Regarding your old post on ketogenic diet and microbiota, why do you think bifidobacterium decreased on low carb? I would generally guess this is a negative...?
I cited two studies on low carbohydrate dieting and gut microbiota composition, one by Duncan et al (1) and the other by Brinkworth et al (2). They showed a negative effect of reducing carbs on gut flora, measured by species composition (16S RNA) and SCFA. They both analyzed fecal samples. In general, fecal samples are reliable and make easier to study colonic SCFA metabolism. However, they are an indirect method of quantification. Of the three main SCFA produced in the colon, only acetate has shown a correlation between fecal concentration and absorption (3):
The data shows a negative correlation (r=-0.834) between acetate absorption from an infusion and fecal acetate concentration. This means that the fecal concentration of acetate might reflect absorption rather than production, in an inverse manner (less acetate in fecal samples equals more absorption). In this study, neither propionate or butyrate showed a correlation between absorption and fecal concentration.
SCFA in the Duncan et al. study
Acetate, butyrate and propionate concentrations in fecal samples from the Duncan et al. study are shown below:
As the intake of carbohydrate decreased, there was a parallel reduction in all three SCFA.
SCFA in the Brinkworth et al. study
Acetate, butyrate and propionate concentrations in fecal samples from the Brinkworth et al. study are shown below:
As seen in the Duncan et al. study, after 8 weeks with a low-carbohydrate diet, SCFA concentrations were reduced, although not as drastically.
Analysis and interpretation of the data
Both studies show a clear correlation between carbohydrate intake and SCFA concentration in fecal samples. The magnitude of the changes between individual SCFA might be due to differences in the intervention time (4 weeks vs. 8 weeks).
As shown by Vogt and Wolever (see above), acetate concentration in fecal samples reflect more precisely acetate absorption rather than production. Thus, lower fecal acetate levels with reduced carbohydrate reflect more acetate absorption (or utilization, see below).
Most focus has been given to the apparent reduction in butyrate levels, which may compromise colonic health. In this regard, low carbohydrate diets might be detrimental for colonic health because of reduced butyrate production. For assessing the validity of this statement, we must look at colonic butyrate metabolism.
Colonic butyrate metabolism
Approximately, 95% of the butyrate produced in the colon is absorbed. This is why fecal concentrations are not a good guide to production rates: a very high proportion of the SCFA is taken up by the colonic mucosa (4). Butyrate is produced from two molecules of acetyl CoA, yielding acetoacetyl CoA, which is further converted to finally butyryl CoA. This metabolite can be converted to butyrate via butyrate kinase or butyryl CoA:acetate CoA transferase.
Butyrogenic substrates include starch, inulin and xylan. But certain species are capable of producing butyrate from acetate. Synthesis of butyrate from acetate is performed via the butyryl CoA:acetate CoA transferase pathway, which seems to be the most prevalent route of butyrate synthesis by human gut bacteria (5). So, while glucose is needed for butyrate synthesis, acetate seems to be the main substrate for butyrate formation. The predominance of butyrate synthesis from the acetate dependent pathway might reflect a selective advantage for bacteria which transform acetate to butyrate in the colon, where acetate concentrations are high.
Overall, the reduction in acetate and butyrate fecal concentrations may be translated to increased absorption and reduced excretion. Butyrate can be synthesized from acetate, which reduces the concentration of both SCFA in feces. The determined Km for butyrate transport in the colon has been found to be 14.8 +/-3.6 mM (6) and 17.5 +/- 4.5 mM in the proximal colon (7). The apparent saturation kinetics showed by butyrate transport across the colonic luminal membrane could further explain the results seen in the studies mentioned above: increasing the carbohydrate content in the diet would augment the number of glucose-dependent butyrogenic bacteria, increasing the colonic production and concentration of butyrate. Because transport of butyrate is saturable, excess butyrate is excreted, producing increased levels in feces.
