Does a ketogenic diet confer the benefits of butyrate without the fibre?

Tenuous arguments from fibre apologists

According to many plant-eating enthusiasts, we must eat fibre to be healthy for the following reasons:

Of the above statements, only one of them seems well-justified to me, but it also seems irrelevant. Let's start from the end.

Without butyrate your colon cells will die off.

This idea (a quote from Wikipedia) seems to to be an exaggerated interpretation of a study by Donohoe et al.. The authors are studying germ-free mice, who don't, of course, have bacteria synthesising butyrate. They describe what looks to them like impaired colon cell energetics in the mice and ultimately autophagy upregulation, meaning the cells are eating themselves. They reverse these effects with butyrate. I've already written about some of the curious paradoxes inherent in the study. To summarise, other studies consistently find germ-free mice to be healthier than wild mice by many a measure, including appearing to be more energetic, and living longer. There seems to have been a conflation of cell energy with mitochondrial energy, by not looking for mitochondrial density changes. So, I'm not convinced the butyrate made things better.

Likewise, the reported evidence of autophagy (increased autophagosomes attributed to upregulation of AMPK), insofar as it indicates autophagy, could equally be a desirable result, given the role of autophagy in maintaining healthy tissues. See, e.g. [Miz2011]. Certainly unrestrained autophagy, with no homeostatic mechanism, should result in total loss of tissue, but that doesn't seem to happen with the germ-free mice. Germ-free rodents have freakishly large caecums, and somewhat reduced small intestines, but so far as I can tell, no colon abnormalities worth mentioning. For an extensive review of the data already available in 1971 on germ-free animals, including the structure and function of various organs, see The gnotobiotic animal as a tool in the study of host microbial relationships..

In any case, if the colons of germ-free mice are at any disadvantage there are clearly more differences that might be attributable to than mere lack of butyrate. Are there other reasons to worry about colon cells that don't get any?

Butyrate is the preferred fuel of the colonocyte, therefore it is essential.

If you haven't read my thoughts on the term "preferred", the point is that what a cell will consume first isn't necessarily the fuel that is the healthiest, though it certainly can be. Other reasons could be to get rid of it, or to access the metabolites. I'm not really suggesting that butyrate is toxic to colon cells. (Though as soon as that thought occurred to me I looked for evidence that it can be, which, of course there is [Pen2007]. Apparently it can accumulate due to maldigestion or bacterial overgrowth and cause serious epithelial damage. But I digress.) All I'm saying is that habitual heavy use doesn't imply something is needed. The same argument has been made about glucose in the brain, and we all know that the brain actually needs only a very small amount of glucose, if β-hydroxybutyrate is in good supply. It's still possible that other fuels are as good or better than butyrate for the colonocyte.

Butyrate in the colon treats colitis.

Normally, colonocytes do metabolise butyrate, mostly into CO2 and ketone bodies, but this is impaired in ulcerative colitis [Roe1980], [Roe1993], [Ahm2000], such that ketogenesis is is inversely proportional to the severity of the disease [Roe1980].

This impairment may explain the mixed results in treatments involving butyrate. Some researchers have tried to treat colitis by adding more butyrate for substrate, by enema. Perhaps unsurprisingly, that has not met with much success. Or has it? I read a somewhat confusing review [Mal2015] that has several citations in it that don't appear to line up with the claims preceded by the citations, including citing the same paper that I've cited above (Roe1980), as showing "that restoration of butyrate levels by intracolonic infusion treats UC", which I can find no mention of in the paper, and citing a single paper twice, ([Ham2010]), once to say that enemas had very limited effect (which I think is correct) and once, later, to say it was a "well demonstrated" "cure". These are probably just simple citation errors on my part or theirs.

There have been some successes using enemas, but the results are mixed [Ham2008]. Insofar as there are successes, it is worth noting that the butyrate was taken in by rectal cells, not colon cells, and so the effect was post-absorptive. In other words, it must have come systemically. In fact, when the butyrate is applied directly to impaired cells it seems to worsen the situation. These points are noted in the review, and motivates their own contribution.

The researchers used intraperitoneal injections of butyrate to apparently almost completely restore colonocyte integrity in rodent models of colitis. At face value, this would suggest that it is not the butyrate that helped, but a metabolite of butyrate, i.e. ketone bodies, since peritoneal injections normally pass through the liver [Tur2011]. If it's systemic ketone bodies we want, we know how to do that! Also, this method is rarely used in humans, so it may not be easy to make any practical use of. In any case, none of this would suggest that eating plant fibre will help colitis in any way, given that the issue appears to depend on inability to use the butyrate.

Ulcerative Colitis UC and Crohn's Disease (CD) constitute the Inflammatory Bowel Diseases (IBD). There is not clear evidence that fibre intake helps with IBD, and in fact, "low residue" or "low fibre" diets are usually recommended (see below). In case you were wondering, "residue" means anything that survives digestion, and comes all the way through the intestines. That includes fibre , but also microorganisms, and secretions and cells shed from the alimentary tract.

While there are studies that support the benefit of fibre in IBD, there are others showing harm. The evidence is mixed enough to be called weak and inconclusive [Kap2016].

Anecdotes such as the "Crohn's Carnivore" suggest a different solution might hold for some:

"Eight years ago I decided to eat nothing but meat for a year. Now I have a perfectly normal colon. If those two events are indeed correlated, and someone could figure out exactly how, a whole lot of people would be able to find relief from a terrible disease."

That experience runs both with and possibly against current dietary guidelines for IBD. In a 2011 review [Bro2011], the authors show that most guidelines advise low fibre intake, especially during flares. Some also advise low fat intake, and in particular, to eat lean meat. I'm not sure whether the Crohn's Carnivore was eating lean or fatty meat during his year of healing. At first blush, the low fat advisory looks like just another "extra-mile" kind of recommendation, in which guideline writers are throwing in other ideas about healthy diet for good measure. However, they state that it comes from the reported reactions of some patients. They also cite patient surveys which list meat as a provoking food in 25% of respondents. (The most common response was vegetables, at 40%). One wonders if there are conflations. Later, the authors specifically say that there is little to support or refute a low fat recommendation.

Another anecdote, this time elevated to "case study" level, because physicians penned it, comes from the Evolutionary Medicine Working Group, in Budapest, Hungary [Tot2016]. They report complete resolution of symptoms in a child with Crohn's and cessation of medications from an essentially meat-only diet. The exception was that patient was allowed some honey, but it was low enough that ketosis was maintained. This was a 2:1 fat:protein diet, so definitely not low fat. The child had previously tried low fat, low fibre, and several medications without improvement. It is interesting to note that even one dose of "paleo approved" fibre caused a flare up.

"Given the patient's severe condition upon the first visit the paleolithic ketogenic diet was started in the strictest form thus containing no vegetables and fruits at all. Such a diet may first sound restrictive but our previous experience indicate that a full fat-meat diet is needed in the most severe cases of Crohn's disease. In addition, our experience shows that even a single occasion of deviation from diet rules may result in lasting relapse. This was the case in the present patient too where breaking the strict rules (eating the "paleo cakes") resulted in a thickening of the bowel wall. Based on our experience this is due to the components of the popular paleolithic diet including coconut oil, oil seeds and sugar alcohols which may trigger inflammation."

In other words, a fibre-free ketogenic diet appears help IBD more than a diet including fibre, even a ketogenic diet including fibre.

Butyrate prevents colon cancer.

The idea that butyrate might be protective of colon cancer seems to have started in the 1980s (see, e.g., [Sen2006].

This area of research is extensive, and I am by no means an expert. If you haven't guessed, that butyrate has a protective effect on colon cancer is the one statement I think is entirely defensible.

It's not known exactly how butyrate exerts its protective effects, but some mechanisms held to be important are also induced by β-hydroxybutyrate. For example butyrate's histone deacelytase (HDAC) inhibition is considered an important mechanism [Hin2002], [Blo2011]. β-Hydroxybutyrate is also an HDAC inhibitor [Shi2013].

Gpr109a receptor activation is a recently identified mechanism [Sin2014]. Gpr109a has many aliases, including hydroxycarboxylic acid receptor 2 (HCA2) or niacin receptor 1 (NIACR1), and HM74a/PUMA-G. Gpr109a is activated by β-hydroxybutyrate [Tag2005], [Rah2014], [Gam2012]. It is sometimes simply called the β-hydroxybutyrate receptor.

In fact, the argument behind the relevance of the Gpr109a discovery is just as strong an argument for a ketogenic diet as for eating fibre! That is, the researchers demonstrated that butyrate could substitute for niacin in activating these receptors, and that just as niacin activation of Gpr109a in fat cells is protective of cardiovascular disease, it may also be in diseases of the colon, and this argues for eating fibre to substitute for pharmalogic doses of niacin. From a press release:

"We think mega-doses of niacin may be useful in the treatment and/or prevention of ulcerative colitis, Crohn's disease, and colorectal cancer as well as familial adenomatous polyposis, or FAP, a genetic condition that causes polyps to develop throughout the gastrointestinal tract"


"Research teams at GlaxoSmithKline and the University of Heidelberg, Germany showed in 2003 that Gpr109a receptors on the surface of fat cells mediate the protective cardiovascular effect of niacin, including increasing good cholesterol, or HDL, while decreasing levels of disease-producing LDL. Their search for other activators identified butyrate, which led Ganapathy to find that not only is the Gpr109a receptor expressed on the surface of colon cells, but that with sufficient fiber intake, butyrate levels in the colon can activate it."

Interestingly, as in the case of colitis, colorectal cancer appears to involve a dysfunction in ability to use butyrate. Specifically, there are detrimental changes in membrane transport that reduce its entry into the cell [Gon2016]. Therefore, it's unclear that once the disease process has begun, increased fibre intake will be of any use. Beta-hydroxybutyrate in the bloodstream, however, might.

There is at least some preliminary evidence that butyrate in the bloodstream has similar effects on intestinal tissue as butyrate coming from the colon itself [Kor1990], [Rol1997], [Bar2004], as does infusion of glutamine and acetoacetate, another ketone body [Rom1990]. Ketogenic diets do increase blood acetoacetate. If bloodstream infusion of butyrate is as effective as absorption of butyrate in the intestines in protecting colon cells from degradation, then it seems reasonable to hypothesise that β-hydroxybutyrate in the bloodstream would also have this effect.

These common mechanisms suggest that much or even all of the benefits obtainable by butyrate are equally achievable simply through ketogenic diets, making additional butyrate in the context of a ketogenic diet potentially superfluous.

