation that pectin might interfere with Even though the fractions of the in jected 57Co recovered in 5-day fecal collec the colorimetrie determination of MMA tions were similar for cellulose- and pectin- since the rats fed 30% of pectin for about fed groups, we cannot conclude that these 2 weeks and the fiber-free diet for 8 weeks two fibers have equivalent effects on re- continued to excrete significantly elevated utilization of biliary Bi2. Neither body pool MMA in urine at 10 weeks. We believe size of Bi2 nor specific activity of Bi2 in that the difference in response to cellulose feces were determined. Therefore, the and to pectin may relate to their digestaamount of radioactivity recovered in the bility by intestinal flora. Williams and feces may represent different proportions Olmsted (21) found that the disappear of total body BJ2 of animals on various ance of hemicellulose from the gut was dietary treatments. In fact, because of the paralleled by an increase in stool volatile extremely high urinary MMA of pectin- fatty acids such as propionate. In our ex fed rats, it seems likely that these animals periment, pectin disappeared from the in were more severely depleted of BJ2 than testinal tract to a far greater extent than were cellulose-fed animals. did cellulose, suggesting that pectin, but not cellulose, might stimulate bacterial Urinary MMA. The immediate explana tion for the increase in urinary MMA ex growth which in turn could increase both cretion in vitamin BJ2 deficiency is the propionate production and Bj2 utilization metabolic block caused by inadequate co- by intestinal bacteria. Urinary 5TCo.Elevated concentrations of factor for the B12-dependent hepatic en zyme methylmalonyl CoA mutase ( 15, 16). precursors of MMCoA, such as excessive MMCoA arises from the metabolism of propionate, also increase the turnover rate propionic acid (3) as well as several amino of tissue B12. Reed and Tarver (16) have acids and pyrimidines. When MMCoA mu shown that propionate feeding exaggerates tase activity is inadequate, MMCoA that depression of MMCoA mutase activity. is not converted to succinyl CoA is hydro- Venkataraman et al. (22) found that feed lyzed to MMA and is excreted. However, ing propionate at 2% of the diet reduced availability of MMA precursors also affects MMCoA mutase activity 80% in rats fed Bi2-deficient diets and 60% in rats fed the amount of MMA excreted. Administra tion of propionic acid in Bi2 deficiency in adequate B12. In our study, pectin-fed rats experienced creases urinary excretion of MMA (6, 7, the highest urinary excretion of "Co, 16, 17). Yang et al. (18) demonstrated that large amounts of propionic acid are cellulose-fed animals the lowest, and fiberproduced by intestinal microorganisms of free control rats were intermediate. If the rat and that a significant proportion is 57Co in urine reflects rate of turnover of absorbed from the cecum and is metabo tissue B]2 and turnover rate is determined by flux of precursor over this B^-depen lized. Williams and Spray (17, 19) re ported that MMA excretion of Bi2-deficient dent pathway, then our urinary "Co data rats varied directly with the level of pro- are consistent with the presumption that
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formation, cellulose appeared to be poorly digested and might therefore have bound or adsorbed biliary Bi2 and carried it out in the feces. Pectin was not recovered in fecal material but might still have bound Bia in the small intestine and carried it past the ileal absorption sites. Additionally, pectin might have served as a substrate for growth of bacteria that require B12. Bacterial overgrowth in the small intestine of rats has been shown to decrease vitamin Bi2 absorption (2). An analogous finding is that antibotics "spare" vitamin B12 in
METABOLISM
646
ROBERT W. CULLEN AND SUSAN M. OACE
ACKNOWLEDGMENTS
We express our appreciation to Dr. Thornton Sargent for the use of the gamma counter, to Larry Citron for assistance with the statistical analysis of our data, and to Karen Leesman for assistance in various aspects of this experiment.
LITERATURE
CITED
1. Burkitt, D. P., Walker, A. R. P. & Painter N. S. (1974) Dietary fiber and disease. J. Am. Med. Assoc. 229, 1068-1074. 2. Donaldson, R. M. (1962) Malabsorption of Co'Mabeled cyanocobalamin in rats with in testinal diverticula. I. Evaluation of possible mechanisms. Gastroenterology 43, 271-281. 3. Kaziro, Y. & Ochoa, S. (1964) The metab olism of propionic acid. Adv. Enzymol. 26, 283-378. 4. Cox, E. V. & White, A. M. (1962) Methylmalonic acid excretion: An index of vitaminBi2 deficiency. Lancet 2, 853-856. 5. Brozovic, M., Hoffbrand, A. V., Dimitriadou, A. & Mollin, D. L. (1967) The excretion of methylmalonic acid and succinic acid in vitamin B,2 and folate deficiency. Br. J. Haematol. 13, 1021-1032. 6. Barness, L. A., Young, D. G. & Nocho, R. ( 1963 ) Methylmalonate excretion in vitamin B,2 deficinecy. Science 140, 76-77. 7. E. Williams, D. L., Spray, G. H., Newman, G. & O'Brien, J. R. P. (1969) Dietary de
8.
