Progress 10/01/05 to 09/30/10
Outputs
OUTPUTS: Over the thirty-two year history of this project a broad series of experiments were conducted primarily using experimental animals. The goals have been to better understand the role of dietary components on cholesterol and bile acid metabolism, lipid absorption and colon function. Seventeen graduate students have been mentored and have completed degrees as a part of this effort along with one postdoctoral trainee. Results of these efforts were presented at a number of scientific meetings as well as many Extension audiences and other non-science groups. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nutrition scientists PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
There have been a number of outcomes from this project that have had an impact on human nutrition and on our understanding of regulation of lipid metabolism. They include: Observation that the sources of dietary fiber that have an impact on cholesterol levels do so, in part, by increasing the amount of bile acids excreted in feces and also alter the relative concentrations of bile acids in the bile acid pool. These changes have been an important part of the data that led to the approval of the health claim for oat fiber as a means of lowering blood cholesterol levels. Our bile acid work also provided an important hypothesis for the mechanism by which these changes in cholesterol in response to certain dietary fiber sources take place. We conducted a series of experiments which examined changes in chylomicron composition in response to changes in the rate of infusion of lipid and cholesterol. Our observations that changes in the size and composition of chylomicrons in response to differences in lipid infusion rates in rats led to a change in our understanding of the relationship between dietary cholesterol and chylomicron appearance of cholesterol. Some of the observations of differences in appearance of lipids in lymph are only now being understood as we learn more about the mechanism of handling of lipids after a meal. We were also involved in a large human study which measured bile acid excretion in response to increased consumption of wheat bran and how these changes related to occurrence of colon polyps. Although wheat bran greatly reduced bile acid concentrations in feces, no relationship with polyp formation in the time frame studied was observed. We were among the first to measure changes in mRNA of enzymes regulating cholesterol balance (cholesterol 7alpha-hydroxylase and HMG CoA reductase) in response to changes in dietary fiber sources. Later we were able to measure a reduction in intestinal bile acid transporter which suggested the mechanism by which bile acid pool sizes were changed in response to dietary fiber sources which lower cholesterol. More recently we have been interested in the effects of conjugated linoleic acid and docosahexaenoic acid on a variety of variables involved in regulating lipid metabolism. These fatty acids are present in some foods and are available as supplements and have been implicated in reduced risk for atherosclerosis. Our studies indicated that in rats, few beneficial changes were observed in response to levels of these fatty acids normally found in foods. Additional work by others has indicated that there are significant species differences. We have been involved with studies of effects of some novel polysaccharides and their effects on bone density and calcium balance in rats and how changes in short chain fatty acids in the colon relate to these changes. Some positive relationships were observed between SCFA concentrations and beneficial changes in bone metabolism.
Publications
- Weaver, C.M., Martin, M.R., Story, J.A., Hutchinson, I., Sanders, L. 2010. Novel fibers increase bone calcium content and strength beyond efficiency of large intestine fermentation. J. Agric. Food. Chem. 58:8952-8957.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The effects of a variety of polysaccharides on short chain fatty acid concentrations in the cecum of rats were examined. Rats were fed one of 9 polysaccharides as part of a nutritionally complete diet for 12 weeks. The polysaccharides used were cellulose, resistant starch (PROMITOR[TM] resistant starch 60 [RS60], resistant starch 75 [RS85]), soluble fiber dextrin (SFD), pullulan PROMITOR[TM] soluble corn fiber (SCF), Sta-Lite(R) polydextrose (PDX), inulin, or inulin/FOS blend. At the termination of the experiment, cecal contents were collected, frozen in liquid nitrogen and stored at -80 degrees C. Analysis of short chain fatty acids was carried out using gas-liquid chromatography. Compared to cellulose, all these polysaccharides resulted in greater concentration and total short chain fatty acids in the cecum. These differences were significantly higher for the groups fed RS60, RS75, inulin, SFD and PDX. Among the individual fatty acids, inulin had among the highest concentrations of butyrate and propionate while PDX had high concentrations of acetate and propionate but very low concentration of butyrate. PARTICIPANTS: Connie Weaver, Berdine Martin TARGET AUDIENCES: Human Nutrition Scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Resistant starches and other polysaccharides increase bacterial formation of short chain fatty acids in the cecum. Increased levels of butyrate in the colon has been suggested to provide increase amounts of energy to the colonic mucosa and to reduce risk for colon cancer. Further evaluation of some of the polysaccharides used in these studies in relation to colonic mucosal health would seem useful.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: An experiment was conducted comparing several sources of soluble dietary fiber sources and oligosaccharides in diets fed to rats. Fecal short chain fatty acids were measured in response to these diet changes. Subsequently we will also measure short chain fatty acid content of cecal and colon contents.
PARTICIPANTS: Connie M. Weaver, PI Training for one undergraduate exchange student provided.
TARGET AUDIENCES: Nutritionists interested in soluble dietary fiber
Impacts
When compared with cellulose, all of the soluble polysaccharides resulted in higher excretion of short chain fatty acids. However, few differences were observed among the various dietary fiber sources.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
Studies similar to that conducted with conjugated linoleic acid (CLA) were conducted with docosahexaenoic acid (DHA). Butter with higher levels of DHA (0.07%) was compared with control butter and with butter fortified with DHA to a level 5 times that in the enriched butter.(0.35%) in Sprague Dawley rats for six weeks. As with CLA increasing dietary DHA resulted in an increase in hepatic DHA levels as a percentage of total fatty acids (control 6.6%, DHA 8.0% and 5XDHA 15.5%), both experimental groups being statistically significantly higher than controls. Feeding DHA decreased (P<0.05) mRNA abundance of acyl-CoA oxidase by 82.4% for 5XDHA, 25.0% for DHA. Lecithin cholesterol acyl transferase was decreased (P<0.05) 80.1% with 5XDHA, 40.4% in DHA, and 3-hydroxy-3-methyl glutaryl CoA synthase was decreased 81.0% for 5XDHA. The data indicate that milkfat that is enriched with DHA has functional food properties on liver fatty acids profile and hepatic gene expression.