Fibre is the only way to get butyrate.

Even though it seems likely that a fibre-free ketogenic diet is not only sufficient for colon health, but better for treating colon disease, we might feel cautious about going without the butyrate from fibre, given the dire pronouncements from nutritional scientists. Is there any other way to get butyrate? The most significant food source, butter, doesn't give much. Only about 3-4% of butter is butyric acid. According to [Sen2006] we produce >200mmol per day. That would take about a pound of butter!

Stepping back, it should be obvious that carnivores such as felines and canines provide an important source of data relevant to this question. Carnivores have colons, and they are not normally in ketosis unless food is scarce. Either their colons don't need butyrate, or they are getting sufficient butyrate from some other source. As it happens, there are microbes that ferment amino acids in to short chain fatty acids (SCFAs), including butyrate. Carnivores are known to get "animal fibre" from their prey. That is, amino acids from incompletely digested animal parts reach their colons and are fermented. In particular, in cheetahs, casein, collagen, and glucosamine have been shown to result in butyrate production comparable to fructo-oligosaccharides [Dep2012].

Beyond poorly digested animal sourced fibre, many amino acids are fermented into SCFAs, including butyrate [Ras1988], and these amino acids are abundant in human intestines and colons and are fermented there [Vit2014], [Dai2015], [Nei2015], [Wie2017]. I was unable to determine how much butyrate this would account for.

I did find research comparing the SCFA levels produced in dogs under conditions of high fibre vs. meat alone showing that they produced almost as much VFA (another word for SCFA) in their colons eating meat alone [Ban1979].

In any case, we certainly do generate butyrate in the absence of dietary fibre.

In sum

Although many in the medical community consider butyrate an essential fuel for colon cells, there may be a parallel to glucose and brain cells, in that some or all of this functionality could be replaceable with β-hydroxybutyrate. This idea is supported by these observations:

  • Carnivores and even germ-free mice have intact, working colons without contributions from fibre-derived butyrate, so it stands to reason that humans may not need it either.
  • Although not discussed in this post, some recent societies thrived on animal-based diets with little and infrequent plant intake.
  • β-hydroxybutyrate triggers many of the same mechanisms that butyrate does; those very mechanisms thought to explain its role in preventing colon cancer and the intestinal degradation seen in diseased colons or the colons of those receiving reduced fibre diets to promote bowel rest.
  • β-hydroxybutyrate may even be the pathway through which butyrate exerts its beneficial effects, given that it is a direct metabolite of butyrate, and that systemic butyrate appears to be as effective or even more effective in treating colitis, than direct application of butyrate to the cells.
  • Even without eating fibre, our intestinal microbes produce butyrate from amino acids. If systemic ketone bodies supplant or even just reduce the need for butyrate, amino acid derived butyrate may supply this need, even if the quantities turn out to be less than we would get from fibre.

End-to-end citations


Evidence type: non-human animal experiment

Ahmad MS, Krishnan S, Ramakrishna BS, Mathan M, Pulimood AB, Murthy SN.
Gut. 2000 Apr;46(4):493-9.



"Impaired colonocyte metabolism of butyrate has been implicated in the aetiopathogenesis of ulcerative colitis. Colonocyte butyrate metabolism was investigated in experimental colitis in mice.


"Colitis was induced in Swiss outbred white mice by oral administration of 4% dextran sulphate sodium (DSS). Colonocytes isolated from colitic and normal control mice were incubated with [(14)C]butyrate or glucose, and production of (14)CO(2), as well as of intermediate metabolites (acetoacetate, beta-hydroxybutyrate and lactate), was measured. The effect of different substrate concentrations on oxidation was also examined.


"Butyrate oxidation (micromol/h per mg protein; mean (SEM)) was significantly reduced in DSS colitis, values on day 7 of DSS administration being 0.177 (0.007) compared with 0.406 (0.035) for control animals (p<0.001). Glucose oxidation (micromol/h per mg protein; mean (SEM)) on day 7 of DSS administration was significantly higher than in controls (0.06 (0.006) v 0.027 (0.004), p<0.001). Production of beta-hydroxybutyrate was decreased and production of lactate increased in DSS colitis compared with controls. Increasing butyrate concentration from 10 to 80 mM enhanced oxidation in DSS colitis (0.036 (0.002) to 0.285 (0.040), p<0.001), although it continued to remain lower than in controls. Surface and crypt epithelial cells showed similar ratios of butyrate to glucose oxidation. When 1 mM DSS was added to normal colonocytes in vitro, it did not alter butyrate oxidation. The initial histological lesion of DSS administration was very patchy and involved crypt cells. Abnormal butyrate oxidation became apparent only after six days of DSS administration, at which time histological abnormalities were more widespread.


"Colonocyte metabolism of butyrate, but not of glucose, is impaired in DSS colitis, and may be important in pathophysiology. Histological abnormalities preceded measurable defects in butyrate oxidation."


Evidence type: non-human animal experiment

Banta, C. A., Clemens, E. T., Krinsky, M. M., and SheiIy, B. E., 1979,
J. Nutr. 109:1592-1600.

"Two commercial type diest, one a cereal based dry food, the other a fortified all meat canned food were fed to male and female adult beagle dogs to evaluate effects of diet on rate of digesta passage and organic acid concentration along the gastrointestinal tract. [...] Concentrations of VFA were highest in the cecum and colon and were not significantly affected by diet."


"Symbols on the abscissa denote sec tions of tract as follows : cranial stomach ( Si ) ; caudal stomach ( 82) ; proximal ( Sii ), middle (SI2) and distal (SL) thirds of the small intestine; cecum (Ce); and proximal (Ci) and distal (C«) halves of the colon ( n = 3 )."


"It was surprising to see high concentrations of VFA produced in the lower gut of dogs fed the meat diet. It was logical to assume that liver and muscle glycogen could serve as the fermentable substrate for lactate production in the stomach, but most of this should have been digested and absorbed by the small intes tine. Another possible source of ferment able substance which could survive passage through the small intestine is the protein- polysaccharides of the connective tissue ground substance found in abundance in the meat by-products and whole ground chicken. The ground substance is made up of chondroitin sulfates and hyaluronic acid. The polysaccharide portion of these substances is composed of long chains of disaccharide units consisting of glucosa- mine or galactosamine and glucuronic acid. The linkages of these polysaccharides are not such that they can be cleaved by the endogenous digestive enzymes found in the gut but they could be split by microbial enzymes."


Evidence type: non-human animal experiment

Bartholome AL1, Albin DM, Baker DH, Holst JJ, Tappenden KA.
JPEN J Parenter Enteral Nutr. 2004 Jul-Aug;28(4):210-22; discussion 222-3.


"Supplementation of total parenteral nutrition (TPN) with a mixture of short-chain fatty acids (SCFA) enhances intestinal adaptation in the adult rodent model. However, the ability and timing of SCFA to augment adaptation in the neonatal intestine is unknown. Furthermore, the specific SCFA inducing the intestinotrophic effects and underlying regulatory mechanism(s) are unclear. Therefore, we examined the effect of SCFA supplemented TPN on structural aspects of intestinal adaptation and hypothesized that butyrate is the SCFA responsible for these effects.


"Piglets (n = 120) were randomized to (1) control TPN or TPN supplemented with (2) 60 mmol/L SCFA (36 mmol/L acetate, 15 mmol/L propionate and 9 mmol/L butyrate), (3) 9 mmol/L butyrate, or (4) 60 mmol/L butyrate. Within each group, piglets were further randomized to examine acute (4, 12, or 24 hours) and chronic (3 or 7 days) adaptations. Indices of intestinal adaptation, including crypt-villus architecture, proliferation and apoptosis, and concentration of the intestinotrophic peptide, glucagon-like pepide-2 (GLP-2), were measured.


"Villus height was increased (p < .029) within 4 hours by supplemented TPN treatments. Supplemented TPN treatments increased (p < .037) proliferating cell nuclear antigen expression along the entire intestine. Indicative of an antiapoptotic profile, jejunal Bax:Bcl-w abundance was decreased (p = .033) by both butyrate-supplemented TPN treatments, and ileal abundance was decreased (p = .0002) by all supplemented TPN treatments, regardless of time. Supplemented TPN treatments increased (p = .016) plasma GLP-2 concentration at all time points.


"Butyrate is the SCFA responsible for augmenting structural aspects of intestinal adaptations by increasing proliferation and decreasing apoptosis within 4 hours postresection. The intestinotrophic mechanism(s) underlying butyrate's effects may involve GLP-2. Ultimately, butyrate administration may enable an infant with short-bowel syndrome to successfully transition to enteral feedings by maximizing their absorptive area."


Evidence type: review

Brown AC, Rampertab SD, Mullin GE.
Expert Rev Gastroenterol Hepatol. 2011 Jun;5(3):411-25. doi: 10.1586/egh.11.29.

"In terms of existing guidelines for dietary modifications, three suggested limiting dairy if lactose intolerant, two suggested limiting excess fat, one indicated decreasing excess carbohydrates, and five suggested avoiding high-fiber foods, especially during flares. The question of whether or not to use probiotics continues to be debated."


"Reducing high-fiber foods during symptoms appears to have generated the most support in the dietary guidelines. It may be important to communicate to IBD patients that high-fiber foods are not recommended, especially for those with CD, during flares or in the presence of active disease states, fistulas or strictures. There appears to be a tendency among the dietary guidelines to restrict foods such as raw fruits, raw vegetables, beans, bran, popcorn, seeds, nuts, corn hulls, whole grains, brown rice and wild rice. Although not mentioned, raw salads would also fall into this category."


"Some patients with IBD react to excess dietary fat and perhaps this is where the recommendation is derived. Few research studies are available to support or refute such a recommendation. The topic needs further investigation because patients with malabsorption may be at risk of not obtaining their necessary essential fatty acids. Perhaps saturated fats should be limited, with more of an emphasis on more healthy fat intakes."