9.
10.
11.
pletion of vitamin Bi2 and the excretion of methylmalonic acid in rats. Br. J. Nutr. 23, 343-352. Oace, S. M. & Abbott, J. M. (1972) Methyl malonate, formiminoglutamate and aminoimidazolecarboxamide excretion of vitamin Bi2-deficient germfree and conventional rats. J. Nutr. 102, 17-25. Oace, S. M. & Chen, S. C. H. (1975) Com parison of automated and manual methods of estimation of methylmalonic acid. Anal. Biochem. 67, 192-197. Giorgio, A. J. & Plaut, G. W. E. (1965) A method for the colorimetrie determination of urinary methylmalonic acid in pernicious anemia. J. Clin. Lab. Med. 66, 667-676. Grafnetter, D., on Janosova, Z. & Cervinkova, (1967) Note Slot's method for the speI.
cific determination of creatinine. Clin. Chim. Acta 17, 493-498. 12. Okuda, K., Grasbeck, R. & Chow, B. F. (1958) Bile and vitamin Bn absorption. J. Lab. Clin. Med. 51, 17-23. 13. Okuda, K. (1960) Vitamin Bi2 absorption in rats, studied by a 'loop' technique. Am. J. Physiol. 199, 84-90. 14. Mickelsen, O. (1956) Intestinal synthesis of vitamins in the non-ruminant. Vitamins Hormones 14, 1-95. 15. Cardinale, G. J., Dreyfus, P. M., Auld, P. & Abeles, R. H. ( 1969 ) Experimental vitamin Bis deficiency: Its effect on tissue vitamin B12 coenzyme levels and on the metabolism of methylmalonyl Co A. Arch. Biochem. Biophys. 131, 92-99. 16. Reed, E. B. & Tarver, H. (1970) Urinary methylmalonate and hepatic methylmalonyl coenzyme A mutase activity in the vitamin Bis-deficient rat. J. Nutr. 100, 935-947. 17. Williams, D. L. & Spray, G. H. (1972) Metabolism of propionate in the vitamin Bizdeficient rat. Br. J. Nutr. 28, 263-268. 18. Yang, M. G., Manoharan, K. & Mickelsen, O. (1970) Nutritional contribution of volatile
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pectin, cellulose, and fiber-free diets sup port the most, least, and intermediate pro duction of propionate, respectively. In conclusion, our data are consistent with the following hypothesis concerning the effects of two components of dietary fiber on vitamin B^ metabolism. Cellulose binds B]2 in the intestine, decreases enterohepatic recycling of biliary B12, and in creases fecal excretion of the vitamin. Be cause cellulose appears not to be hydrolyzed in the gut, it would not stimulate microbial propionate production and there fore would neither increase turnover of tissue Bi2 nor exaggerate MMA excretion. Pectin may also bind B12, but because it is hydrolyzed to an appreciable extent by in testinal microorganisms, it may also stimu late growth of Bia-requiring bacteria and increase production of propionate. The absorbed propionate would stress the B12containing enzyme MMCoA mutase, re sulting in increased rate of turnover, loss from tissues, and urinary excretion of vita min Bi2 and a considerable increase in urinary excretion of MMA. Thus, both cellulose and pectin result in a net de terioration of B,2 status, but pectin, by virtue of its availability as an intestinal microbial substrate, has a more dramatic effect. Experiments designed to verify and to quantify the relative importance of these suggested mechanisms are currently being conducted. We have presented evidence that di etary fiber supplementation can have a negative effect on the utilization of a micronutrient. Furthermore, in view of the greatly differing effects of pectin and of cellulose, it is apparent that conclusions about physiological effects of fibers cannot be generalized and must take into account the heterogeneity and individual proper ties of dietary fibers.
DIETARY FIBER AND B,2 METABOLISM
The manner in which food controls the bulk of the feces. Ann. Int. Med. 10, 717-727. 22. Venkataraman, S., Biswas, D. K. & Johnson, B. C. (1967) Effect of propionate on the induction of vitamin Bis deficiency in chicks and rats. J. Nutr. 93, 131-134. 23. Zar, J. H. (1974) Biostatistical Analysis, Prentic Hall, Inc., Englewood Cliffs, N.J. 24. Dünnet,C. W. (1955) A multiple compari son procedure for comparing several treat ments with a control. J. Amer. Statist. Assoc. 50, 1096-1121.