Impacts
Similar to our work with CLA, feeding DHA has an impact on liver levels of the fatty acid in relation to the amount fed. DHA also has an effect on some of the genes involved in lipid metabolism. Although this is encouraging, use of this information for improvement of human health will require a great deal more research.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
We have continued our work with conjugated linoleic acid (CLA) and its effects on lipid metabolism and gene expression. Our recent experiment involved thirty-two female Sprague Dawley rats (21 days of age) which were randomized to diets containing either 10% control butter (control, c9, t11-CLA was 0.6% of total fatty acids) or 10% CLA enriched butter (CLA, c9, t11-CLA was 4.1% of total fatty acid; 90.8% of CLA). After six weeks, rats were killed and liver samples adipose tissue samples were taken for lipid analysis and mRNA transcript profiling. There was no difference between the two groups (P less than 0.05) in body weight gain. Liver lipid analysis showed that CLA as a percentage of total liver fatty acids was elevated 5-fold (P less than 0.0001) in CLA group (0.98+/-0.06%) compared with the controls (0.21+/-0.06%). Feeding CLA elevated mRNA abundance of hepatic fatty acid synthase by more than 400% (P less than 0.0001), and acyl-CoA oxidase by 227% (P less than
0.05). In adipose tissue, however, mRNA abundance of neither enzyme was different between the two groups (P less than 0.05). The data indicate that milkfat that is enriched with CLA acts as a functional food to impart biological effects to change liver fatty acid profile. Its effect on expression of genes involved in lipid metabolism in growing rats appears to be specific to liver.
Impacts
Evidence has indicated an role for CLA in reducing risk for development of atherosclerosis. The mechanism for such an effect has not been elucidated. Our studies suggest that the effects of CLA are not consistent in the various strains of experimental animals or in various tissues. These data suggest that we should proceed with caution in making recommendations for inclusion of CLA in human diets until we understand if benefits should be expected from such dietary changes.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/29/04
Outputs
Our recent studies have focused on our second objective, the role of fatty acids on lipid metabolism. Conjugated linoleic acid (CLA) has been shown to modify experimentally induced atherosclerosis but has variable effects on cholesterol metabolism that appear to be dependent on the degree of saturation of the other lipids in the diet. Rats were fed a semipurified diet (AIN93M) with either butter or corn oil supplying the lipid (4%) with and without 0.25% cholesterol added. Control butter (C/B) was compared with butter naturally high (about 0.16%) in CLA (CLA/B), butter with 0.16% commercial CLA (B/lowCLA), butter with 1% commercial CLA (B/highCLA), corn oil control (C/CO), corn oil with 0.16% CLA (CO/lowDLA), and corn oil with 1% CLA (CO/highCLA). After 4 weeks of feeding, serum and liver cholesterol, hepatic abundance of cholesterol 7[alpha] hydroxylase (CYP7A) mRNA and bile acid excretion were measured. Serum cholesterol was not significantly altered by any of the
diet treatments. Liver cholesterol levels were significantly higher when cholesterol was added to the diet and higher with corn oil as a source of lipid when compared to butter. None of the CLA treatments had an effect on liver cholesterol levels. As was shown in our earlier studies without added dietary cholesterol, mRNA abundance of CYP7A higher with added dietary cholesterol but was not significantly altered by addition of CLA. Bile acid excretion was higher with corn oil as a source of dietary lipids and tended to be higher with the highest level of CLA. These results would suggest that the "background" dietary lipids have an impact on the effect of CLA on cholesterol homeostasis.
Impacts
CLA and other polyunsaturated fatty acids which occur in our diets in limited quantities have been suggested to have an impact on cholesterol metabolism and/or atherosclerosis. This has led to development of functional foods containing higher levels of these fatty acids, some naturally occurring and some supplemented with the fatty acids. Our data would suggest that the impact of these fatty acids may be modified by the other lipids in the diet and that the levels achievable by natural means are not likely to be sufficient to have an impact on this disease.
Publications
- Story, J.A. New insights into deitary fibre. Canadian Journal of Dietetic Practice and Research 64(4), 2003.
- Spiller, G.A., Story, J.A., Lodics, T.A., Pollack, M., Monyan, S., Butterfield, G., Spiller, M. Effect of sun-dried raisins on bile acid excretion, intestinal transit time, and fecal weight: a dose-response study. J. Med. Food 6:87-91, 2003.
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Progress 10/01/02 to 09/30/03
Outputs
Earlier studies demonstrated the ability of psyllium (PSY) to lower serum cholesterol in experimental animals and in humans. Studies from our laboratory observed changes in the abundance of mRNA for enzymes involved in cholesterol and bile acid synthesis as well as the active transport reabsorption of bile acids from the ileum. The objective of this study was to investigate the impact of PSY on hepatic gene expression, using microarray. Two groups of male Wistar rats (10 in each group) were fed semipurified diets containing 10% dietary fiber from cellulose (control) or PSY for 21d. Rats were killed by exsanguination. Total liver RNA samples of three rats selected at random from each group were subjected to mircoarray analysis. The order of each hybridization was randomly determined. The analysis was controlled for microarray filter (GF300, Research Genetics), day of hybridization, and trt*filter effect. mRNA abundance of 35 of 4432 named genes were significantly
different between the two groups. mRNA levels of genes involved in cholesterol synthesis and metabolism were significantly higher in PSY group: HMG CoA synthase, 1.6592-fold (P<0.01); lanosterol 14-a-demethylase, 1.391-fold (P<0.05); lanosterol 14-alpha-demethylase, 1.4852-fold (P<0.05). Northern blot analysis was carried out on these four mRNAs and only HMG CoA synthase was significantly increased (32-fold). In spite of earlier findings of increased abundance of CYP7A, no change was observed in microarray analysis. These changes suggest additional mechanisms for the effects of PSY on cholesterol metabolism need to be explored.