Evidence type: review

Zhaolai Dai Zhenlong Wu Suqin Hang Weiyun Zhu Guoyao Wu
MHR: Basic science of reproductive medicine, Volume 21, Issue 5, 1 May 2015, Pages 389–409

"Recent studies with the human colonic bacteria have shown that protein- and AA-fermenting bacteria are abundant and diverse in the colon. The abundance of the AA-fermenting bacteria in the large intestine is very high and their number can reach up to 1011 per gram dry feces (Smith and Macfarlane, 1998). Using the traditional plate counting technique, the authors have also reported that the dominant bacterial species for the utilization of single AA or pairs of AA are very different. For instance, Clostridium bifermentans is the predominant bacteria for the utilization of lysine or proline, and pairs of AA (e.g. phenylalanine/leucine, isoleucine/tryptophan and alanine/glycine), whereas Peptostreptococcus spp. bacteria are predominant for the utilization of glutamate or tryptophan. Many species of bacteria utilize the same AA as substrates for growth (Smith and Macfarlane, 1998). Overall, bacteria belonging to the Clostridium spp. dominate in AA fermentation in the human large intestine, but other bacterial species, such as Fusobacterium spp., Bacteroides spp., Veillonella spp., Megasphaera elsdenii and Selenomonas ruminantium, may also be important for AA metabolism in the large intestine (Smith and Macfarlane, 1998; Dai et al., 2011)."


Evidence type: non-human animal experiment

Depauw, S., G. Bosch, M. Hesta, K. Whitehouse-Tedd, W. H. Hendriks, J. Kaandorp, and G. P. J. Janssens. 2012.
J. Anim. Sci. 90:2540-2548. doi:10.2527/jas.2011-4377

"End-product profile per unit of OM differed among substrates (Table 3). The greatest total SCFA production was recorded for FOS (P < 0.05), followed by collagen, casein, and glucosamine (P < 0.05). The FOS and collagen showed comparable acetate production. Collagen not only had a high production of total SCFA but also resulted in a greater acetate to propionate ratio relative to all other substrates (8.41:1 for collagen and 1.67:1–2.97:1 for other substrates). Chicken cartilage and glucosamine-chondroitin produced similar total SCFA production, which was moderate compared with FOS (P < 0.05). Total SCFA production from incubated rabbit bone and skin was low (P < 0.05), whereas total SCFA production from rabbit hair was negligible and comparable with the negative control cellulose. Butyrate production was greatest for casein and glucosamine (P < 0.05). Incubation with casein resulted in the greatest total BCFA production (P < 0.05), which was more than double compared with all other substrates that had similar total BCFA production. Considerable variation in BCFA ratios was observed among substrates. In all animal substrates, isovalerate was the main BCFA, whereas fermentation of FOS, glucosamine, and glucosamine-chondroitin led to valerate as the main BCFA. The greatest amount of ammonia production was observed for casein, collagen, and rabbit bone (P < 0.05), whereas the least ammonia production was detected for FOS, cellulose, and rabbit hair (P < 0.05)."


Evidence type: non-human animal and human cell in vitro experiments

Gambhir D, Ananth S, Veeranan-Karmegam R, et al.
Investigative Ophthalmology & Visual Science. 2012;53(4):2208-2217. doi:10.1167/iovs.11-8447.

"GPR109A is the G-protein–coupled receptor responsible for mediating the antilipolytic actions of niacin (nicotinic acid), a B-complex vitamin and also a drug used widely to lower blood lipid levels.1 β-hydroxybutyrate (β-HB) is the physiologic ligand for this receptor.2 GPR109A expression was initially thought to be limited to adipocytes, the cell type in which its antilipolytic functions are most warranted, and immune cells.3–5 Recent reports, however, have described expression of the receptor in a number of other cell types, including hepatocytes6 and epithelial cells of the small intestine and colon.7,8 In addition, we demonstrated GPR109A expression in the retinal pigment epithelium (RPE), localized specifically to the basolateral membrane.9 Although GPR109A is most noted functionally for its antilipolytic effects in adipocytes, recent studies suggest that activation of the receptor also is associated with novel immunomodulatory responses.10–12 We have characterized expression of GPR109A in RPE; however, the functional significance of receptor expression in this cell type remains unknown."


Evidence type: review

Pedro Gonçalves and Fátima Martel
Porto Biomedical Journal Volume 1, Issue 3, July–August 2016, Pages 83-91

"The most important molecular mechanisms involved in the anticarcinogenic effect of BT are dependent on its intracellular concentration (because HDAC expression is overregulated,41,42 while BT membrane receptors (GPR109A and GPR43) are silenced or downregulated in CRC34,38). So, knowledge on the mechanisms involved in its membrane transport is relevant to both its physiological and pharmacological benefits. Also, changes in transporter expression or function will have an obvious impact on the effect of BT, and therefore, knowledge on the regulation of its membrane transport seems particularly important.


"[D]ifferences in MCT1, SMCT1 and BCRP expression between normal colonocytes and tumoral cells contribute to the different effects of BT in these cells (‘the BT paradox’). More specifically, BT is transported into normal colonic epithelial cells by both MCT1 and SMCT1, but its intracellular concentration is kept low because it is efficiently metabolized and effluxed from these cells by BCRP-mediated transport. In contrast, colonic epithelial tumoral cells show a decrease in SMCT1 protein expression, and BT is taken up by these cells through MCT1. In these cells, BT accumulates intracellularly because it is inefficiently metabolized (due to the fact that glucose becomes the primary energy source of these cells) and because there is a reduction in BCRP expression."


Evidence type: human cell in vitro experiment

Brian F. Hinnebusch, Shufen Meng, James T. Wu, Sonia Y. Archer, and Richard A. Hodin
J. Nutr. May 1, 2002 vol. 132 no. 5 1012-1017

"The short-chain fatty acid (SCFA) butyrate is produced via anaerobic bacterial fermentation within the colon and is thought to be protective in regard to colon carcinogenesis. Although butyrate (C4) is considered the most potent of the SCFA, a variety of other SCFA also exist in the colonic lumen. Butyrate is thought to exert its cellular effects through the induction of histone hyperacetylation. We sought to determine the effects of a variety of the SCFA on colon carcinoma cell growth, differentiation and apoptosis. HT-29 or HCT-116 (wild-type and p21-deleted) cells were treated with physiologically relevant concentrations of various SCFA, and histone acetylation state was assayed by acid-urea-triton-X gel electrophoresis and immunoblotting. Growth and apoptotic effects were studied by flow cytometry, and differentiation effects were assessed using transient transfections and Northern blotting. Propionate (C3) and valerate (C5) caused growth arrest and differentiation in human colon carcinoma cells. The magnitude of their effects was associated with a lesser degree of histone hyperacetylation compared with butyrate. Acetate (C2) and caproate (C6), in contrast, did not cause histone hyperacetylation and also had no appreciable effects on cell growth or differentiation. SCFA-induced transactivation of the differentiation marker gene, intestinal alkaline phosphatase (IAP), was blocked by histone deacetylase (HDAC), further supporting the critical link between SCFA and histones. Butyrate also significantly increased apoptosis, whereas the other SCFA studied did not. The growth arrest induced by the SCFA was characterized by an increase in the expression of the p21 cell-cycle inhibitor and down-regulation of cyclin B1 (CB1). In p21-deleted HCT-116 colon cancer cells, the SCFA did not alter the rate of proliferation. These data suggest that the antiproliferative, apoptotic and differentiating properties of the various SCFA are linked to the degree of induced histone hyperacetylation. Furthermore, SCFA-mediated growth arrest in colon carcinoma cells requires the p21 gene."


Evidence type: in vitro experiments

Blouin, J.-M., Penot, G., Collinet, M., Nacfer, M., Forest, C., Laurent-Puig, P., Coumoul, X., Barouki, R., Benelli, C. and Bortoli, S. (2011)
Int. J. Cancer, 128: 2591–2601. doi:10.1002/ijc.25599

"Butyrate, a short-chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO2, they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate-stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC."


Evidence type: armchair


"Butyric acid has two contrasting functional roles. As a product of fermentation within the human colon, it serves as the most important energy source for normal colorectal epithelium. It also promotes the differentiation of cultured malignant cells. A switch from aerobic to anaerobic metabolism accompanies neoplastic transformation in the colorectum. The separate functional roles for n-butyrate may reflect the different metabolic activities of normal and neoplastic tissues. Relatively low intracolonic levels of n-butyrate are associated with a low fibre diet. Deficiency of n-butyrate, coupled to the increased energy requirements of neoplastic tissues, may promote the switch to anaerobic metabolism. The presence of naturally occurring differentiating agents, such as n-butyrate, may modify the patterns of growth and differentiation of gastrointestinal tumours."


Evidence type: review

Alimentary Pharmacology & Therapeutics, 27: 104–119. doi:10.1111/j.1365-2036.2007.03562.x

"Although some controlled studies with enemas containing butyrate or SCFA mixtures in UC patients did not find beneficial effects121 or only trends towards clinical improvement,46, 118, 119 various other studies revealed a significant improvement in clinical and inflammatory parameters.45, 115, 120, 124, 126 Studies in patients with diversion colitis reported inconsistent results with regard to improvement in clinical symptoms and inflammatory parameters in response to administration of mixtures of SCFAs vs. placebo.96, 114 Two other human intervention studies determined mucosal cell proliferation in patients after Hartmann’s procedure and found trophic effects of SCFA mixtures in the mucosa of the closed rectal and sigmoid segment.73, 116"

"The effects of butyrate containing enemas on radiation proctitis113, 117, 122, 125 and pouchitis123 have been studied in small groups and besides one report125 that showed that butyrate was an effective treatment of radiation proctitis, other studies did not report clear-cut beneficial effects of SCFA irrigation in these two patient groups."


Evidence type: human experiment

Hamer HM, Jonkers DM, Vanhoutvin SA, Troost FJ, Rijkers G, de Bruïne A, Bast A, Venema K, Brummer RJ.
Clin Nutr. 2010 Dec;29(6):738-44. doi: 10.1016/j.clnu.2010.04.002. Epub 2010 May 15.



"Butyrate, produced by colonic fermentation of dietary fibers is often hypothesized to beneficially affect colonic health. This study aims to assess the effects of butyrate on inflammation and oxidative stress in subjects with chronically mildly elevated parameters of inflammation and oxidative stress.


"Thirty-five patients with ulcerative colitis in clinical remission daily administered 60 ml rectal enemas containing 100mM sodium butyrate (n=17) or saline (n=18) during 20 days (NCT00696098). Before and after the intervention feces, blood and colonic mucosal biopsies were obtained. Parameters of antioxidant defense and oxidative damage, myeloperoxidase, several cytokines, fecal calprotectin and CRP were determined.