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fatty acids from the caecum of rats. J. Nutr. 100, 545-550. 19. Williams, D. L. & Spray, G. H. (1970) The effect of antibiotics on rats receiving a vitamin Bi2-deficient diet. Br. J. Nutr. 24, 405-412. 20. Armstrong, B. K. & Curnow, D. H. (1967) The effect of streptomycin and erythromycin on vitamin Bi2 nutrition in rats in which coprophagy was prevented. Br. J. Nutr. 21, 527-535. 21. Williams, R. D. & Olmsted, W. H. (1936)
647
aeveiopmenr thlS theory
nf
Alrhrmcrh or fVipcp mese rlicpacp«; diseases. Aitnougn
has not
been
proved,
dietary
fiber supplements currently are very popular. However, little is known about potential
interactions
of dietary
vitamin status or about comparative effects of fibers of differing composition, Fiber, by virtue of its indigestibility by mammalian enzymes, is available in the lower intestine to support growth of the microbial population. Previous work in our Receivedfor publicationAugust25, 1977.
* Portions of these data were presented at the annual meetlng of the Federation of American so-
dettes for Experimental Biology, April 1977, Chicago,
ÕS^^^^uS^^»£^ration^roc.
faber With
g ins ¿*g
"To whom reprint requests should be sent. 640
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Methylmalonic Acid and Vitamin B,, Excretion of Rats Consuming Diets Varying in Cellulose and Pectin1
DIETARY FIBER AND B^ METABOLISM
MATERIALS
AND METHODS
Male, 28-day old Fischer rats4 were housed individually in mesh-bottomed me tabolism cages. After consuming a closed formula, cereal based stock diet5 for 3 days, groups of six rats were assigned to dietary treatments such that group average weights were not significantly different (ca. 70 g). Food and tap water were al lowed ad libitum throughout the study. Fresh food was given every 2 to 3 days; water bottles were changed two times a week. A vitamin Bi2-deficient purified basal diet ( table 1 ) served as our fiber-free con trol (FF). It was made in batches of less than 15 kg as needed throughout the study. Cellulose8 (C) was added to the FF diet to final concentrations of 10%, 20%, 30%,
TABLE l Composition of basai fiber-free diet diet20.070.05.03.51.00.5 g Soy-bean protein1Glucose monohydrate2Corn oil3Salt mix4Vitamin mix6D.L-Methionine6g/100 1Soya Assay Protein ; Teklad Test Diets, Madi son, Wisconsin. 2Cerelose; Corn Products, CPC International, Inc., Englewood Cliffs, New Jersey. 3Mazóla; Best Foods, CPC International, Inc., Englewood Cliffs, New Jersey. 4Provides : (in mg/100 g diet) CaCO3) 725; CaHPO4. 1130; Na2HPO4, 651; KC1, 730; MgS04, 230; MnS04 •H20,15.4; CuS04, 1.3; ferric citrate, 15.1; ZnC03, 2.1 ; KIOS, 0.1. 'In glucose, provides : (in mg/100 g diet) D-biotin, 0.2 ; folie acid, 1 ; thiamin • HC1, 1.5; riboflavin, 1.5; pyridoxine • HC1, 1.5; menadione, 1.5; nicotinic acid, 5; o-calcium pantothenate, 5.0; choline bitartrate, 100; (in IU/100 g diet) D,L-Otocopherol, 5; ergocalciferol, 125; retinyl acetate, 1,000. 6Nutrional Biochemical«Corp., Cleveland, Ohio.
40%, and 50%, and unhydrated pectin7 (P) was added to final concentrations of 5%, 10%, 15%, 20%, and 30%. All diets were stored in a freezer. Body weight and food intake were re corded three times a week. Twenty-fouror 48-hour fecal samples were collected periodically during the study for all rats. Twenty-four-hour urine samples were collected in vials containing 3 ml of 0.72 N H2SO4 (approximately 2 meq) in order to maintain an acid pH for stability of MMA and to inhibit bacterial growth. Funnels with a screen baffle and a small screen over the collection vial separated urine and feces. Urine samples were fil tered through Whatman number 1 filter paper into graduated cylinders and di luted to a standard volume, usually 30 ml. Urinary methylmalonic acid was analyzed colorimetrically 8 by the method developed 3 Chen, S. C. H. & Oaee, S. M. (1974) Decreased depletion rate and prolonged biogloglcal half-life of vitamin B12 in germfree rats. Federation Proc. 33, 605 (abstract). 4 Charles River Breeding Laboratories, Inc., Wil mington, Massachusetts. 6 Purina Rat Chow, Ralston Purina Company, St. Louis, Missouri. • Alpini rol. ICN Pharmaceuticals, Inc., Cleveland, Ohio. ' Citrus Pectin, ICN Pharmaceuticals, Inc., Cleve land. Ohio. 8 Spectronic 20 spectrophotometer ; Bausch and Lomb, Rochester, New York.