Impacts
Dietary fiber has been epidemiologically linked to a number of chronic diseases, but we are just beginning to understand the mechanisms involved in these relationships. Our work will begin to identify mechanistic links between dietary fiber and enzymes which may be involved in these disease states. Subsequent research will be needed to allow more specific recommendations to the public which will alter disease risk.
Publications
- Spiller, G.A., J.A. Story, E.J. Furumoto, J.C. Chezem and M. Spiller. Effect of tartaric acid and dietary fiber from sun-dried raisins on colonic function, bile acids, and volatile fatty acids excretion in healthy adults. Br. J. Nutr. 90:803-807, 2003.
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Progress 10/01/01 to 09/30/02
Outputs
Earlier studies demonstrated the ability of psyllium (PSY) to lower serum cholesterol in experimental animals and in humans. Studies from our laboratory observed changes in the abundance of mRNA for enzymes involved in cholesterol and bile acid synthesis as well as the active transport reabsorption of bile acids from the ileum. The objective of this study was to investigate the impact of PSY on hepatic gene expression, using microarray. Two groups of male Wistar rats (10 in each group) were fed semipurified diets containing 10% dietary fiber from cellulose (control) or PSY for 21d. Rats were killed by exsanguination. Total liver RNA samples of three rats selected at random from each group subjected to mircoarray analysis. The order of each hybridization was randomly determined. The analysis was controlled for microarray filter (GF300, Research Genetics), day of hybridization, and trt*filter effect. mRNA abundance of 35 of 4432 named genes were significantly different
between the two groups. mRNA levels of genes involved in cholesterol synthesis and metabolism were significantly higher in PSY group: HMG CoA synthase, 1.6592-fold (P<0.01); lanosterol 14-a-demethylase, 1.391-fold (P<0.05); lanosterol 14-alpha-demethylase, 1.4852-fold (P<0.05). Other genes with significantly different mRNA levels involved functions such as cell cycle and growth, immunoresponse, transport, etc. PSY seems to have more comprehensive impact on gene expression than already known. Further investigation may reveal new mechanisms of PSY's function.
Impacts
Dietary fiber has been epidemiologically linked to a number of chronic diseases but we are just beginning to understand the mechanisms involved in these relationships. Our work will begin to identify mechanistic links between dietary fiber and enzymes which may be involved in these disease states. Subsequent research will be needed to allow more specific recommendations to the public which will alter disease risk.
Publications
- Jenkins, D.J.A., C.W. Kendall, D.G. Popovich, E. Vidgen, C.C. Mehling, V. Vuksan, T.P. Ransom, A.V. Rao, R. Rosenberg-Zand, N. Tariq, P. Corey, P.J. Jones, M. Raeini, J.A. Story, E.J. Furumoto, D.R. Illingworth, A.S. Pappu, P.W. Connelly. Effect of a very-high-fiber vegetable, fruit, and nut diet on serum lipids and colonic function. Metabolism 50:494-503, 2001.
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Progress 10/01/00 to 09/30/01
Outputs
In our continuing effort to understand the role of diet in regulation of cholesterol and bile acid metabolism we have examined the role of conjugated linoleic acid (CLA). CLA has been shown to modify experimentally induced atherosclerosis but has variable effects on cholesterol metabolism that appear to be dependent on the degree of saturation of the other lipids in the diet. Rats were fed a semipurified diet (AIN93M) with either butter or corn oil supplying the lipid (4%) and 0.25% cholesterol added. Control butter (C/B) was compared with butter naturally high (about 0.16%) in CLA (CLA/B), butter with 0.16% commercial CLA (B/lowCLA), butter with 1% commercial CLA (B/highCLA), corn oil control (C/CO), corn oil with 0.16% CLA (CO/lowCLA), and corn oil with 1% CLA (CO/highCLA). After 4 weeks of feeding, serum and liver cholesterol, hepatic abundance of cholesterol 7[alpha]hydroxylase (CYP7A) mRNA and bile acid excretion were measured. With butter as a source of lipid
there was little change in serum or liver cholesterol levels. Serum cholesterol was not changed with corn oil as a lipid source but there was a significant increase in liver cholesterol level (almost 3-fold) with the highest intake of CLA. As was shown in our earlier studies without added dietary cholesterol, CYP7A was not significantly altered by addition of CLA. Unlike those earlier studies, with added dietary cholesterol CYP7A mRNA levels were generally higher with butter as a source of lipid in comparison to corn oil. These results would suggest that the "background" dietary lipids have an impact on the effect of CLA on cholesterol homeostasis. The impact of CLA on experimental atherosclerosis employing variations in fatty acid saturation of dietary lipids has not been examined.
Impacts
As we increase our understanding of the mechanisms by which dietary components regulate cholesterol balance, it will be possible to make broader and more effective recommendations for prevention of risk factors for chronic disease.
Publications
- Jenkins, D.J.A., C.W. Kendall, D.G. Popovich, E. Vidgen, C.C. Mehling, V. Vuksan, T.P. Ransom, A.V. Rao, R. Rosenberg-Zand, N. Tariq, P. Corey, P.J. Jones, M. Raeini, J.A. Story, E.J. Furumoto, D.R. Illingworth, A.S. Pappu, P.W. Connelly. Effect of a very-high-fiber vegetable, fruit, and nut diet on serum lipids and colonic function. Metabolism 50:494-503, 2001.