"Butyrate enemas induced minor effects on colonic inflammation and oxidative stress. Only a significant increase of the colonic IL-10/IL-12 ratio was found within butyrate-treated patients (p=0.02), and colonic concentrations of CCL5 were increased after butyrate compared to placebo treatment (p=0.03). Although in general butyrate did not affect colonic glutathione levels, the effects of butyrate enemas on total colonic glutathione appeared to be dependent on the level of inflammation.


"Although UC patients in remission were characterized by low-grade oxidative stress and inflammation, rectal butyrate enemas showed only minor effects on inflammatory and oxidative stress parameters."


Evidence type: review

Gilaad G. Kaplan, MD, MPH, FRCPC
Clinical Gastroenterology and Hepatology , Volume 14 , Issue 8 , 1137 - 1139

"After reviewing the study from Brotherton et al and prior literature, information for patients with IBD on the effects of fiber on the risk of flaring is unclear. The current article adds to this discussion but does not definitively answer the question. Overall, the data suggest that in the absence of a known fibrostenotic stricture with obstructive symptoms, a high fiber diet is likely safe in patients with IBD and may impart a weak benefit. Yet, answering these clinically relevant questions with more confidence and detail is within our grasp. The advent of e-cohorts offers the potential to transform research in the future by allowing investigators to design cost-efficient Web-based clinical studies, particularly for interventional environmental clinical trials."


Evidence type: non-human animal experiment

Koruda MJ1, Rolandelli RH, Bliss DZ, Hastings J, Rombeau JL, Settle RG.
Am J Clin Nutr. 1990 Apr;51(4):685-9.


"When enteral nutrition is excluded from animals maintained solely with total parenteral nutrition (TPN), atrophy of the intestinal mucosa is observed. Short-chain fatty acids (SCFAs) are produced in the colon by the fermentation of dietary carbohydrates and fiber polysaccharides and have been shown to stimulate mucosal-cell mitotic activity in the intestine. This study compared the effects of an intravenous and an intracecal infusion of SCFAs on the small-bowel mucosa. Rats received standard TPN, TPN with SCFAs (sodium acetate, propionate, and butyrate), TPN with an intracecal infusion of SCFAs, or rat food. After 7 d jejunal and ileal mucosal weights, DNA, RNA, and protein were determined. Standard TPN produced significant atrophy of the jejunal and ileal mucosa. Both the intracecal and intravenous infusion of SCFAs significantly reduced the mucosal atrophy associated with TPN. The intravenous and intracolonic infusion of SCFAs were equally effective in inhibiting small-bowel mucosal atrophy."


Evidence type: non-human animal experiment

Joshua J. Malago and Catherine L. Sangu
Zhejiang Univ Sci B. 2015 Mar; 16(3): 224–234. doi: 10.1631/jzus.B1400191

"Earlier studies that linked the development of UC and butyrate levels in the colon, observed that deficiency of butyrate leads to disease development and that restoration of butyrate levels by intracolonic infusion treats UC (Roediger, 1980). Since then, butyrate enemas have popularly been used as medicaments stemming from their potential to impart beneficial attributes to the colon. This potential involves an increase in mechanical strength of injured colonic mucosa to hasten the healing process (Bloemen et al., 2010; Mathew et al., 2010), suppression of IL-8 production by intestinal epithelial cells to protect against the inflammatory process (Malago et al., 2005), and clinical remission of UC by protecting against inflammatory and oxidative stress parameters of the disease (Hamer et al., 2010b). Much as butyrate tends to impart a protective effect, several authors have indicated failures or limited success of butyrate to relieve IBD patients (Harig et al., 1989; Sanderson, 1997; Hamer et al., 2010b)."


"Topical administration of butyrate to cure colitis has been fairly well demonstrated (Scheppach et al., 1992; Hamer et al., 2010a; 2010b). This is done mainly through intrarectal administration of enemas that contain butyrate. The procedure is one of the earliest approaches to treat UC even in patients who had been unresponsive to or intolerant of standard therapy (Scheppach et al., 1992). The intrarectally administered butyrate needs to be absorbed before it works. Normally butyrate absorption mainly occurs in proximal colon whose function is impaired during UC. This hinders absorption of topically administered butyrate and may not benefit UC patients. However, butyrate absorption in the colon can be increased by manipulating electrolyte composition in the rectal lumen (Holtug et al., 1995) since rectal butyrate absorption remains normal during UC (Hove et al., 1995). Thus, topical butyrate, given intrarectally in form of SB, plays a double role; firstly by employing sodium ions, it accelerates rectal absorption of SB and secondly, the absorbed butyrate imparts healing to the colonocytes. The end result is epithelial proliferation to restore the damaged epithelium, especially the lost colonic epithelial continuity."


"We have demonstrated the potential of intraperitoneally administered butyrate to prevent the severity of AA-induced UC lesions. To the best of our knowledge, this finding has not been reported before. However, the systemic effect of butyrate to other body systems and organs has been reported. For instance, intraperitoneal injection of butyrate at 50–200 mg/kg body weight decreases gentamicin-induced nephrotoxicity in rats by enhancing renal antioxidant enzyme activity and expression of prohibitin protein (Sun et al., 2013). When given at 1200 mg/kg, intraperitoneal butyrate ameliorates an aging-associated deficit in object recognition memory in rats (Reolon et al., 2011). Silingardi et al. (2010) further demonstrated that chronic intraperitoneal administration of butyrate to long-term monocularly deprived adult rats causes a complete recovery of visual acuity. A more recent study has also reported that intraperitoneal injections of butyrate for 28 d to adult C57BL/6 mice prevent repressed contextual fear memory caused by isoflurane (Zhong et al., 2014). All these facts and our own study affirm that butyrate has a potential to impart protective roles to various body organs and systems through systemic administration."


Evidence type: non-human animal experiment

Miles JP, Zou J, Kumar MV, Pellizzon M, Ulman E, Ricci M, Gewirtz AT, Chassaing B.



"Lack of dietary fiber has been suggested to increase the risk of developing various chronic inflammatory diseases, whereas supplementation of diets with fiber might offer an array of health-promoting benefits. Consistent with this theme, we recently reported that in mice, compositionally defined diets that are made with purified ingredients and lack fermentable fiber promote low-grade inflammation and metabolic syndrome, both of which could be ameliorated by supplementation of such diets with the fermentable fiber inulin.


"Herein, we examined if, relative to a grain-based mouse diet (chow), compositionally defined diet consumption would impact development of intestinal inflammation induced by dextran sulfate sodium (DSS) and moreover, whether DSS-induced colitis might also be attenuated by diets supplemented with inulin.


"Analogous to their promotion of low-grade inflammation, compositionally defined diet of high- and low-fat content with cellulose increased the severity of DSS-induced colitis relative to chow. However, in contrast to the case of low-grade inflammation, addition of inulin, but not the insoluble fiber cellulose, further exacerbated the severity of colitis and its associated clinical manifestations (weight loss and bleeding) in both low- and high-fat diets.


"While inulin, and perhaps other fermentable fibers, can ameliorate low-grade inflammation and associated metabolic disease, it also has the potential to exacerbate disease severity in response to inducers of acute colitis."


Evidence type: review

Mizushima N, Komatsu M.
Cell. 2011 Nov 11;147(4):728-41. doi: 10.1016/j.cell.2011.10.026.

"Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the lysosome. However, the purpose of autophagy is not the simple elimination of materials, but instead, autophagy serves as a dynamic recycling system that produces new building blocks and energy for cellular renovation and homeostasis. Here we provide a multidisciplinary review of our current understanding of autophagy's role in metabolic adaptation, intracellular quality control, and renovation during development and differentiation. We also explore how recent mouse models in combination with advances in human genetics are providing key insights into how the impairment or activation of autophagy contributes to pathogenesis of diverse diseases, from neurodegenerative diseases such as Parkinson disease to inflammatory disorders such as Crohn disease."


Evidence type: review

Neis EPJG, Dejong CHC, Rensen SS.
Nutrients. 2015;7(4):2930-2946. doi:10.3390/nu7042930.

"Although protein breakdown followed by amino acid absorption in the small intestine is a rather efficient process, substantial amounts of amino acids seem to escape assimilation in the small intestine in humans [38]. These amino acids can subsequently be used by the microbiota in the colon, or transported from the lumen into the portal blood stream. In addition, the host itself produces substrates such as glycoproteins (e.g., mucins) which contribute to the available amino acids within the colon [39]. "


"Regarding the large intestine, it appears that amino acids are not significantly absorbed by the colonic mucosa, but rather are intensively metabolized by the large intestinal microbiota [23]. This higher rate of bacterial protein fermentation has been related to high pH and low carbohydrate availability in the large intestine [22]. The preferred amino acid substrates of colonic bacteria include lysine, arginine, glycine, and the BCAA leucine, valine, and isoleucine [32], resulting in the generation of a complex mixture of metabolic end products including among others ammonia, SCFA (acetate, propionate, and butyrate), and branched-chain fatty acids (BCFA; valerate, isobutyrate, and isovalerate). "


Evidence type: non-human animal experiment

Luying Peng, Zhenjuan He, Wei Chen, Ian R Holzman and Jing Lin
Pediatric Research (2007) 61, 37–41; doi:10.1203/01.pdr.0000250014.92242.f3

"In premature infants, the maturation of the intestinal barrier function does not develop properly in the absence of enteral nutrients (6). Intestinal barrier function is significantly less developed in full-term newborn piglets receiving total parental nutrition compared with those receiving enteral nutrition (7). Production of SCFA in the bowel may be crucial for gastrointestinal adaptation and maturation in the early stage of postnatal life (8). However, overproduction and/or accumulation of SCFA in the bowel due to maldigestion and bacterial overgrowth may be toxic to mucosal cells and cause intestinal mucosal injury (9,10). Overproduction and/or accumulation of SCFA in the bowel and inability to clear the intraluminal SCFA because of poor gastrointestinal motility in premature infants have been hypothesized to play a role in the pathogenesis of neonatal NEC (11)."


Evidence type: non-human animal experiment

Rahman M, Muhammad S, Khan MA, Chen H, Ridder DA, Müller-Fielitz H, Pokorná B, Vollbrandt T, Stölting I, Nadrowitz R, Okun JG, Offermanns S, Schwaninger M.
Nat Commun. 2014 May 21;5:3944. doi: 10.1038/ncomms4944.