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laboratory 3 demonstrated that the vitamin Bi2 depletion rate is significantly greater in the presence than in the absence of in testinal microorganisms. It has also been shown that intestinal bacterial overgrowth, such as in blind loop syndrome, is accom panied by Bi2 depletion, presumably due to competition for available B12 between the gut flora and the host (2). It therefore seemed possible that fiber, by stimulating intestinal bacterial growth, might increase the rate of vitamin Bi2 utilization of the host animal. To test this, groups of rats were assigned to Bi2-deficient diets differ ing in level and type of two dietary fiber constituents, cellulose and pectin. Two approaches were used to study the interaction of dietary fiber with B12 metab olism. First, urinary methylmalonic acid (MMA) was measured at intervals during the first 10 weeks of B12 depletion. MMA accumulates when there is a block in the propionate metabolic pathway at the BJ2containing enzyme methylmalonyl CoA (MMCoA) mutase (3). Urinary MMA ex cretion is elevated in pernicious anemia in humans ( 4, 5 ) and in experimental vitamin B12 deficiency in rats (6-8). Relatively higher urinary MMA of a particular fiber level would be taken as an indicator of greater vitamin Bi2 depletion. Secondly, a small tracer dose of 57Co-cyanocobalamin was injected. Urinary and fecal excretion of radioactivity would reflect any alteration in the rate of vitamin elimination.
641
642
ROBERT W. CULLEN
AND SUSAN M. OACE
TABLE 2 Responses to cellulose and pectin diets Diet1
Weight gain
Food efficiency consumed0.335 total food consumed
weeks128 FF (12)!IOC
(6)10P (6)15P (6)20P (6)30P (6)g/4
±0.0040.352 ±0.00840.238±0.00740.202±0.00440.177±0.00340.322 ±0.0100.340±0.0100.336
±5108 ±64129±791
±0.0060.354±0.0060.340±0.0040.318±0.0090
±0.0040.286±0.00840.197±0.01340.129±0.0234rats
±7*50 ±4431 ±64(allgam:
±0.015»0.161 ±0.0294and lost weight during first0.335 second weeks)
1FF = fiber-free control diet (described in table 1) ; C = cellulose; P = pectin; number preceding letter indicates percent of fiber in diet (see text). 2 Number of animals per group. *Mean±SEM. 4 Group means which differ significantly from the control mean, P < 0.05. Nested analysis of variance (réf.23, pp. 144-147) and Dunnet's multiple range test (24).
by Oace and Chen (9) using automated sampling and pumping equipment.9 It is an adaptation of the original colorimetrie method of Giorgio and Flaut (10). Onetenth of each urine sample was applied to an ion exchange resin column 10 after ad justment to pH 6.5 with NaOH. Columns were rinsed twice with 50 ml volumes of deionized water. MMA was eluted with 30 ml of 0.1 N HC1. The eluant was ana lyzed directly or diluted appropriately with 0.1 N HC1 so that it fell on the stan dard curve. Creatinine was used as a basis to ex press MMA excretion in order to correct for occasional incomplete or spilled col lections. An automated modification of the Jaffe reaction utilizing picric acid and NaOH (11) was used to assay creatinine. An autoanalyzer was used for this analy sis.8'9 After 10 weeks of B12 deprivation, repre sentative groups (FF, 20C, 40C, 5P, and 10P) were injected intraperitoneally with 0.1 jug of B12 containing 0.87 /«Ciof 57Cocyanocobalamin (179 ^.Ci/^g in 0.9% benzyl alcohol).11 All other groups were injected with an equivalent amount of nonradioactive vitamin Bia.12 Urine and feces were collected quantitatively from "Co-Bi2-injected rats for 5 days. Urine samples were filtered and diluted to 30 ml as for MMA analyses. Three-milliliter
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(6)20C (6)30C (6)40C (6)50C (6)5P
gain: fiber-free diet
±53137±7128±6118±2112 ±0.0040.308±0.00440.282 ±0.0040.342
samples were counted in 16 X 25 mm poly styrene counting vials.11 Feces from rats consuming FF, 5P, and 10P diets were counted directly in the counting vials since volumes were less than or equal to 3 ml. Counting efficiency varied little with this range of volumes. Feces from rats fed 20C and 40C diets were weighed, thoroughly ground in a mortar and pestle, and a weighed portion was counted. Samples were counted in a well-type gamma counter.13 All values were expressed as a percentage of the injected dose by com paring samples to standards made from the injection solution. RESULTS The growth and food efficiency of the experimental groups during the first 4 weeks of the study varied with type and amount of dietary fiber. Up to 40% cellu lose did not affect body weight gain sig nificantly during this period (table 2). Weight gain averaged 4 to 5 g/day. Fifty percent cellulose slightly depressed body • Proportioning Pump and Sampler ; Technicon Instruments Corporation, Chauncey, New York. 10AG-3, 200-400 mesh chloride form ; Bio-Rad. Lab., Richmond, California. "Amersham Corporation, Arlington Heights, Illinois. " Merck and Company, Inc., Rahway, New Jersey. u Nuclear-Chicago, Model 1085 ; G. D. Searle and Company, Des Plaines, Illinois.