- Savaiano, D.A. and J.A. Story. Cardiovascular disease and fiber: Is insulin resistance the missing link? Nutrition Reviews 58:356-358, 2000.
- Story, J.A. and D.A. Savaiano. Dietary fiber and colorectal cancer: what do we recommend? Nutrition Reviews 59:84-86, 2001.
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Progress 10/01/99 to 09/30/00
Outputs
Our work with psyllium has found that the reduction in blood cholesterol level appears to be as a result of a reduction in reabsorption of bile acids from the small intestine. This appears to be related to the viscosity of intestinal contents which alters both the amount and the identity of bile acids returning to the liver via the portal system. We observed and increase in mRNA for the ileal bile acid transporter in response to psyllium feeding in rats. Since the active transport of bile acids favors the more hydrophillic bile acids, we hypothesize that this would alter the relative size of the bile acid pools in line with an earlier observation in or lab. This effect modifies cholesterol metabolism in several ways: 1) By reducing the amount of bile acids reabsorbed more cholesterol will be used for bile acid synthesis; 2) Changes in the hydrophobicity of bile acids regulates the rate of cholesterol and bile acid synthesis; and 3) Reduced reabsorption will increase
excretion of bile acids.
Impacts
Recommendations of increased consumption of sources of dietary fiber that have the benefits suggest above can be used as a means of reducing the risk of chronic disease.
Publications
- Tariq, N., D.J.A. Jenkins, E. Vidgen, N. Fleshner, C.W.C. Kendall, J.A. Story, W. Singer, M. D'Costa and N. Struthers. Effect of soluble and insoluble fiber diets on serum prostate specific antigen levels in men. J. Urology 163:114-118, 2000.
- Buhman, K.K., E.J. Furumoto, S.S. Donkin and J.A. Story. Dietary psyllium increases expression of ileal apical sodium dependent bile acid transporter mRNA coordinately with dose response changes in bile acid metabolism in rats. J. Nutr. 130:2137-2142, 2000.
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Progress 10/01/98 to 09/30/99
Outputs
The bile acid pool is maintained by an efficient enterohepatic circulation with 97% of bile acids being reabsorbed by the intestine and returned to the liver. The majority of conjugated bile acid reabsorption occurs via the apical sodium bile acid transporter (ASBT) located in the ileal mucosa; however, little is known about its regulation by substrate. Four groups of ten rats were fed a semipurified diet which contained 10% cellulose ( C), 3.33% psyllium and 6.67% cellulose (P3), 6.67% psyllium and 3.33% cellulose (P6), or 10% psyllium (P10) as the fiber source for 21 days and total intestinal bile acids and ASBT gene expression in the ileum measured. Total intestinal bile acid levels were significantly greater in all rats fed psyllium containing diets compared to C-fed rats in a dose responsive manner (1.2, 4.3, 5.4,8.0 mg for C, P3, P6 and P10, respectively). Levels of mRNA for ASBT followed a similar pattern with all rats fed psyllium containing diets having 1.5
-2 fold increases in expression compared to C-fed rats. These data indicate positive feedback regulation of ASBT mRNA expression by bile acids in the intestine in response to the increase in the bile acid pool resulting from dietary psyllium.
Impacts
(N/A)
Publications
- Chezem, J.C., E.J. Furumoto and J.A. Story. Effects of resistant potato starch on cholesterol and bile acid metabolism in the rat. Nutr. Res. 17:1671-1682, 1997.
- Buhman, K. K., E.J. Furumoto, S.S. Donkin, and J.A. Story. Dietary psyllium increases fecal bile acid excretion, total steroid excretion and bile acid biosynthesis in rats. Journal of Nutrition 128:1199-1203, 1998.
- Haack, V. S., J. G. Chesters, N. W. Vollendorf, J. A. Story and J. A. Marlett. Increasing amounts of dietary fiber provided by foods normalizes physiologic response of the large bowel without altering calcium balance or fecal steroid excretion. American Journal of Clinical Nutrition 68:615-622, 1998.
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Progress 10/01/96 to 09/30/97
Outputs
Sources of dietary fiber that have the ability to reduce cholesterol levels in rats diet have been shown to increase bile acid synthesis and the activity of the rate limiting enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase (C7H). Psyllium hydrocolloid has been shown to have these effects when fed to rats as a part of a semipurified diet. In order to better define this effect, we measured changes in hepatic activity and mRNA for C7H and hepatic portal blood bile acid concentrations in rats fed three levels of psyllium (3.3, 6.7 and 10 g/100g). Portal blood concentrations of bile acids were not significantly altered by any of the levels of psyllium but the hydrophobicity of the bile acids was significantly increased by the two higher levels of psyllium. C7H activity was also increased by these two higher levels of intake of psyllium. Hepatic C7H mRNA levels were increased by psyllium feeding in relation to the activity. Our hypothesis suggests that these
changes in hydrophobicity are, in part, responsible for changes in activity of C7H through changes in the bile acid response element that provides partial control of the expression of the C7H gene. How these sources of dietary fiber alter the hydrophobicity of bile acids returning to the liver remains to be elucidated.
Impacts
(N/A)
Publications
- Chezem, J.C. and J.A. Story. Development of an updated method for fecal bile acid and neutral steroid analysis. Am. Clin. Lab. 16:20-21, 1997.
- Wolever, T.M.S., R.A. Hegele, P.W. Connelly, T.P.P. Ransom, J.A. Story, E.J. Furumoto and D.J.A. Jenkins. Long-term effect of soluble-fiber foods on postprandial fat metabolism in dyslipidemic subjects with apo E3 and apo E4 genotypes. Am. J. Clin. Nutr. 66:584-590, 1997.
- Story, J.A.,, E.J. Furumoto and K.K. Buhman. Dietary fiber and bile acid metabolism - an update. In: Dietary Fiber in Health and Disease. Kritchevsky, D. and C. Bonfield, editors. Plenum Press, New York, NY, 1997, pp. 266.