"The ketone body β-hydroxybutyrate (BHB) is an endogenous factor protecting against stroke and neurodegenerative diseases, but its mode of action is unclear. Here we show in a stroke model that the hydroxy-carboxylic acid receptor 2 (HCA2, GPR109A) is required for the neuroprotective effect of BHB and a ketogenic diet, as this effect is lost in Hca2(-/-) mice. We further demonstrate that nicotinic acid, a clinically used HCA2 agonist, reduces infarct size via a HCA2-mediated mechanism, and that noninflammatory Ly-6C(Lo) monocytes and/or macrophages infiltrating the ischemic brain also express HCA2. Using cell ablation and chimeric mice, we demonstrate that HCA2 on monocytes and/or macrophages is required for the protective effect of nicotinic acid. The activation of HCA2 induces a neuroprotective phenotype of monocytes and/or macrophages that depends on PGD2 production by COX1 and the haematopoietic PGD2 synthase. Our data suggest that HCA2 activation by dietary or pharmacological means instructs Ly-6C(Lo) monocytes and/or macrophages to deliver a neuroprotective signal to the brain."


Evidence type: in vitro experiment

Rasmussen HS, Holtug K, Mortensen PB.
Scand J Gastroenterol. 1988 Mar;23(2):178-82.

"Short-chain fatty acids (SCFA) originate mainly in the colon through bacterial fermentation of polysaccharides. To test the hypothesis that SCFA may originate from polypeptides as well, the production of these acids from albumin and specific amino acids was examined in a faecal incubation system. Albumin was converted to all C2-C5-fatty acids, whereas amino acids generally were converted to specific SCFA, most often through the combination of a deamination and decarboxylation of the amino acids, although more complex processes also took place. This study indicates that a part of the intestinal SCFA may originate from polypeptides, which apparently are the major source of those SCFA (isobutyrate, valerate, and isovalerate) only found in small amounts in the healthy colon. Moreover, gastrointestinal disease resulting in increased proteinous material in the colon (exudation, mucosal desquamation, bleeding, and so forth) may hypothetically influence SCFA production."

[Roe1980](1, 2)

Evidence type: human experiment

"The view that UC might be due to a metabolic defect in the epithelial cells[5,6] has received little general recognition. The present study was undertaken to assess the metabolic performance of the mucosa in UC and especially to explore whether a metabolic abnormality could be detected. To facilitate this approach a method of preparing suspensions of colonocytes was devised.[7] Colonocytes have been used to determine the utilisation of respiratory fuels by the non-diseased ascending and descending colon in man.[8] The results showed that short-chain fatty acid (SCFA), especially n-butyrate of bacterial origin, was the predominant contributor to cellular oxidation and that a large proportion of the carbon atoms of colonocyte respiration was derived from SCFAs. Mucosa of the distal colon depended metabolically mostly on n-butyrate, whereas the proximal colonic mucosa depended mostly on glucose and glutamine for respiratory fuel.[8] These same respiratory fuels were chosen for the investigation of colonocytes prepared from the mucosa of patients with ulcerative colitis.


"Generation of 14C02 from radioactively labelled butyrate was observed for at least 40 min. Production of 14C02 was linear whenever this could be tested for 60 min. Generation of 14C02 was significantly less in quiescent and acute-colitis cells than in controls (p = <0.001) (table II). Some of the oxidised butyrate appeared as ketone bodies (acetoacetate and β-hydroxybutyrate, table III). The diminished production of ketone bodies mirrors the decreased oxidation of butyrate to CO2. Ketogenesis was significantly lower in the quiescent-colitis group than the control group and lower still in the acute-colitis group.


"The metabolism of colonocytes from patients with UC seemed to differ in three respects from the metabolism of colonocytes prepared from non-ulcerated and apparently normal mucosa. In UC: 1. Butyrate oxidation to C02 and ketone bodies was significantly impaired, and the impairment correlated with the acute or chronic involvement of the mucosa. 2. Glucose oxidation was increased. 3. Glutamine oxidation was increased."


"Ketogenesis was significantly lower in the quiescent-colitis group than the control group and lower still in the acute-colitis group."


Evidence type: non-human animal experiment

Roediger WE, Duncan A, Kapaniris O, Millard S.
Clin Sci (Lond). 1993 Nov;85(5):623-7.


"Isolated colonic epithelial cells of the rat were incubated for 40 min with [6-14C]glucose and n-[1-14C]butyrate in the presence of 0.1-2.0 mmol/l NaHS, a concentration range found in the human colon. Metabolic products, 14CO2, acetoacetate, beta-hydroxybutyrate and lactate, were measured and injury to cells was judged by diminished production of metabolites. 2. Oxidation of n-butyrate to CO2 and acetoacetate was reduced at 0.1 and 0.5 mmol/l NaHS, whereas glucose oxidation remained unimpaired. At 1.0-2.0 mmol/l NaHS, n-butyrate and glucose oxidation were dose-dependently reduced at the same rate. 3. To bypass short-chain acyl-CoA dehydrogenase activity necessary for butyrate oxidation, ketogenesis from crotonate was measured in the presence of 1.0 mmol/l NaHS. Suppression by sulphide of ketogenesis from crotonate (-10.5 +/- 6.1%) compared with control conditions was not significant, whereas suppression of ketogenesis from n-butyrate (-36.00 +/- 5.14%) was significant (P = < 0.01). Inhibition of FAD-linked oxidation was more affected by NaHS than was NAD-linked oxidation. 4. L-Methionine (5.0 mmol/l) significantly redressed the impaired beta-oxidation induced by NaHS. Methionine equally improved CO2 and ketone body production, suggesting a global reversal of the action of sulphide. 5. Sulphide-induced oxidative changes closely mirror the impairment of beta-oxidation observed in colonocytes of patients with ulcerative colitis. A hypothesis for the disease process of ulcerative colitis is that sulphides may form persulphides with butyryl-CoA, which would inhibit cellular short-chain acyl-CoA deHydrogenase and beta-oxidation to induce an energy-deficiency state in colonocytes and mucosal inflammation."


Evidence type: non-human animal experiment

Rolandelli RH, Buckmire MA, Bernstein KA.
Dis Colon Rectum. 1997 Jan;40(1):67-70.


"Intracolonic infusions of short chain fatty acids promote healing of colonic anastomoses. Because the intravenous route may have wider clinical application, we studied the effect of intravenous n-butyrate on the mechanical strength of colonic anastomoses in the rat.


"After placement of an indwelling intravenous catheter, the descending colon was transected and an anastomosis was performed. Rats were then randomized to receive total parenteral nutrition (TPN group; n = 15) or total parenteral nutrition plus 130 mM/l of n-butyrate (TPN+BUT group; n = 13). On the fifth postoperative day, bursting pressure and bowel wall tension of the anastomoses were measured in situ. Anastomotic tissues were analyzed for hydroxyproline.


"The TPN+BUT group had a significantly higher bursting pressure (107.5 +/- 30.3 vs. 83 +/- 41.0 mmHg; P = 0.04) and bowel wall tension (20.7 +/- 7.6 vs. 14.1 +/- 9.9 Newton; P = 0.03). Tissue hydroxyproline was not different between the two groups (TPN, 45.8 +/- 9.2, and TPN+BUT, 47.9 +/- 2.9 microg/mg tissue nitrogen).


"We conclude that intravenous butyrate improves mechanical strength of a colonic anastomosis without a detectable change in total collagen content."


Evidence type: review

Rombeau J.L., Kripke S.A., Settle R.G. (1990)
In: Kritchevsky D., Bonfield C., Anderson J.W. (eds) Dietary Fiber. Springer, Boston, MA

"As mentioned previously hepatic metabolism of butyrate and acetate results in the production of glutamine and the ketone bodies acetoacetate and which are the preferred oxidative fuels of enterocytes (Windmueller and Spaeth, 1978). The enteral or parenteral provision of glutamine and acetoacetate has been shown to be trophic to both small and large intestinal mucosa (Fox et al., 1987; Kripke et al., 1988a)."

[Sen2006](1, 2)

Evidence type: review

Sengupta S, Muir JG, Gibson PR.
J Gastroenterol Hepatol. 2006 Jan;21(1 Pt 2):209-18.


"Butyrate, the four-carbon fatty acid, is formed in the human colon by bacterial fermentation of carbohydrates (including dietary fiber), and putatively suppresses colorectal cancer (CRC). Butyrate has diverse and apparently paradoxical effects on cellular proliferation, apoptosis and differentiation that may be either pro-neoplastic or anti-neoplastic, depending upon factors such as the level of exposure, availability of other metabolic substrate and the intracellular milieu. In humans, the relationship between luminal butyrate exposure and CRC has been examined only indirectly in case-control studies, by measuring fecal butyrate concentrations, although this may not accurately reflect effective butyrate exposure during carcinogenesis. Perhaps not surprisingly, results of these investigations have been mutually contradictory. The direct effect of butyrate on tumorigenesis has been assessed in a number of in vivo animal models, which have also yielded conflicting results. In part, this may be explained by methodological differences in the amount and route of butyrate administration, which are likely to significantly influence delivery of butyrate to the distal colon. Nonetheless, there appears to be some evidence that delivery of an adequate amount of butyrate to the appropriate site protects against early tumorigenic events. Future study of the relationship between butyrate and CRC in humans needs to focus on risk stratification and the development of feasible strategies for butyrate delivery."


Evidence type: non-human animal experiment

Shimazu T, Hirschey MD, Newman J, et al.
Science (New York, NY). 2013;339(6116):211-214. doi:10.1126/science.1227166.

"Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress."


Evidence type: non-human animal experiment

"The most widely studied function of butyrate is its ability to inhibit histone deacetylases. However, cell surface receptors have been identified for butyrate; these receptors, GPR43 and GPR109A (also known as hydroxycarboxylic acid receptor 2 or HCA2), are G protein coupled and are expressed in colonic epithelium, adipose tissue, and immune cells (Blad et al., 2012, Ganapathy et al., 2013). GPR43-deficient mice undergo severe colonic inflammation and colitis in DSS-induced colitis model and the GPR43 agonist acetate protects germ-free mice from DSS-induced colitis (Maslowski et al., 2009). Although GPR43 is activated by all three SCFAs, GPR109A (encoded by Niacr1) is activated only by butyrate (Blad et al., 2012, Taggart et al., 2005). GPR109A is also activated by niacin (vitamin B3) (Blad et al., 2012, Ganapathy et al., 2013). In colonic lumen, butyrate is generated at high concentrations (10–20 mM) by gut microbiota and serves as an endogenous agonist for GPR109A (Thangaraju et al., 2009). We have shown that Gpr109a expression in colon is induced by gut microbiota and is downregulated in colon cancer (Cresci et al., 2010, Thangaraju et al., 2009). Gpr109a in immune cells plays a nonredundant function in niacin-mediated suppression of inflammation and atherosclerosis (Lukasova et al., 2011). Gut microbiota also produce niacin. Niacin deficiency in humans results in pellagra, characterized by intestinal inflammation, diarrhea, dermatitis, and dementia (Hegyi et al., 2004). It is of great clinical relevance that lower abundance of GPR109A ligands niacin and butyrate in gut is associated with colonic inflammation."