DIETARY FIBER AND B* METABOLISM
TABLE 3 Effect of dietary fiber consumption on fecal mass Diet1FF
weightg/48
consumedg/48
hr1.54±0.1335.03±0.424 hr—3.72±0.254 (12)2IOC (6) 20C (6) 300 (6) 40C (6) 50C (6)5P 10P 15P 20P 30P
(6) (6) (6) (6) (3)Fecal
8.88±0.344 7.67±0.224 15.2 ±0.70* 13.5 ±0.594 20.9 ±0.554 19.4 ±0.544 27.8 ±1.442.17±0.28 25.8 ±1.7«1.88±0.08< 1.60±0.11 1.22 ±0.18 1.88±0.37 0.53±0.18Fiber
2.9C±0.144 3.22 .^0.154 4.23±0.244 4.30±0.204
1See footnote 1, table 2. 2 Number of animals per group. 3MeanisEM. 4Group means which differ significantly from the control mean, P < 0.05. Nested analysis of variance (réf.23, pp. 144-147) and Dunnet's multiple range test (24).
Rats offered the 5% pectin diet experi enced the same weight gain and food ef ficiency as did the fiber-free controls dur ing the first 4 weeks (table 2). Subse quently, their weight gain was depressed significantly compared to the FF controls, perhaps due to intermittent diarrhea. Higher levels of pectin progressively and significantly depressed weight gain and food efficiency as well as food intake. The fiber-free food efficiency of these groups indicates a decreased utilization of the food consumed as pectin levels increase. In contrast to cellulose, pectin disappeared from the intestinal tract. Pectin did not increase fecal weight (table 3), indicating that it was metabolized by intestinal bac teria. Rats fed the highest level of pectin failed to gain weight. After 1 week, one rat died and survivors were transferred to a fiber-free diet during the second and third weeks. Methylmalonic acid excretion was mea sured after 6 and 10 weeks of vitamin Bt2 deprivation. Among groups assigned cellu lose diets, very little difference in MMA excretion occurred during the first 5 weeks (table 4). Even at 10 weeks, only the highest level of cellulose, 50%, was asso ciated with a significant increase in MMA excretion. In contrast, each level of pectin was ac companied by a significantly greater in-
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weight compared with the fiber-free and other cellulose groups. When calculated using the weight of diet consumed, food efficiency of the cellu lose groups decreased proportionally with increasing fiber; food efficiency based on the amount of fiber-free diet consumed was equivalent for all cellulose groups (table 2). Since the fiber-free food effi ciency ratios were the same, the observed differences in body weight appeared to be due to a lower intake of energy and not to a deficiency of other essential nutrients. The high-cellulose groups did not increase food intake sufficiently to overcome the energy dilution of their diet. These rats appeared to be less fat but not smaller than the rats fed lower levels of cellulose. However, body composition data were not collected. Another implication of the fiberfree food efficiency data is that our source of cellulose was not appreciably metab olized by intestinal bacteria. This conclu sion is further supported by an analysis of the fecal weights of rats fed cellulose. Compared with the group average 48-hour fecal weight of rats consuming FF diet, fecal weights of cellulose-fed rats increased equivalent to the amount of cellulose con sumed (table 3). When fecal weight is plotted versus cellulose consumed, the simple linear regression equation is: Y = 1.01 X + 1.44, where Y = fecal weight in g, and X = g of cellulose consumed The slope, which is near unity, predicts com plete excretion of cellulose; and the Y-intercept (1.44), which predicts fecal weight when cellulose consumption is zero, is rea sonably close to the observed data from the FF group (1.54 g). The correlation coefficient ( r = 0.996 ) indicates that linear ity was significant. Since cellulose content of feces was not measured directly, it is possible that some cellulose was digested by bacteria and that the increased fecal weight and decreased gross food efficiency of cellulose-fed rats resulted from increased excretion of other carbohydrate, protein and lipid. However, the appearance of the fecal material, which was progressively whiter and more powdery as the cellulose content of the diet increased, suggests that at least a substantial proportion of the cellulose was not digested.