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Progress 10/01/95 to 09/30/96
Outputs
Sources of dietary fiber that have the ability to reduce cholesterol accumulation in rats fed a cholesterol-containing diet have been shown to increase bile acid synthesis and the activity of the rate limiting enzyme in bile acid synthesis, cholesterol 7 -hydroxylase. In an effort to better understand the mechanism for this change, we measured changes in the amounts of mRNA for this enzyme in liver RNA. Rats were fed diets containing cellulose, psyllium, cholestyramine (a bile acid sequestrant) or cholic acid, total liver RNA isolated and the amount of cholesterol 7 -hydroxylase mRNA measured by Northern blot techniques. As would be expected, feeding cholestyramine dramatically increased the level of mRNA in comparison to cellulose. Psyllium also increased mRNA levels for this enzyme. Further experiments are being conducted to determine if the amount of mRNA synthesized in animals fed psyllium and other sources of dietary fiber with similar effects on bile acid
synthesis cause this change through and increase in the rate of mRNA synthesis or reduced degradation of that mRNA.
Impacts
(N/A)
Publications
- Alberts, D.S., C. Ritenbaugh, J.A. Story, M. Aickiin, S. Rees-McGee, M.K. Buller, J. Atwood, J. Phelps, P.S. Ramanujam, S. Bellapravalu, J. Patel, L. Bettinger and L. Clark. Randomized, double-blinded, placebo-controlled study of effect of
- Jenkins, D. J. A., V. Vuksan, T. M. S. Wolever, T.P.P. Ransom, E. Vidgen, R.A. Hegele, L. Leiter, R.G. Josse, M. Abdolell, R. Patten, A.V. Rao, C.W.C. Kendall,J. A. Story, D.L. Boctor, and P.N. Corey. The potential effect of high fibre die
- Hosig, K.W., F.L. Shinnick, M.D. Johnson, J.A. Story and J.A. Marlett. Comparison of large bowel function and calcium balance during soft wheat bran and oat bran consumption. Cereal Chemistry 73:392-398, 1996.
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Progress 10/01/94 to 09/30/95
Outputs
Investigation into the mechanism by which dietary fiber alters cholesterol balance have let us to examine the effect of several sources of dietary fiber on two enzymes which control the spectrum of bile acids synthesized by rats. The activity of the 12a-hydroxylation of 7a-hydroxycholesten-3-one determines the ratio of cholic acid to chenodeoxycholic acid synthesized. Our earlier studies have indicated a change in this ration in response to pectin and psyllium, two sources of dietary fiber which lower cholesterol accumulation in rats. Activity of 12a-hydroxylase were measured in rats fed diets containing these sources of dietary fiber and compared to rats fed diets with cellulose as a source of dietary fiber. Neither pectin nor psyllium had activities of this enzyme which were any greater than that observed in animals fed cellulose. Activities of the 6b-hydroxylase enzyme found in rats was also measured in these animals. Bile acids with a 6b-hydroxyl group are almost
exclusively excreted and thus are an important mechanism by which rats regulate their cholesterol balance. In these experiments, rats fed pectin and psyllium both displayed activities of the 6b-hydroxylase which were significantly higher than found in animals fed cellulose. These results suggest that these sources of dietary fiber prevent cholesterol accumulation in rats by altering the spectrum of bile acids synthesized.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
We have completed work on a revised method for analysis of fecal bile acids which applies newer technology to avoid time consuming steps and improve recovery of bile acids. The method involves chloroform:methanol (1:1) extraction, extraction of neutral steroids with petroleum ether, enzymatic hydrolysis of conjugated bile acids (chololylglycine hydrolase), purification with a C-18 liquid chromatography column, methylation using dimethoxypropane/HC1, derivatization of hydroxyl groups to trimethylsilyl ethers, and quantification of these derivatives by gas-liquid chromatography (30m DB1701 capillary column). The greatest differences in recoveries of bile acids standards were observed when extraction with chloroform/methanol (>90% for all bile acids) was compared with acetic acid/toluene (51-72%) and when column chromatography (27-49%) was compared with thin layer chromatography &72-80%). The newer method offers increase recovery and reduced labor. A comparison of the
effects of ungelatinized potato starch (raw) with retrograded starch (cooked and cooled) on cholesterol and bile acid metabolism in rats was conducted. Raw potatoes resulted in little change in cholesterol levels or bile acid excretion in comparison to cellulose, the control. Rats fed cooked potatoes excreted higher levels of bile acids and had lower levels of liver cholesterol.
Impacts
(N/A)
Publications
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Progress 10/01/92 to 09/30/93
Outputs
In a series of studies in which the we examined potential mechanisms for changesin cholesterol balance in response to psyllium or pectin, we found that both sources of dietary fiber resulted in changes in bile acid metabolism that might account for their ability to reduce cholesterol accumulation in rats. In comparison to cellulose, both pectin and psyllium caused significant increases in bile acid pool size and secretion rate and increased synthesis rate, although the latter change was not statistically significant. A change in the relative size of the bile acid pools was also noted, namely an increase in the derivatives of chenodeoxycholic acid. As a follow-up to this study we measured the activity of cholesterol 7(alpha)-hydroxylase, the rate limiting enzyme in bile acid synthesis, in the livers of these same animals. Both pectin and psyllium increased the activity of this enzyme, indicating that the change in pool size and synthesis resulted in increased activity
of this key enzyme. In an effort to quantify changes in relative size of the bile acid pools as changes in another key enzyme, activity of 12(alpha)-hydroxylase was measured. This enzyme is the step which differentiates between cholic and chenodeoycholic acids. No difference in the activity of this enzyme was observed in response to pectin or psyllium in comparison to cellulose. These changes may result from reduced reabsorption of bile acid from the distal small intestine which would cause increased excretion.