"Activation of Gpr109a Suppresses Colonic Inflammation and Carcinogenesis in the Absence of Gut Microbiota or Dietary Fiber

"We then examined the relevance of niacin, a pharmacologic agonist for GPR109A, to colonic inflammation. For this, we first depleted gut microbiota with antibiotics, which reduces the production of butyrate, the endogenous agonist for GPR109A. Antibiotic treatment resulted in >300-fold reduction in aerobic and anaerobic bacterial counts in the stool (data not shown). Antibiotic treatment increased DSS-induced weight loss, diarrhea, and bleeding in WT mice (Figures 7B and S6A). Consistent with increased inflammation, we found that antibiotic treatment increased the number of polyps (8.2 ± 2.2 polyps/mouse with antibiotics; 1.6 ± 1.5 polyps/mouse without antibiotics) in WT mice (Figures 7C and 7D). We then tested whether administration of niacin protects antibiotic-treated mice against colonic inflammation and carcinogenesis. Niacin was added to drinking water along with antibiotic cocktail. Niacin ameliorated AOM+DSS-induced weight loss, diarrhea, and bleeding and reduced colon cancer development in antibiotic-treated WT mice (Figures 7B–7D and S6A). Consistent with a role of niacin in IL-18 induction, the protective effect of niacin in DSS-induced weight loss and diarrhea in antibiotic-treated Il18−/− mice was significantly blunted (Figure S6B). Niacin did not alter the development of weight loss, diarrhea, rectal bleeding, and colon cancer in antibiotic-treated Niacr1−/− mice, suggesting an essential role of Gpr109a in niacin-mediated promotion of colonic health (Figures 7B–7D and S6A). Antibiotic treatment reduced colonic inflammation and number of polyps in Niacr1−/− mice. This may be due to the presence of altered colitogenic gut microbiota in Niacr1−/− animals. "


"Although it has been known for decades that the commensal metabolite butyrate suppresses inflammation and carcinogenesis in colon, the exact identity of molecular target(s) of butyrate in this process remained elusive. The present studies identify Gpr109a as an important mediator of butyrate effects in colon and also as a critical molecular link between colonic bacteria and dietary fiber and the host. These findings have important implications for prevention as well as treatment of inflammatory bowel disease and colon cancer and suggest that under conditions of reduced dietary fiber intake and/or decreased butyrate production in colon, pharmacological doses of niacin might be effective to maintain GPR109A signaling and consequently protect colon against inflammation and carcinogenesis."


Evidence type: in vitro non-human animal experiment

Taggart AK1, Kero J, Gan X, Cai TQ, Cheng K, Ippolito M, Ren N, Kaplan R, Wu K, Wu TJ, Jin L, Liaw C, Chen R, Richman J, Connolly D, Offermanns S, Wright SD, Waters MG.
J Biol Chem. 2005 Jul 22;280(29):26649-52. Epub 2005 Jun 1.

"Here we show that the fatty acid-derived ketone body (d)-β-hydroxybutyrate ((d)-β-OHB) specifically activates PUMA-G/HM74a at concentrations observed in serum during fasting. Like nicotinic acid, (d)-β-OHB inhibits mouse adipocyte lipolysis in a PUMA-G-dependent manner and is thus the first endogenous ligand described for this orphan receptor. These findings suggests a homeostatic mechanism for surviving starvation in which (d)-β-OHB negatively regulates its own production, thereby preventing ketoacidosis and promoting efficient use of fat stores."


Evidence type: human case study

Tóth C, Dabóczi A, Howard M, Miller NJ, Clemens Z.
Int J Case Rep Images 2016;7(10):570–578.

"Given the ineffectiveness of standard therapies the parents of the child were seeking for alternative options. When we first met the patient he reported bilateral pain and swelling of the knee, frequent episodes of fever and night sweats as well as fatigue. He looked pale. We offered the paleolithic ketogenic diet along with close monitoring of the patient. The patient started the diet on 4 January 2015. The diet is consisting of animal fat, meat, offal and eggs with an approximate 2:1 fat : protein ratio. Red and fat meats instead of poultry as well as regular intake of organ meats from pork and cattle were encouraged. Grains, milk, dairy, refined sugars, vegetable oils, oilseeds, nightshades and artificial sweeteners were excluded. Small amount of honey was allowed for sweetening. The patient was not taking any supplements. Regular home monitoring of urinary ketones indicated sustained ketosis. Regular laboratory follow-up was used to monitor the course of the disease as well as for giving feedback how to fine tune the diet. The patient was under our close control and gave frequent feedbacks and so we could assess the level of dietary compliance. The patient maintained a high level dietary adherence on the long-term, yet on his birthday, he made a mistake: he has eaten two pieces of commercially available "paleo" cake which contained coconut oil, flour from oilseeds as well as sugar alcohol. Clinical consequences are discussed later. From July 2015 onwards he also consumed small amounts of vegetables and fruits. Given the persistence of certain alterations in laboratory values (mild anemia) on 10 November 2015, despite 10 months on the paleolithic ketogenic diet, we suggested to tighten the diet again. From this time on he did neither consume vegetables and fruits nor vegetable oil containing spices such as cumin and cinnamon.

"Discontinuing medication

"Within two weeks after diet onset the patient discontinued azathioprine, the only medicine he was taking at this time. Currently, he is without medicines for 15 months.


"The frequent night sweats of the patient disappeared within three weeks after diet onset and thus his sleep improved significantly. The knee pains of the patient began to lessen at 4th week on the diet and completely disappeared by the third month. From this time onwords he regularly went to school by bike (20 km daily). He reported restored energy and increased physical and mental fitness. Although during the eight months before diet onset his weight was declining, following diet onset he began to gain weight. At diet onset his weight was 41 kg and was 152 cm tall (BMI = 17.7). At 12 months after diet onset, his height was 160 cm and weighted 50 kg (BMI: 19.5). The change in his height and weight is depicted in Figure 5. At the time of writing the article he is on the diet for 15 months and is free of symptoms as well as side effects."


Evidence type: review

Patricia V Turner, Thea Brabb, Cynthia Pekow, and Mary Ann Vasbinder
J Am Assoc Lab Anim Sci. 2011 Sep; 50(5): 600–613.

"Intraperitoneal administration.

"Injection of substances into the peritoneal cavity is a common technique in laboratory rodents but rarely is used in larger mammals and humans. Intraperitoneal injection is used for small species for which intravenous access is challenging and it can be used to administer large volumes of fluid safely (Table 1) or as a repository site for surgical implantation of a preloaded osmotic minipump. Absorption of material delivered intraperitoneally is typically much slower than for intravenous injection. Although intraperitoneal delivery is considered a parenteral route of administration, the pharmacokinetics of substances administered intraperitoneally are more similar to those seen after oral administration, because the primary route of absorption is into the mesenteric vessels, which drain into the portal vein and pass through the liver.74 Therefore substances administered intraperitoneally may undergo hepatic metabolism before reaching the systemic circulation. In addition, a small amount of intraperitoneal injectate may pass directly across the diaphragm through small lacunae and into the thoracic lymph."


Experiment type: metagenomic analysis

Marius Vital, Adina Chuang Howe, and James M. Tiedje
mBio. 2014 Mar-Apr; 5(2): e00889-14. Published online 2014 April 22. doi: 10.1128/mBio.00889-14

"Diet is a major external force shaping gut communities (33). Good reviews of studies investigating the influence of diet on butyrate-producing bacteria exist (11 and 34) and suggest that plant-derived polysaccharides such as starch and xylan, as well as cross-feeding mechanisms with lactate-producing bacteria, are the main factors governing their growth. Our metagenomic analysis supports the acetyl-CoA pathway as the main pathway for butyrate production in healthy individuals (Fig. 4), implying that a sufficient polysaccharide supply is probably sustaining a well-functioning butyrate-producing community, at least in these North American subjects. However, the detection of additional amino acid-fed pathways, especially the lysine pathway, indicates that proteins could also play an important role in butyrate synthesis and suggests some flexibility of the microbiota to adapt to various nutritional conditions maintaining butyrate synthesis. Whether the prevalence of amino acid-fed pathway is associated with a protein-rich diet still needs to be assessed. It should be noted that those pathways are not restricted to single substrates, as displayed in Fig. 1, i.e., glutarate and lysine, but additional amino acids, such as aspartate, can be converted to butyrate via those routes as well (26). Furthermore, the acetyl-CoA pathway also can be supplied with substrates derived from proteins either by cross-feeding with the lysine pathway (as discussed above) or by direct fermentation of amino acids to acetyl-CoA (35). However, whereas diet-derived proteins are probably important for butyrate synthesis in the ileum, where epithelial cells use butyrate as a main energy source as well (36), it still needs to be assessed whether enough proteins reach the human colon to serve as a major nutrient source for microorganisms. Another possible colonic protein source could originate with lysed bacterial cells. Enormous viral loads have been detected in this environment, suggesting fast cell/nutrient turnover, which might explain the presence of corresponding pathways in both fecal isolates and metagenomic data (Fig. 1, 4, and 5). Detailed investigations of butyrate-producing communities in the colon of carnivorous animals will add additional key information on the role of proteins in butyrate production in that environment. It should be noted that diet provides only a part of the energy/carbon sources for microbial growth in the colon, since host-derived mucus glycans serve as an important nutrient source as well. Several butyrate-producing organisms do specifically colonize mucus (37), and for some, growth on mucus-derived substrates was shown (38). "


Evidence type: review

van der Wielen N, Moughan PJ, Mensink M.
J Nutr. 2017 Aug;147(8):1493-1498. doi: 10.3945/jn.117.248187. Epub 2017 Jun 14.

"Protein digestion and fermentation in the large intestine. Intact proteins that escape the small intestine or produced in the large intestine (mucus, cells, microbial proteins) are digested further in the large intestine by bacterial enzymes and the surviving pancreatic proteases and peptidases (35, 36). This protein degradation has been reported to be highest in the distal large intestine and is m ost likely related to the pH in the different regions (37). The di gested proteins can be used by the microbiota, which produce several metabolites such as SCFAs, ammonia, and amines. These metabolites may be linked to several health outcomes (38)."