643
644
ROBERT W. CULLEN AND SUSAN M. OACE TABLE 4 Urinary methylmalonic acid excretion Length of vitamin BJ2 depletion 5 weeks
(12)2
timóleMM A/mg creatinine 7.6± 1.0s 14± 2.0 1.26.6± 0.56.6± 0.86.1± 0.76.6± 0.655
(6)20C IOC (6)30C (6)40C (6)50C (6)5P (6)10P (6)15P (6)20P (6)30P/FF (5)8.6±
±11*131 8.44221 ± ±18"293 ±34'84 ±24413±
10 weeks
Response to 0.5 f vitamin Bn % depression at 5 days 67.3
2.517± 2.617± 3.221± 4.831 3.5«122± ± 5.1«165±264300±554 (4)73.8 (4)777» (1)62.9 (1)117±32462.850.665.456.851.551.774.8
1See footnote 1, table 2. 2Number of animals per group at 5 weeks; number of survivors at other collection periods in parentheses if different. 3Mean±sEM. 4Group means which differ significantly from the control mean, P < 0.01. Single-factor analysis of variance (réf.23, pp. 133-137) and Dunnet's multiple range test (24).
crease in MM A excretion (table 4). The former 30P group, which consumed pectin for less than 3 weeks and was thereafter fed a diet lacking fiber, excreted MM A at a level significantly greater than the fiberfree controls. The fecal and urinary "Co excretion data of the representative groups (FF, 20C, 40C, 5P, 10P) injected with radio active BIOare presented in table 5. Urinary "Co excretion during the 5-day collection period was only about 2% to 3% of the dose for all groups. However, variability TABLE 5 Cumulative excretion of "Co for five days following injection
Diet1
Urine
Feces
FF (5)2
% of injected dose 2.32±0.04'.« 9.75±0.85°
20C (6) 40C (6)5P
±1.0' 20±0.02' 2.02±0.04b2.39±0.09° ±0.78C22.5 22.9
(6) 10P (6)2.
±1.5« 2.91±0.10£15.021.3 ±2.9«
1See footnote 1, table 2. 2Number of animals per group. 3Mean±sEM.Means in same vertical column not sharing a common superscript letter differ significantly, P < 0.05. Nested analysis of variance (réf. 23, pp. 144-147) and Newman-Keuls' multiple range test (.réf. 23, pp. 151-155).
within groups was small and compared with the FF group, rats fed 10% of pectin excreted a significantly greater and those consuming 40% of cellulose excreted a significantly smaller proportion of the dose. In contrast, both fibers signifi cantly increased fecal excretion of "Co. Over 20% of the "Co was recovered in the 5-day fecal collection of the 40C, 5P, and 10P groups, whereas control FF rats ex creted only about 10% of the dose by this route. The final phase of this experiment is pre sented in table 4. All rats were injected with 0.5 /xg of vitamin B1212 and again urinary MMA was measured. A significant decrease in urinary MMA occurred in all groups. DISCUSSION Fecal "Co. Normal Bi2 metabolism in the rat involves secretion of the vitamin into the intestine with bile (12) and fairly efficient reabsorption by an intrinsic factormediated mechanism in the ileum (13). Body stores of Bi2 could become depleted if this enterohepatic recycling were pre vented. In our study, both cellulose and pectin increased fecal excretion of Bi2, presum ably by interfering with reabsorption of the vitamin. Although we have no direct in-
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FF
Diet1
DIETARY FIBER AND
645
pionate in the cecum. Furthermore, in situ ations where bacterial fermentation and production of propionate would be ex pected to be minimal, such as germfree rats (8) or conventional rats that have been fasted (7) or treated with antibiotics (20), excretion of MMA is very low, de spite B12 deprivation. In our study, vitamin B12-responsive urinary MMA excretion was increased by both cellulose- and by pectin-containing diets but this occurred much earlier and to a greater extent with pectin than with rats (14). cellulose. We have ruled out a trivial ex planation that pectin might interfere with Even though the fractions of the in jected 57Co recovered in 5-day fecal collec the colorimetrie determination of MMA tions were similar for cellulose- and pectin- since the rats fed 30% of pectin for about fed groups, we cannot conclude that these 2 weeks and the fiber-free diet for 8 weeks two fibers have equivalent effects on re- continued to excrete significantly elevated utilization of biliary Bi2. Neither body pool MMA in urine at 10 weeks. We believe size of Bi2 nor specific activity of Bi2 in that the difference in response to cellulose feces were determined. Therefore, the and to pectin may relate to their digestaamount of radioactivity recovered in