The case for Bifidobacteria
The change in Bifidobacteria concentrations after the low carbohydrate diet is due to the presence of an important number of bacteria capable of degrading glucose/starch. In this scenario, reduced carbohydrate availability would reduce the number of total Bifidobacteria (at least certain species). This does not mean that this is bad per se. It is important to determine the specific Bifidobacteria which are responsive to diet. For instance, B.longum seem to be capable of catabolizing not only dietary oligosaccharides, but also glycoproteins and glycoconjugates from the host; as well as nucleotides (8). Moreover, gut Bifidobacteria (as shown by the genomic analysis of B.longum), are capable of adapting to different carbohydrate substrates depending on their availability (9). In addition, metabolic-crossfeeding occurs between Bifidobacteria and other species. For example, E.hallii, a butyrogenic bacterium, is unable to grow on pure starch by itself. Co-culture of this bacterium with B.adolescentis stimulates its growth and butyrate synthesis, paralleled by a reduction in lactate levels (10). The scheme is pretty simple: B.adolescentis is capable of fermenting starch, producing lactate which serves as substrate to E.hallii. Other lactate-independent mechanisms of cross-feeding have also been observed in FOS and oligofructose-only co-cultures of B.longum with Roseburia intestinalis or Anaerostipes caccae, which are cabaple of producing butyrate and consuming acetate (11).
Because of its complexity, the specific mechanisms by which certain Bifidobacteria could be beneficial are unknown, although there is evidence of health benefits from increasing gut Bifidobacteria (12, 13, 14). There are some issues with interpreting the evidence in this topic:
Having this in mind, I cant assure either that a low carbohydrate diet is not harmful to the gut microbiota. As far as the evidence goes, we can only speculate and formulate hypotheses. And useful hypotheses should be based on logic and evolutionary inference. We should ask not only "how" but also "why". In this case, we are not going to focus on the "how" but on the "why"; to put it formally, why an increased intake of starch is associated with an increase in Bifidobacteria? What is the evolutionary basis?
One important protective role of Bifidobacteria is preventing colonization of enteropathogens by reducing their adhesion to intestinal epithelial cells. This has been shown directly for E. coli and S. typhimurium (15). Other commonly problematic Enterobacteriaceae include Klebsiella and Shigella. Growth of these pathogens is stimulated by high glucose-low oxygen conditions. The selective advantage of having responsive Bifidobacteria in the gut might be protection. As increased glucose concentrations favor the development of an adequate environment for growth of these pathogens, there has to be a mechanism by which the composition of the normal microbiota is maintained. So there is a parallel increase in Bifidobacteria with increasing concentrations of dietary carbohydrates to restrain colonization of pathogenic anaerobes. The fact that certain species of Bifidobacteria can metabolize different oligosaccharides and adapt to the substrate availability supports this hypothesis. I might elaborate more on this in subsequent posts.
Conclusions
Low carbohydrate diets seem to reduce the fecal concentration of SCFA in the short term. Some adaptation seems to occur, judging by the differences between the study periods (4 weeks vs. 8 weeks). Fecal concentrations of SCFA are not good indicators of SCFA colonic production. Conversely, they rather reflect excretion (butyrate) and absorption (acetate). Butyrate can be produced from different substrates, of which acetate is the main precursor in the human gut. There is a reduction in the levels of Bifidobacteria detected in stool samples, proportional to the decrease in carbohydrate in the diet. Although no individual species where identified, studies have shown that Bifidobacteria are capable of adapting to substrate availability and cross-feed with other bacteria. The evolutionary basis for increased Bifidobacteria in response to sugar might involve a protective mechanism against colonization of enteropathogenic bacteria, such as E. coli, Klebsiella, Shigella and Salmonella.
Duncan SH, Belenguer A, Holtrop G, Johnstone AM, Flint HJ, & Lobley GE (2007). Reduced dietary intake of carbohydrates by obese subjects results in decreased concentrations of butyrate and butyrate-producing bacteria in feces. Applied and environmental microbiology, 73 (4), 1073-8 PMID: 17189447
Brinkworth GD, Noakes M, Clifton PM, & Bird AR (2009). Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty acids and bacterial populations. The British journal of nutrition, 101 (10), 1493-502 PMID: 19224658
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| Copyright © 2011 by the American Society for Nutrition |
The data shows a negative correlation (r=-0.834) between acetate absorption from an infusion and fecal acetate concentration. This means that the fecal concentration of acetate might reflect absorption rather than production, in an inverse manner (less acetate in fecal samples equals more absorption). In this study, neither propionate or butyrate showed a correlation between absorption and fecal concentration.