"The large intestine is important for whole-body protein and nitrogen metabolism, in particular via bacterial metabolism. Both small and large intestinal microbiota are capable of synthesizing AAs, and absorption of microbial AAs has been demonstrated to take place in the intestine."


Ketogenic Diets, Vitamin C, and Metabolic Syndrome

This is an excerpt from today's guest post on breaknutrition.com:

The Recommended Daily Allowances (RDA) for different nutrients were developed on Western diets, and therefore, high-carb diets. Given that a ketogenic metabolism uses different metabolic pathways and induces cascades of drastically different metabolic and physiological effects, it would be astonishing if any of the RDAs are entirely applicable as is.

One micronutrient that seems to be particularly warranting reassessment is vitamin C, because vitamin C is biochemically closely related to glucose. Most animals synthesize it themselves out of glucose. It shares cellular uptake receptors with glucose. Some argue that because we don’t make vitamin C, we need to ensure a large exogenous supply. I will argue the opposite: so long as we are eating a low-carb diet, we actually need less. On our way, we’ll briefly re-examine the relationship between vitamin C deficiency and insulin resistance.

End-to-End Citations:

Evidence type: review
Louis Rosenfeld
Clinical Chemistry Vol. 43, Issue 4 April 1997
"In 1911, Casimir Funk isolated a concentrate from rice polishings that cured polyneuritis in pigeons. He named the concentrate “vitamine” because it appeared to be vital to life and because it was probably an amine. Although the concentrate and other “accessory food substances” were not amines, the name stuck, but the final “e” was dropped. "
Evidence type: review
Drouin, Guy, Jean-Rémi Godin, and Benoît Pagé.
Current Genomics 12.5 (2011): 371–378. PMC. Web. 19 Dec. 2016.
"Vitamin C (ascorbic acid) plays important roles as an anti-oxidant and in collagen synthesis. These important roles, and the relatively large amounts of vitamin C required daily, likely explain why most vertebrate species are able to synthesize this compound. Surprisingly, many species, such as teleost fishes, anthropoid primates, guinea pigs, as well as some bat and Passeriformes bird species, have lost the capacity to synthesize it. Here, we review the genetic bases behind the repeated losses in the ability to synthesize vitamin C as well as their implications. In all cases so far studied, the inability to synthesize vitamin C is due to mutations in the L-gulono-γ-lactone oxidase (GLO) gene which codes for the enzyme responsible for catalyzing the last step of vitamin C biosynthesis. The bias for mutations in this particular gene is likely due to the fact that losing it only affects vitamin C production. Whereas the GLO gene mutations in fish, anthropoid primates and guinea pigs are irreversible, some of the GLO pseudogenes found in bat species have been shown to be reactivated during evolution. The same phenomenon is thought to have occurred in some Passeriformes bird species. Interestingly, these GLO gene losses and reactivations are unrelated to the diet of the species involved. This suggests that losing the ability to make vitamin C is a neutral trait."
Evidence type: observation
Bánhegyi Gábor,Csala Miklós,Braun László,Garzó Tamás and Mandl József
FEBS Letters, 381, doi: 10.1016/0014-5793(96)00077-4 (1996)
"Ascorbic acid and glutathione are involved in the antioxidant defense of the cell. Their connections and interactions have been described from several aspects: they can substitute each other [1], dehydroascorbate can be reduced at the expense of GSH [2] and glutathione depletion results in the stimulation of ascorbate synthesis [3]. In ascorbate-synthesising animals, the formation of ascorbate from gulonolactone catalysed by microsomal gulonolactone oxidase is accompanied by the stoichiometric consumption of 02 and production of the oxidant hydrogen peroxide [4]. Metabolism of hydrogen peroxide by glutathione peroxidase requires reduced glutathione. Therefore, we supposed that synthesis of ascorbate should decrease the intracellular glutathione level. To prove our hypothesis, experiments were undertaken to investigate the effect of ascorbate synthesis stimulated by the addition of gulonolactone on the oxidation of GSH in isolated mouse hepatocytes and liver microsomal membranes."
"In this paper, a new connection between ascorbate and GSH metabolism is described. Our data show that the synthesis of ascorbate leads to consumption of GSH, the other main intracellular antioxidant (Fig. 1). We suppose that the formation of hydrogen peroxide is underlying the increased GSH consumption. First, oxidation of GSH caused by increased ascorbate synthesis was prevented by the addition of catalase in microsomal membranes (Table 1). Second, inhibition of glutathione peroxidase by mercaptosuccinate moderated the gulonolactone-dependent glutathione consumption in microsomes (Table 2). Third, the inhibition of catalase by aminotriazole deepened the ascorbate synthesis-dependent GSH depletion in isolated hepatocytes (Table 3). This interaction may be one of the causes why primates and some other species have lost their ascorbate-synthesising ability. This event occurred in the ancestors of primates about 70 million years ago, owing to mutation(s) in the gulonolactone oxidase gene [14]. Despite the well-known benefits [15] of ascorbate, the mutation(s) had to be advantageous, as this metabolic error did not remain an enzymopathy affecting only a minority of the population, but spread widely amongst the species (and individuals) of primates and became exclusive [16]. There is no explanation for this unexpected outcome. Based on these analytical data, the following conceptual evolutionary hypothesis can be outlined: in the tropical jungle of the Cretaceous Period, when exogenous ascorbate was abundant [17,18], the loss of gulonolactone oxidase activity could have proved to be advantageous. It saved the reduced GSH, the main defence system against oxidants, while the access to ascorbate was not hindered. Later, the evolutionary gains of these periods allowed the conservation of the genetic disorder manifested in the loss of ascorbate synthesis."
Evidence type: experiment
Reactive oxygen species (ROS)-induced mitochondrial abnormalities may have important consequences in the pathogenesis of degenerative diseases and cancer. Vitamin C is an important antioxidant known to quench ROS, but its mitochondrial transport and functions are poorly understood. We found that the oxidized form of vitamin C, dehydroascorbic acid (DHA), enters mitochondria via facilitative glucose transporter 1 (Glut1) and accumulates mitochondrially as ascorbic acid (mtAA). The stereo-selective mitochondrial uptake of D-glucose, with its ability to inhibit mitochondrial DHA uptake, indicated the presence of mitochondrial Glut. Computational analysis of N-termini of human Glut isoforms indicated that Glut1 had the highest probability of mitochondrial localization, which was experimentally verified via mitochondrial expression of Glut1-EGFP. In vitro mitochondrial import of Glut1, immunoblot analysis of mitochondrial proteins, and cellular immunolocalization studies indicated that Glut1 localizes to mitochondria. Loading mitochondria with AA quenched mitochondrial ROS and inhibited oxidative mitochondrial DNA damage. mtAA inhibited oxidative stress resulting from rotenone-induced disruption of the mitochondrial respiratory chain and prevented mitochondrial membrane depolarization in response to a protonophore, CCCP. Our results show that analogous to the cellular uptake, vitamin C enters mitochondria in its oxidized form via Glut1 and protects mitochondria from oxidative injury. Since mitochondria contribute significantly to intracellular ROS, protection of the mitochondrial genome and membrane may have pharmacological implications against a variety of ROS-mediated disorders.
Evidence type: non-human animal experiment
"Effect of starvation and subsequent feeding. The effect of starvation was then investigated, and it appeared that a 24 hr. period of starvation was enough to decrease the synthesis of ascorbic acid (Table 2). Since Caputto et al. (1958) had shown that the maximum effect of vitamin-E deficiency on the synthesis of ascorbic acid was reached as shortly as 3-4 days after deprivation, the possibility was considered that the effect of starvation was actually due to lack of vitamin E. This was discounted by giving starved animals enough vitamin E to prevent formation of peroxides; there was no effect on the synthesis of ascorbic acid. The effect ofstarving was quickly reversed by feeding the rats again for 24 hr."
"Effect of omission of carbohydrates from the diet and of administration of precursors: The effect of starvation could be attributed either to the stress or to the lack of some dietary components. A strong impairment of the synthesis of ascorbic acid was observed in rats given a carbohydrate-free diet for 24 hr., whereas values significantly higher but still below normal ones were obtained by giving this same diet for 6 days (Table 3). Rats on this ration had a lower content of ascorbic acid in the liver, but showed an enhanced excretion of ascorbic acid in the urine. Since carbohydrates are precursors of ascorbic acid in the rat, this observation led to the hypothesis of an adaptive response of the enzyme system to lack of substrates, and evidence was sought by giving glucuronolactone to rats. Administration of glucuronolactone did not affect the rate of synthesis in normal rats, but caused a moderate but significant enhancement in starved animals. However, a similar enhancement followed the administration of an equal amount of glucose. All rats receiving glucuronolactone had a higher liver content and an enhanced urinary excretion of ascorbic acid."
Evidence type: non-human animal experiment
Braun L1, Garzó T, Mandl J, Bánhegyi G.
FEBS Lett. 1994 Sep 19;352(1):4-6.
"The role of the hepatic glycogen content in ascorbic acid synthesis was investigated in isolated mouse hepatocytes. The cells were prepared from fed or 48 h-starved mice and the ascorbic acid content was measured in the suspension (cells+medium). After 48 h starvation hepatocytes did not contain measurable amounts of glycogen. The initial concentration of ascorbic acid was lower in the suspension of glycogen-depleted hepatocytes compared to the fed controls (Fig. 1) and only a moderate synthesis could be observed under both nutritional conditions. The effects of dibutyryl CAMP and glucagon on ascorbate synthesis were examined. Glucagon or dibutyryl cyclic AMP caused a stimulation of ascorbic acid synthesis in hepatocytes from fed mice, while in hepatocytes from 48 hstarved animals ascorbic acid production was not increased significantly by the two agents (Fig. 1). The addition of glucose and gluconeogenic precursors to the incubation medium did not result in a significant increase in ascorbic acid production (Fig. 1). In another series of experiments glucose and ascorbic acid production of the cells was measured simultaneously. The rate of glucose production (in the absence of gluconeogenic precursors mainly via glucogenolysis) and ascorbic acid synthesis showed a close correlation (r = 0.9091) (Fig. 2). As ascorbic acid synthesis and glycogenolysis seemed to be connected, we examined the effect on ascorbic acid synthesis of various agents known to increase glycogenolysis. The al agonist phenylephrine, the protein phosphatase inhibitor okadaic acid and vasopressin all increased the rate of ascorbic acid production in isolated hepatocytes prepared from fed mice similarly to glucagon (Table 1).