Impacts
(N/A)
Publications
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Progress 10/01/91 to 09/30/92
Outputs
Our work with psyllium, rice bran and oat bran has indicated a reduction in accumulation of liver cholesterol in response to all three dietary fiber sources in comparison to cellulose when cholesterol was added to the diet. Serum cholesterol levels were lower in response to psyllium when compared to any of the other sources of dietary fiber, including cellulose. Fecal steroid excretion was not significantly altered by any of the dietary fiber sources. In comparison to cellulose, rice bran and oat bran increase the concentration of fecal bile acids while psyllium did not. Activity of cholesterol 7alpha-hydroxylase were measured in microsome isolated from livers of rats fed cellulose, psyllium, oat bran, pectin or cholestyramine. Cholestyramine resulted in the greatest activity while psyllium and pectin activities were significantly lower than cholestyramine but higher than oat bran and cellulose. These activities correspond fairly well with bile acid synthesis
measurements we have made using bile duct cannulation in rats. However this change in bile acid synthesis does not always appear to be caused by increased excretion of bile acids in feces. We have worked to develop a new, faster method for analysis of fecal bile acids. Metabolism of bile acids is an important link to our understanding of the ability of diet to lower serum cholesterol levels and in colon cancer risk. Our new method will be less time consuming but yield more precise results, especially in analyzing rat bile acids from feces.
Impacts
(N/A)
Publications
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Progress 10/01/90 to 09/30/91
Outputs
We continue to investigate the mechanism by which changes in bile acid pool sizeand excretion occur in response to some sources of dietary fiber. We have observed an increase in bile acid synthesis in response to psyllium and oat bran which are coupled with increase fecal excretion of bile acid. Currently we are assaying the activity of cholesterol 7(alpha)-hydroxylase in hepatic microsomes from animals fed various sources of dietary fiber as part of a semipurified diet. Differences in enzyme activity will be followed up by quantification of protein using an antibody for cholesterol 7(alpha)-hydroxylase. Our studies with the effects of fat saturation on lipid absorption and chylomicron composition have indicated that saturated fatty acid containing lipids (butter oil) result in smaller chylomicrons but do not seem to alter the total amount of lipid absorbed in comparison to polyunsaturated lipids. This may result from absorption of lipid over a longer portion of the
intestine when saturated lipids are fed. Larger amounts of phospholipid appear in lymph and in chylomicrons in response to saturated lipid.
Impacts
(N/A)
Publications
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Progress 10/01/89 to 09/30/90
Outputs
In a recent study which examined the effects of oat bran, rice bran, psyllium, and cellulose on serum and liver cholesterol level in rats fed a semipurified diet with and without added cholesterol, few changes in serum cholesterol levels were observed. Psyllium was the only source of dietary fiber which lowered serum cholesterol and did so in both cholesterol-containing and cholesterol-free diets. We have also observed that psyllium alters the rate of synthesis of bile acids and the relative sizes of the bile acid pools, resulting in an increase in the more hydrophilic chenodeoxycholic acid and its derivatives. We are currently examining this change in bile acid pools as a mechanism for changes in serum and liver cholesterol levels. Effects of dietary fatty acid saturation and cholesterol load on chylomicron size and composition in rats has been examined. Cholesterol load does not result in an increase in chylomicron cholesterol at low levels of cholesterol in
relation to lipid. Increasing the amount of cholesterol causes an increase in esterified cholesterol present on the chylomicron. Chylomicron size does not change with changes in infused cholesterol. Saturation of fatty acids infused with cholesterol does not seem to alter chylomicron size. Lipids with more saturated fatty acids (butter oil) result in smaller chylomicrons when compared with olive or corn oils.
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Progress 10/01/88 to 09/30/89
Outputs
In comparing the effects of these sources of dietary fiber on individual bile acids excreted, we have found that excretion of derivatives of chenodeoxycholic acid was increased t a greater extent that derivatives of cholic acid. If thiis change in excretion was indicative of changes in the pool sizes of these bile acids, several effects which would alter cholestero balance might be expected. As the size of the chenodeoxycholic acid pool increases, evidence would suggest that cholesterol synthesis and absorption as well as bile acid reabsorption would decrease, changes that would all result in negative cholesterol balance. In an effort to examine this effect more closely, we have carried out several experiments which have measured bile acid pool sizes in rats in response to some of the sources of dietary fiber used earlier. Total bile acid pool size was much higher when corn bran was included as the source of dietary fiber and much lower with oat bran when compared
to cellulose. This was reflected in the synthetic and secretion rates. Both pectin and psyllium caused increases in all three variables in comparison to cellulose. Composition of the bile acid pool was changed by the source of dietary fiber, the percent chenodeoxycholic acid derivatives was increased by oat bran, pectin and psyllium in comparison to cellulose, the three sources of dietary fiber which have been shown to reduce cholesterol accumulation.
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Progress 10/01/87 to 09/30/88
Outputs
In a continuation of our efforts to understand the mechanisms involved in the ability of some sources of dietary fiber to lower cholesterol levels and reduce fecal bile acid concentrations, we have examined the effects of a variety of food sources of dietary fiber and isolated polysaccharides on bile acid excretion and bile acid pool sizes in rats. Lyophilized carrots, apples or potatoes were fed to rats as part of a semipurifed diet for four weeks and serum and liver cholesterol and fecal steroid excretion measured. Apple and carrot seemed somewhat more effective at preventing the increase in serum cholesterol which was observed with added dietary cholesterol that potato or cellulose (control). Liver cholesterol levels were much higher with potato added to the diet. Daily bile acid and neutral steroid excretion was significantly increased with apple added to the diets suggesting this as a possible reason for the ability of apple to prevent some of the increase in
cholesterol levels resulting from cholesterol feeding. Potato caused a dramatic increase in alpha- and beta-muricholic acid concentrations and daily excretion both with and without cholesterol feeding. Excretion of muricholic acids has been suggested as a mechanism by which the rat maintains cholesterol homeostasis. We have also compared the effects of pectin, cellulose and psyllium hydrocolloid on bile acid pool sizes in rats.