SCFA in the Duncan et al. study
Acetate, butyrate and propionate concentrations in fecal samples from the Duncan et al. study are shown below:
 |
| SCFA concentrations (mM) for fecal samples. M=Maintenace; HPMC= High-protein, moderate-carbohydrate; HPLC = High-protein, low-carbohydrate. Mean values. |
As the intake of carbohydrate decreased, there was a parallel reduction in all three SCFA.
SCFA in the Brinkworth et al. study
Acetate, butyrate and propionate concentrations in fecal samples from the Brinkworth et al. study are shown below:
 |
| Fecal SCFA concentrations (mM) after 8 weeks of either a low carbohydrate (LC) or high carbohydrate (HC) diet. Mean values. |
As seen in the Duncan et al. study, after 8 weeks with a low-carbohydrate diet, SCFA concentrations were reduced, although not as drastically.
Analysis and interpretation of the data
Both studies show a clear correlation between carbohydrate intake and SCFA concentration in fecal samples. The magnitude of the changes between individual SCFA might be due to differences in the intervention time (4 weeks vs. 8 weeks).
As shown by Vogt and Wolever (see above), acetate concentration in fecal samples reflect more precisely acetate absorption rather than production. Thus, lower fecal acetate levels with reduced carbohydrate reflect more acetate absorption (or utilization, see below).
Most focus has been given to the apparent reduction in butyrate levels, which may compromise colonic health. In this regard, low carbohydrate diets might be detrimental for colonic health because of reduced butyrate production. For assessing the validity of this statement, we must look at colonic butyrate metabolism.
Colonic butyrate metabolism
Approximately, 95% of the butyrate produced in the colon is absorbed. This is why fecal concentrations are not a good guide to production rates: a very high proportion of the SCFA is taken up by the colonic mucosa (4). Butyrate is produced from two molecules of acetyl CoA, yielding acetoacetyl CoA, which is further converted to finally butyryl CoA. This metabolite can be converted to butyrate via butyrate kinase or butyryl CoA:acetate CoA transferase.
Butyrogenic substrates include starch, inulin and xylan. But certain species are capable of producing butyrate from acetate. Synthesis of butyrate from acetate is performed via the butyryl CoA:acetate CoA transferase pathway, which seems to be the most prevalent route of butyrate synthesis by human gut bacteria (5). So, while glucose is needed for butyrate synthesis, acetate seems to be the main substrate for butyrate formation. The predominance of butyrate synthesis from the acetate dependent pathway might reflect a selective advantage for bacteria which transform acetate to butyrate in the colon, where acetate concentrations are high.
Overall, the reduction in acetate and butyrate fecal concentrations may be translated to increased absorption and reduced excretion. Butyrate can be synthesized from acetate, which reduces the concentration of both SCFA in feces. The determined Km for butyrate transport in the colon has been found to be 14.8 +/-3.6 mM (6) and 17.5 +/- 4.5 mM in the proximal colon (7). The apparent saturation kinetics showed by butyrate transport across the colonic luminal membrane could further explain the results seen in the studies mentioned above: increasing the carbohydrate content in the diet would augment the number of glucose-dependent butyrogenic bacteria, increasing the colonic production and concentration of butyrate. Because transport of butyrate is saturable, excess butyrate is excreted, producing increased levels in feces.
The case for Bifidobacteria
The change in Bifidobacteria concentrations after the low carbohydrate diet is due to the presence of an important number of bacteria capable of degrading glucose/starch. In this scenario, reduced carbohydrate availability would reduce the number of total Bifidobacteria (at least certain species). This does not mean that this is bad per se. It is important to determine the specific Bifidobacteria which are responsive to diet. For instance, B.longum seem to be capable of catabolizing not only dietary oligosaccharides, but also glycoproteins and glycoconjugates from the host; as well as nucleotides (8). Moreover, gut Bifidobacteria (as shown by the genomic analysis of B.longum), are capable of adapting to different carbohydrate substrates depending on their availability (9). In addition, metabolic-crossfeeding occurs between Bifidobacteria and other species. For example, E.hallii, a butyrogenic bacterium, is unable to grow on pure starch by itself. Co-culture of this bacterium with B.adolescentis stimulates its growth and butyrate synthesis, paralleled by a reduction in lactate levels (10). The scheme is pretty simple: B.adolescentis is capable of fermenting starch, producing lactate which serves as substrate to E.hallii. Other lactate-independent mechanisms of cross-feeding have also been observed in FOS and oligofructose-only co-cultures of B.longum with Roseburia intestinalis or Anaerostipes caccae, which are cabaple of producing butyrate and consuming acetate (11).