the bility by intestinal flora. Williams and feces may represent different proportions Olmsted (21) found that the disappear of total body BJ2 of animals on various ance of hemicellulose from the gut was dietary treatments. In fact, because of the paralleled by an increase in stool volatile extremely high urinary MMA of pectin- fatty acids such as propionate. In our ex fed rats, it seems likely that these animals periment, pectin disappeared from the in were more severely depleted of BJ2 than testinal tract to a far greater extent than were cellulose-fed animals. did cellulose, suggesting that pectin, but not cellulose, might stimulate bacterial Urinary MMA. The immediate explana tion for the increase in urinary MMA ex growth which in turn could increase both cretion in vitamin BJ2 deficiency is the propionate production and Bj2 utilization metabolic block caused by inadequate co- by intestinal bacteria. Urinary 5TCo.Elevated concentrations of factor for the B12-dependent hepatic en zyme methylmalonyl CoA mutase ( 15, 16). precursors of MMCoA, such as excessive MMCoA arises from the metabolism of propionate, also increase the turnover rate propionic acid (3) as well as several amino of tissue B12. Reed and Tarver (16) have acids and pyrimidines. When MMCoA mu shown that propionate feeding exaggerates tase activity is inadequate, MMCoA that depression of MMCoA mutase activity. is not converted to succinyl CoA is hydro- Venkataraman et al. (22) found that feed lyzed to MMA and is excreted. However, ing propionate at 2% of the diet reduced availability of MMA precursors also affects MMCoA mutase activity 80% in rats fed Bi2-deficient diets and 60% in rats fed the amount of MMA excreted. Administra tion of propionic acid in Bi2 deficiency in adequate B12. In our study, pectin-fed rats experienced creases urinary excretion of MMA (6, 7, the highest urinary excretion of "Co, 16, 17). Yang et al. (18) demonstrated that large amounts of propionic acid are cellulose-fed animals the lowest, and fiberproduced by intestinal microorganisms of free control rats were intermediate. If the rat and that a significant proportion is 57Co in urine reflects rate of turnover of absorbed from the cecum and is metabo tissue B]2 and turnover rate is determined by flux of precursor over this B^-depen lized. Williams and Spray (17, 19) re ported that MMA excretion of Bi2-deficient dent pathway, then our urinary "Co data rats varied directly with the level of pro- are consistent with the presumption that
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formation, cellulose appeared to be poorly digested and might therefore have bound or adsorbed biliary Bi2 and carried it out in the feces. Pectin was not recovered in fecal material but might still have bound Bia in the small intestine and carried it past the ileal absorption sites. Additionally, pectin might have served as a substrate for growth of bacteria that require B12. Bacterial overgrowth in the small intestine of rats has been shown to decrease vitamin Bi2 absorption (2). An analogous finding is that antibotics "spare" vitamin B12 in
METABOLISM
646
ROBERT W. CULLEN AND SUSAN M. OACE
ACKNOWLEDGMENTS
We express our appreciation to Dr. Thornton Sargent for the use of the gamma counter, to Larry Citron for assistance with the statistical analysis of our data, and to Karen Leesman for assistance in various aspects of this experiment.
LITERATURE
CITED
1. Burkitt, D. P., Walker, A. R. P. & Painter N. S. (1974) Dietary fiber and disease. J. Am. Med. Assoc. 229, 1068-1074. 2. Donaldson, R. M. (1962) Malabsorption of Co'Mabeled cyanocobalamin in rats with in testinal diverticula. I. Evaluation of possible mechanisms. Gastroenterology 43, 271-281. 3. Kaziro, Y. & Ochoa, S. (1964) The metab olism of propionic acid. Adv. Enzymol. 26, 283-378. 4. Cox, E. V. & White, A. M. (1962) Methylmalonic acid excretion: An index of vitaminBi2 deficiency. Lancet 2, 853-856. 5. Brozovic, M., Hoffbrand, A. V., Dimitriadou, A. & Mollin, D. L. (1967) The excretion of methylmalonic acid and succinic acid in vitamin B,2 and folate deficiency. Br. J. Haematol. 13, 1021-1032. 6. Barness, L. A., Young, D. G. & Nocho, R. ( 1963 ) Methylmalonate excretion in vitamin B,2 deficinecy. Science 140, 76-77. 7. E. Williams, D. L., Spray, G. H., Newman, G. & O'Brien, J. R. P. (1969) Dietary de
8.
9.
10.