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Progress 01/01/87 to 12/30/87
Outputs
Comparison of the effects of cellulose with wheat bran, corn bran and barley bran on liver cholesterol accumulation and fecal steroid excretion indicated similar liver cholesterol levels when cellulose wheat bran or barley bran were used and dietary fiber sources. However corn bran resulted in a significantly higher level than any of the other sources. Steroid excretion was highest in response to cellulose, corn bran and barley bran but was significantly lower in wheat bran fed animals. The highest level of excretion of steroids was observed in the animals which exhibited the highest liver cholesterol accumulation indicating the lack of correlation between steroid excretion and liver cholesterol accumulation in response to all sources of dietary fiber. Concentrations of bile acids in feces, a possible risk factor for colon cancer susceptibility, were lowest in response to corn bran in both cholesterol free and cholesterol fed animals. Corn bran may be more
effective than these other sources of dietary fiber in reducing bile acid concentrations in spite of its inability to alter cholesterol accumulation indicating the importance of inclusion of a variety of dietary fiber sources in our diets and the inaccuracy of a single variable in determining the usefulness of a diet ingredient.
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Progress 01/01/86 to 12/30/86
Outputs
We have completed a study comparing effects of a wide variety of dietary fiber sources on cholesterol levels and intestinal transit time in rats. We compared cellulose, wheat bran, corn bran, barley bran, and bamboo shoots as part of semipurified diet with and without added cholesterol. Bamboo shoots caused a dramatic reduction in food intake and reduced weight gain in comparison to all the other sources of dietary fiber. However animals did not display reductions in liver weight as a percent of body weight or other signs of food restriction. They did have greatly reduced serum and liver cholesterol levels. Both with and without added cholesterol, bamboo shoots resulted in serum cholesterol levels of about 15% of control animals and significant reductions in liver cholesterol also. We are now investigating the relative input of the reduced food intake in causing these effects. Transit time as measured by excretion of an oral dose of Cr was more rapid in the three
sources of "bran" than with cellulose and was increased by bamboo shoots, probably as a result of reduced food intake. We also completed a group of studies aimed at improving our understanding of the role of dietary cholesterol on regulation of hepatic enzymes which regulate cholesterol and bile acid metabolism. Chylomicron remnants were prepared and incubated with hepatocytes and activities of cholesterol 7-alpha-hydroxylast, hydroxymethylglutaryl coenzyme A reductase and acyl CoA:cholesterol acyl transferase were measured.
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Progress 01/01/85 to 12/30/85
Outputs
We have been involved in studies which measured the effects of several sources of dietary fiber on cholesterol levels and bile acid excretion in human subjects. Oat bran and beans (100 g per day) were fed to hypercholesterolemic human subjects as part of an otherwise normal diet (38% of kcal as fat). Serum cholesterol levels, especially LDL cholesterol levels, were reduced by both treatments by about 20% after 2 weeks of feeding. Fecal bile acid excretion was increased significantly by oat bran, as has been reported previously. This suggests that oat bran may reduce cholesterol levels by increasing steroid excretion to an extent sufficient to result in negative cholesterol balance. Beans, in spite of having substantial water soluble dietary fiber components much like oat bran, caused a decrease in bile acid excretion. Thus, in spite of similar effects on serum cholesterol and similar dietary fiber composition, these two sources of dietary fiber have different
mechanisms for imparting these effects. We are also investigating the effects of cholesterol, delivered to the liver on chylomicron remnants, on hydroxymethylglutaryl coenzyme A reductase and cholesterol 7-alpha-hydroxylase. The importance of dietary cholesterol in regulating these key enzymes in cholesterol metabolism is of interest in our understanding of the importance of dietary cholesterol in regulating cholesterol balance in general.
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Progress 01/01/84 to 12/30/84
Outputs
We have completed development of a method for measuring cholesterol 7 alpha-hydroxylase which employs phospholipid liposomes to solubilize the cholesterol substrate. This avoids the inhibitory effects of detergents used for solubilization previously and greatly reduces variability in the assay. We have also measured the effects of alfalfa, oat bran, corn bran and cellulose as sources of dietary fiber on cholesterol levels and steroid excretion in rats. Liver cholesterol was reduced by oat bran, and alfalfa in comparison to cellulose. Fecal excretion of steroids was not significantly higher in response to these dietary fiber sources but was much higher in animals fed corn bran. Cholesterol levels in response to these dietary fiber sources are obviously mediated by mechanisms in addition to changes in steroid excretion.
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Progress 01/01/83 to 12/30/83
Outputs
We have continued our study of the dietary regulation of cholesterol metabolism by examining the rate limiting step in bile acid synthesis, the 7a-hydroxylation of cholesterol. We are now examining the effect of the phosphorylation state of the enzyme on its activity in an effort to examine the coupling of its activity to that of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Our results suggest that these two enzymes are regulated by phosphorylation but in opposite directions, i.e., 7a-hydroxylase being active in the phosphorylated state and the reductase in the dephosphorylated state. We plan to examine the effects of change in diet on the activities of these enzymes through effectors such as insulin and glucagon and also in response to lipoproteins. Our work with dietary fiber has been directed toward development of a method for use in estimating the bile acid adsorption capacity of water soluble materials such as gums. We have observed substantial adsorption
of cholic and deoxycholic acids by gums from guar and oats and other commercially used gums. Hemicellulose was also found to adsorb significant amounts of these bile acids.