Because of its complexity, the specific mechanisms by which certain Bifidobacteria could be beneficial are unknown, although there is evidence of health benefits from increasing gut Bifidobacteria (12, 13, 14). There are some issues with interpreting the evidence in this topic:
- Many authors don't determine the exact species being studied (take all Bifidobacteria as a group).
- Supplementation is done with different strains and the long-term effects are not known, because bacteria supplemented via diet are treated as allochthonous.
- Genomic inspection has shown that Bifidobacteria are metabolically very flexible. Adaptation to substrate variations might take longer than 8 weeks.
- There is metabolic-crossfeeding occuring between bacteria. This is a highly complex network of connections for which we are only starting to get an initial picture.
Having this in mind, I cant assure either that a low carbohydrate diet is not harmful to the gut microbiota. As far as the evidence goes, we can only speculate and formulate hypotheses. And useful hypotheses should be based on logic and evolutionary inference. We should ask not only "how" but also "why". In this case, we are not going to focus on the "how" but on the "why"; to put it formally, why an increased intake of starch is associated with an increase in Bifidobacteria? What is the evolutionary basis?
One important protective role of Bifidobacteria is preventing colonization of enteropathogens by reducing their adhesion to intestinal epithelial cells. This has been shown directly for E. coli and S. typhimurium (15). Other commonly problematic Enterobacteriaceae include Klebsiella and Shigella. Growth of these pathogens is stimulated by high glucose-low oxygen conditions. The selective advantage of having responsive Bifidobacteria in the gut might be protection. As increased glucose concentrations favor the development of an adequate environment for growth of these pathogens, there has to be a mechanism by which the composition of the normal microbiota is maintained. So there is a parallel increase in Bifidobacteria with increasing concentrations of dietary carbohydrates to restrain colonization of pathogenic anaerobes. The fact that certain species of Bifidobacteria can metabolize different oligosaccharides and adapt to the substrate availability supports this hypothesis. I might elaborate more on this in subsequent posts.
Conclusions
Low carbohydrate diets seem to reduce the fecal concentration of SCFA in the short term. Some adaptation seems to occur, judging by the differences between the study periods (4 weeks vs. 8 weeks). Fecal concentrations of SCFA are not good indicators of SCFA colonic production. Conversely, they rather reflect excretion (butyrate) and absorption (acetate). Butyrate can be produced from different substrates, of which acetate is the main precursor in the human gut. There is a reduction in the levels of Bifidobacteria detected in stool samples, proportional to the decrease in carbohydrate in the diet. Although no individual species where identified, studies have shown that Bifidobacteria are capable of adapting to substrate availability and cross-feed with other bacteria. The evolutionary basis for increased Bifidobacteria in response to sugar might involve a protective mechanism against colonization of enteropathogenic bacteria, such as E. coli, Klebsiella, Shigella and Salmonella.
Duncan SH, Belenguer A, Holtrop G, Johnstone AM, Flint HJ, & Lobley GE (2007). Reduced dietary intake of carbohydrates by obese subjects results in decreased concentrations of butyrate and butyrate-producing bacteria in feces. Applied and environmental microbiology, 73 (4), 1073-8 PMID: 17189447Brinkworth GD, Noakes M, Clifton PM, & Bird AR (2009). Comparative effects of very low-carbohydrate, high-fat and high-carbohydrate, low-fat weight-loss diets on bowel habit and faecal short-chain fatty acids and bacterial populations. The British journal of nutrition, 101 (10), 1493-502 PMID: 19224658
What do you think about the role of exogenous butyrate? Butyric acid is quite high in butter, for example. It is also high in some less common foods, such as fermented meat, but they might have played a more important role in diets of the past. I've posted some studies in my blog showing orally consumed butyric acid had a similar effect on IBD patients as BA enemas did.
ReplyDeleteThe amout of butyric acid (BA) produced by colonic bacteria is about 2 times higher then amount of preformed exogenous BA present in butter. Theferore, I prefere supplementation of foods with high content of resistent starches in low carb. or ketogenic diets.