11.
pletion of vitamin Bi2 and the excretion of methylmalonic acid in rats. Br. J. Nutr. 23, 343-352. Oace, S. M. & Abbott, J. M. (1972) Methyl malonate, formiminoglutamate and aminoimidazolecarboxamide excretion of vitamin Bi2-deficient germfree and conventional rats. J. Nutr. 102, 17-25. Oace, S. M. & Chen, S. C. H. (1975) Com parison of automated and manual methods of estimation of methylmalonic acid. Anal. Biochem. 67, 192-197. Giorgio, A. J. & Plaut, G. W. E. (1965) A method for the colorimetrie determination of urinary methylmalonic acid in pernicious anemia. J. Clin. Lab. Med. 66, 667-676. Grafnetter, D., on Janosova, Z. & Cervinkova, (1967) Note Slot's method for the speI.
cific determination of creatinine. Clin. Chim. Acta 17, 493-498. 12. Okuda, K., Grasbeck, R. & Chow, B. F. (1958) Bile and vitamin Bn absorption. J. Lab. Clin. Med. 51, 17-23. 13. Okuda, K. (1960) Vitamin Bi2 absorption in rats, studied by a 'loop' technique. Am. J. Physiol. 199, 84-90. 14. Mickelsen, O. (1956) Intestinal synthesis of vitamins in the non-ruminant. Vitamins Hormones 14, 1-95. 15. Cardinale, G. J., Dreyfus, P. M., Auld, P. & Abeles, R. H. ( 1969 ) Experimental vitamin Bis deficiency: Its effect on tissue vitamin B12 coenzyme levels and on the metabolism of methylmalonyl Co A. Arch. Biochem. Biophys. 131, 92-99. 16. Reed, E. B. & Tarver, H. (1970) Urinary methylmalonate and hepatic methylmalonyl coenzyme A mutase activity in the vitamin Bis-deficient rat. J. Nutr. 100, 935-947. 17. Williams, D. L. & Spray, G. H. (1972) Metabolism of propionate in the vitamin Bizdeficient rat. Br. J. Nutr. 28, 263-268. 18. Yang, M. G., Manoharan, K. & Mickelsen, O. (1970) Nutritional contribution of volatile
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pectin, cellulose, and fiber-free diets sup port the most, least, and intermediate pro duction of propionate, respectively. In conclusion, our data are consistent with the following hypothesis concerning the effects of two components of dietary fiber on vitamin B^ metabolism. Cellulose binds B]2 in the intestine, decreases enterohepatic recycling of biliary B12, and in creases fecal excretion of the vitamin. Be cause cellulose appears not to be hydrolyzed in the gut, it would not stimulate microbial propionate production and there fore would neither increase turnover of tissue Bi2 nor exaggerate MMA excretion. Pectin may also bind B12, but because it is hydrolyzed to an appreciable extent by in testinal microorganisms, it may also stimu late growth of Bia-requiring bacteria and increase production of propionate. The absorbed propionate would stress the B12containing enzyme MMCoA mutase, re sulting in increased rate of turnover, loss from tissues, and urinary excretion of vita min Bi2 and a considerable increase in urinary excretion of MMA. Thus, both cellulose and pectin result in a net de terioration of B,2 status, but pectin, by virtue of its availability as an intestinal microbial substrate, has a more dramatic effect. Experiments designed to verify and to quantify the relative importance of these suggested mechanisms are currently being conducted. We have presented evidence that di etary fiber supplementation can have a negative effect on the utilization of a micronutrient. Furthermore, in view of the greatly differing effects of pectin and of cellulose, it is apparent that conclusions about physiological effects of fibers cannot be generalized and must take into account the heterogeneity and individual proper ties of dietary fibers.
DIETARY FIBER AND B,2 METABOLISM
The manner in which food controls the bulk of the feces. Ann. Int. Med. 10, 717-727. 22. Venkataraman, S., Biswas, D. K. & Johnson, B. C. (1967) Effect of propionate on the induction of vitamin Bis deficiency in chicks and rats. J. Nutr. 93, 131-134. 23. Zar, J. H. (1974) Biostatistical Analysis, Prentic Hall, Inc., Englewood Cliffs, N.J. 24. Dünnet,C. W. (1955) A multiple compari son procedure for comparing several treat ments with a control. J. Amer. Statist. Assoc. 50, 1096-1121.
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fatty acids from the caecum of rats. J. Nutr. 100, 545-550. 19. Williams, D. L. & Spray, G. H. (1970) The effect of antibiotics on rats receiving a vitamin Bi2-deficient diet. Br. J. Nutr. 24, 405-412. 20. Armstrong, B. K. & Curnow, D. H. (1967) The effect of streptomycin and erythromycin on vitamin Bi2 nutrition in rats in which coprophagy was prevented. Br. J. Nutr. 21, 527-535. 21. Williams, R. D. & Olmsted, W. H. (1936)
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