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Progress 01/01/82 to 12/30/82
Outputs
Our studies concerning regulation of cholesterol metabolism by dietary fiber have led us to examine the short-term regulation of 3-hydroxy-3-methylglutaryl Coenzyme A reductase. We have found that changes in enzyme activity can be detected within 15 minutes following initiation of feeding in rats. The active form of the enzyme (measured in the presence of fluoride to prevent phosphorylation) decreases during the first hour and then increases by one hour after the beginning of a meal. We have examined dietary cholesterol and dietary oat bran in this system since both these dietary variables have been shown to inhibit reductase during long term experiments. Cholesterol feeding resulted in a greater reduction in the active form of the enzyme and a continuation of this lower activity through 4 hours after the meal began. Oat bran caused a similar inhibition in enzyme activity which remained lower than control animals but was higher than cholesterol-fed animals. Oat
bran appears to inhibit enzyme activity during the short-term which may be a part of the mechanism responsible for its observed hypocholesteremic effect. Inhibition of reductase activity by cholesterol feeding appears to involve phosphorylation/dephosphorylation which may be controlled by the composition of returning chylomicron remnants.
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Progress 01/01/81 to 12/30/81
Outputs
Our studies investigating the effects of sources of dietary fiber and some isolated polysaccharides on bile acid metabolism were continued using rats in which large amounts of liver cholesterol had accumulated in response to diet and following the regression of this cholesterol in response to dietary lignin, cellulose or pectin. Pectin resulted in the fastest regression of liver cholesterol but little difference was seen in the rate of regression of any of the dietary groups. Bile acid excretion increased rapidly in response to lignin and more slowly to pectin. Both remained high while liver cholesterol levels remained elevated. Through examination of the fate of newly synthesized and previously synthesized cholesterol in these animals indicated that chenodeoxcholic acid is synthesized primarily from cholesterol esterified possibly as a result of an inhibition of the 12a-hydroxylation step or bile acid synthesis. We have also examined the effect of saturation of
dietary lipid of bile acid excretion in rats. More saturated fats result in lower excretion of fecal bile acids than unsaturated fats. Mixtures of dietary fats do not seem to result in levels of excretion which would be predicted from the saturation of the mixture. Individual fatty acids are thought to alter cholesterol and bile acid metabolism rather than the total fatty acid saturation.
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Progress 01/01/80 to 12/30/80
Outputs
In a continuation of our investigations into the components responsible for the hypocholesteremic effects of alfalfa, we compared the effects of alfalfa with saponin-free alfalfa (extracted with 50% ethanol) and alfalfa sprouts, a product of alfalfa often consumed by humans. Alfalfa and saponin-free alfalfa reduced the accumulation of liver cholesterol in cholesterol-fed rats. Alfalfa sprouts did not prevent a significant liver cholesterol accumulation. This tends to agree with our earlier experiments which indicated that saponins were not involved in bile acid adsorption, a suggested mechanism for the hypocholesteremic effects of alfalfa. We have also found an alternate mechanism for the increased bile acid excretion observed with some sources of dietary fiber. Oats and alfalfa increase bile acid excretion in rats. In addition, the spectrum of bile acids excreted is changed from approximately 50% chenodeoxycholic (CDC) acid derivatives in fiber-free or cellulose
fed animals to 60% CDC in alfalfa or oats fed animals. Derivatives of CDC are not reabsorbed as efficiently and are also thought to inhibit cholesterol synthesis. Both these effects may be an additional mechanism by which some dietary fibers reduce cholesterol levels and increase bile acid excretion.
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Progress 01/01/79 to 12/30/79
Outputs
The hypocholesteremic effects of alfalfa have been examined to find which component is responsible. We examined the in vitro bile acid adsorption of alfalfa and several fractions of alfalfa generated by sequential extraction of known components. The main components extracted were saponins, pectins and gums, and lignin. Cellulose and hemicellulose (holocellulose) were the only components remaining after completion of this extraction sequence. Removal of saponins, which have been shown to be hypocholesteremic, did not alter bile acid binding capacity. Likewise, removal of pectins and gums did not alter bile acid adsorption. Delignification, however, dramatically reduced adsorption. These data indicate that saponins are not responsible for the adsorption of bile acids and that their hypocholesteremic effects result from some other mechanism. The adsorption of bile acids by saponin-free preparations indicates that saponins may not be solely responsible for the
hypocholesteremic effects of whole alfalfa. We are now in the process of examining the effects of these components which display bile acid adsorption on in vivo cholesterol metabolism.
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Progress 10/01/78 to 12/30/78
Outputs
A comparison of the effects of various sources of dietary cholesterol on the accumulation of liver cholesterol in the rat has been completed. Male, Wistar rats (100 g) were fed a semipurified diet (B) to which 0.5% cholesterol was added as crystalline cholesterol, fresh eggs, lyophilized fresh eggs or dried whole egg. Dietary lipids used were corn oil, lard or hydrogenated coconut oil. Liver cholesterol levels were signigicantly increased by the addition of any form of cholesterol. However, liver levels were higher when crystalline cholesterol was used than when fresh or lyophilized fresh eggs supplied the cholesterol. The source of lipid used in the diets also influenced cholesterol accumulation on the liver. Corn oil and lard yielded similar levels while coconut oil resulted in a lower level of liver cholesterol. Serum high density lipoprotein (HDL) cholesterol was also measured. HDL cholesterol levels were significantly higher in both saturated fat (lard)
and egg fed groups than when crystalline cholesterol and corn oil were used in the diet. These results indicate dietary interactions which result in a cariation of the widely accepted role of eggs in determining cholesterol level. Coupled with recently done human experiments eleswhere, it appears that a thorough reevaluation the role of dietary cholesterol is in order. We are also examining the interaction of dietary fiber and its components on cholesterol metabolism in order to determine its role in the development of atherosclerosis.
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