DeleteIndeed, carbohydrate restriction doesn't entail fiber restriction. For example Inulin, one of the resistant starch fibers occurs naturally in some "root" foods (underground vegetables) such as jicama, onions, Jerusalem artichokes and chicory roots.
Relatively high content of resistent starch is also present in green banana, cassava and sweet potatoes. Another one type of restent starch fiber is made from a hybrid form of corn, the brand name is Hi maize 260.
Hi Melissa,
ReplyDeleteI think the main problem with dietary butyric acid is absorption. From the studies I have read using oral butyrate supplementation, most use modified butyric acid (such as tributyrin) or coated tabs to resist degradation. Other studies are done in vitro.
"Bovine milk fat contains from 7.5 to 13.0 mol/100 mol butyric acid. Because dibutyrylacylglycerols are present in trace amounts only, this means that about one third of milk fat triacylglycerols contain one molecule of butyrate. On ingestion, lipase-mediated hydrolysis of butyrate commences in the stomach and will be complete on reaching the proximal small intestine. Liberated butyrate is absorbed from the intestinal lumen to the enterocytes; it then passes directly to the portal circulation for transport to the liver where most is metabolized." (Parodi, 1997)
Im not sure what the level of tributyrin in dairy is or if the digestion/absorption is modified with co-ingestion of LCFA and other lipids present in dairy fat. From the data available, it seems unlikely that dietary butyric acid could reach significant levels in the colon.
So, assuming there is a correlation between bifidobacteria population and health, is it simply that there aren't enough low carbers to "disrupt" the correlation, kind of like glucose tolerance and health/longevity?...what happens is that we end up comparing crap carb eaters with whole-food carb eaters, and the whole-food carb eaters have more bifidobacteria?
ReplyDeleteHi john,
ReplyDeleteI really don't know. Low Bifidobacteria counts have been observed in many pathologies, "how low" depends on the threshold. There is no clear cut, as most of information related to gut microbiota is merely speculative. According to my hypothesis, in a context of a high carbohydrate diet, high gut Bifidobacteria would correlate strongly with health, while low levels with increased disease risk. I would also expect better composition in whole-food carb eaters, given that inflammation and certain dietary agressors can debilitate and alter the gut microbiota. So it is not only about the amount of carbohydrates but also the sources.
On perfect health diet they mentioned this post with "Lucas Tafur gives us a reason to put vinegar in our foods: gut bacteria can convert acetate to butyrate." Does dietary acetate work for this? I got the impression that it was acetate produced by other bacteria that was at play here?
ReplyDeleteI may be wrong but when they reduce carbohydrate don't they also reduce fiber? And wouldn't that play a role? One of the fastest ways to increase butrate production is to consume inulin after all.
ReplyDeleteIf indeed I'm right, the reduction in fiber has confounded the effect of carbohydrate restriction, we can't really know which one produced the effects.
Carbohydrate restriction doesn't entail fiber restriction, there is jicama and chicory root and other sources of it. A good experiment would then be high carb high fiber, low carb high fiber, high carb low fiber and low carb low fiber. Then we would know.
Hi Melissa,
ReplyDeleteAs with dietary butyrate, seems unlikely. See:
http://www.ncbi.nlm.nih.gov/pubmed/1248680
http://www.ncbi.nlm.nih.gov/pubmed/16482
I am talking about acetate produced by the microbiota. This is what cross-feeding means: the final product of one bacteria serves as substrate for another bacteria.
Hi Stabby,
The differences in fiber consumption can play a role. However, glucose is a substrate for certain bacteria, so reducing carbohydrate would have an effect over this population. This seems to be the case with some Bifidobacteria.
You say that 95% of butyrate is absorbed by the intestine. Where does this number come from? Reference 4 makes the same claim but also gives no reference for this number. Really enjoyed reading! Thanks!
ReplyDeleteHi John,
DeleteI think it comes from this reference.
http://www.ncbi.nlm.nih.gov/pubmed/9508842
Nevertheless, I should revise the numbers.
OK, thanks!
ReplyDeleteI am curious, I have had a GI test done and my Acetate was considered high at 73, Butyrate was 12, Propoionate 15 and Valerate .8 (low), what does this mean? I had been on a low carb diet for about a week at the time of the test.
ReplyDelete