"Glycogenolysis was stimulated by the in vivo addition of glucagon. Glucagon elevated the blood glucose level of mice by 50%; at the same time a more than fifteenfold increase of plasma ascorbic acid concentration could be observed (Table 2). The concentration of ascorbic acid in the liver was also increased, indicating a stimulated hepatic synthesis (Table 2).
Glycogen content is considered to be a sensitive marker showing the actual metabolic state of the liver. Observations described in this paper suggest that ascorbic acid synthesis in murine liver is tightly connected with the glycogen pool; the source of ascorbic acid is glycogen. The following results gained in isolated hepatocytes support this assumption: first, in hepatocytes isolated from glycogen-depleted animals the ascorbic acid level as well as the rate of synthesis is lower than that in hepatocytes from control fed mice (Fig. 1); second, different glycogen-mobilizing agents acting via different mechanisms enhance ascorbic acid production in hepatocytes from fed but not from fasted animals (Fig. 1, Table 1); third, addition of glucose to hepatocytes prepared from glycogen-depleted mice failed to increase the formation of ascorbic acid (Fig. 1). The results gained under in vitro conditions in isolated hepatocytes were confirmed by in vivo experiments: a single i.p. injection of glucagon elevated both the plasma and liver ascorbic acid levels within 15 min (Table 2). "
"The finding that the source of ascorbate production is glycogenolysis is in according with the fact that liver and kidney -the main sites of glycogen storage - are responsible for the ascorbic acid supply in most animal species [2]. The increased hepatic ascorbic acid production after glucagon administration can be explained as a compensatory mechanism of the missing intake of ascorbate, i.e. adaptation of ascorbic acid supply from external to internal sources. Considering the fifteenfold elevation of plasma ascorbate levels, in the light of recent findings concerning the effect of ascorbate on insulin secretion [18] and on the calcium channels in pancreatic beta cells [19] it might be also regarded as a possible intercellular messenger. "
Evidence type: experiment
Drew KL, Tøien Ø, Rivera PM, Smith MA, Perry G, Rice ME.
Comp Biochem Physiol C Toxicol Pharmacol. 2002 Dec;133(4):483-92.
"During hibernation plasma ascorbate concentrations w(Asc)px were found to increase 3–5 fold in two species of ground squirrels, AGS and 13-lined ground squirrels (TLS); S. tridecemlineatus and cerebral spinal fluid (CSF) ascorbate concentration w(Asc)CSFx doubled in AGS (CSF was not sampled in TLS) (Drew et al., 1999). During arousal, however, when oxygen consumption peaks and the generation of reactive oxygen species is thought to be maximal, plasma ascorbate concentrations progressively decrease to levels typical for euthermic animals (Fig. 3)."
Evidence type: observation
George Mann and Pamela Newton
Ann N Y Acad Sci. 1975 Sep 30;258:243-52.
"We have formulated two hypotheses. The first proposes that the transport of ascorbate across cell membranes may be impaired by glucose. The second proposes that the transport of ascorbate in certain tissues is facilitated by insulin. If either hypothesis is valid, those species requiring exogenous ascorbate would be in double jeopardy if they were also hyperglycemic. Carbohydrate intolerance resulting from eithcr a lack of or a resistance to insulin is common in Western man. Gore et al. have shown with electron microscopy that the vascular lesion of scurvy involves collagenous structures in the basement membranes, and this is also the site of the lesion in diabetic microangiopathy. These hypotheses, which propose that the intracellular availability of dehydroascorbate (DHA), the transportable form of vitamin C, would be impaired in certain tissues by either hypcrglycemia or lack of insulin, suggest that diabetic microangiopathy, the main complication of human diabetes, may be a consequence of local ascorbate deficiency. The laboratory investigations described here deal with the first and somewhat simpler of these hypotheses: Glucose will impair the transport of dehydroascorbate into cells. The data collected show that D-glUCOSe does inhibit the transport of dehydroascorbate into human red blood cells, a noninsulin-dependent tissue. Trials wiih other sugars show a hierarchy of sugars that inhibit transport, suggesting that DHA and D-glucose share a carrier mechanism."
Evidence type: review
"Hyperglycemia-induced ascorbic acid deficiency
Vitamin C is a derivative of glucose and Mann [138] proposed that the structural similarity between these two molecules may account for many of the complications of diabetes. Glucose has been shown to inhibit vitamin C transport in several mesenchymal cell types, including endothelial cells [139], mononuclear leukocytes [140], neutrophils [141,142], fibroblasts [143,144], and erythrocytes [145]. Facilitative glucose transporters (GLUTs) bind dehydroascorbic acid and are thought to be the primary transporters of vitamin C in mammalian cells [146]. After transport, dehydroascorbic acid is quickly reduced to ascorbic acid. Glucose competitively inhibits the uptake of dehydroascorbic acid but does not affect ascorbic acid transport. Ascorbic acid is transported by a family of membrane-bound proteins that are Na+-dependent and whose function is not directly inhibited by elevated extracellular concentrations of glucose [146,147]. This latter system is prevalent in bulk-transporting epithelia (e.g. kidney and small intestine) and have been recently isolated in both human [148] and rat [149] biological systems. Many cell types, of course, [150,151] express both transport systems.
High blood glucose concentrations mimic the conditions of vitamin C deficiency. Acute hyperglycemia, for example, impairs endothelium-dependent vasodilation in healthy humans [152], an effect which can be reversed by acute administration of vitamin C [153]. Ascorbic acid plays an important role in extracellular matrix regulation and has a stimulatory effect on sulfate incorporation in mesangial cell and matrix proteoglycans; high glucose concentrations have been shown to impair this effect [154]. Endothelial surface proteoglycans help prevent thrombus formation and also inhibit smooth-muscle growth [1]. High glucose concentrations also have been shown to inhibit the stimulatory effect of ascorbic acid on collagen and proteoglycan synthesis in cultured fibroblasts [114]. Moreover, a high concentration of glucose can induce the expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells [155]. Endothelial cells express these and other membrane-bound proteins to enable leukocyte adhesion and transmigration across the endothelium during an inflammatory response. Atherosclerosis is one such inflammatory response.
Experimental and clinical studies suggest that latent scurvy is characterized by IGT [16,24] and diabetes mellitus is a disease complex characterized by impaired glucose and vitamin C metabolism [27,28]. Diabetic patients are prone to hyperglycemia, prolonged wound healing, infection, increased synthesis of cholesterol, decreased liver glycogen, and notably, diffuse vascular disease. All of these findings are consistent with latent scurvy [16]. Diabetic platelets have been shown to have low intracellular ascorbic acid concentrations and display hypercoagulability [156]. Long-term vitamin C administration has beneficial effects on glucose and lipid metabolism in aged NIDDM patients [157]. It has also been suggested that vitamin C consumption above the RDA may provide important health benefits for individuals with IDDM [158]. This latter recommendation is supported by recent evidence. For example, mesenchymal cells from patients with IDDM have an impaired uptake of dehydroascorbic acid that persists in culture [159] and ascorbic acid has been shown to prevent the inhibition of DNA synthesis induced by high glucose concentrations in cultured endothelial cells [160]. Diabetic patients have been observed to have a lowered ascorbic acid/dehydroascorbic acid plasma ratio, indicating a decreased vitamin C status [161]. Therefore, diabetic patients may benefit from vitamin C supplementation to alleviate multiple physiologic and metabolic impairments in a variety of cell types."
Evidence type: review
Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta A, Dutta SK, Levine M.
J Am Coll Nutr. 2003 Feb;22(1):18-35.
"Problems in Demonstrating Antioxidant Benefit of Vitamin C in Clinical
"Studies Despite epidemiological and some experimental studies, it has not been possible to show conclusively that higher than anti-scorbutic intake of vitamin C has antioxidant clinical benefit. This is despite the fact that vitamin C is a powerful antioxidant in vitro. It is of course possible that the lack of antioxidant effect of vitamin C in clinical studies is real. It seems more likely that vitamin C has antioxidant or other benefits. Detection of these benefits has remained elusive due to the vicissitudes of experimental design. Vitamin C may be a weak antioxidant in vivo, or its antioxidant actions may have no physiological role, or its role may be small. The oxidative hypothesis is unproven, and oxidative damage may have a smaller role than anticipated in some diseases. Further, antioxidant actions of vitamin C may occur at relatively low plasma vitamin C concentrations. Thus additional clinical benefits that occur at higher vitamin C concentrations may be difficult to demonstrate. Although all these are possible explanations, it seems unlikely that these are the real reasons for the lack of detectable effects of vitamin C in clinical studies. Many factors may contribute to the failure so far to demonstrate clear antioxidant benefits of vitamin C in clinical studies. The antioxidant actions of vitamin C may be specific to certain reactions or occur only at specific locations. In either case, beneficial effects can be shown only in disorders where such reactions or sites are the focus of disease process. There may be many different antioxidants that are active at the same time. In the face of such redundancy, only multiple antioxidant deficiencies will have detectable clinical effects. Antioxidant deficiency may have to be of long duration for accumulated damage to be noticeable. Antioxidant effects may be of importance only in those with oxidant stress. Thus, normal subjects or those with mild disease may have no need for high antioxidant concentrations. In a way analogous to the effect of acetaminophen on fever, antioxidants may have no effect in the absence of marked oxidant stress. A further problem is presented by the sigmoidal dose concentration curve for vitamin C. Small changes in oral intake of vitamin C produce large changes in plasma vitamin C concentrations. This makes it difficult to conduct controlled studies such that the plasma vitamin C concentrations of the control and study groups differ sufficiently to have physiological meaning."
Evidence type: review
Padayatty SJ, Levine M
Oral Dis. 2016 Sep;22(6):463-93. doi: 10.1111/odi.12446. Epub 2016 Apr 14.
(Emphasis mine)
"Collagen hydroxylation
"Common symptoms of scurvy include wound dehiscence, poor wound healing and loosening of teeth, all pointing to defects in connective tissue (Crandon et al, 1940; Lind, 1953; Hirschmann and Raugi, 1999). Collagen provides connective tissue with structural strength. Vitamin C catalyzes enzymatic (Peterkofsky, 1991) posttranslational modification of procollagen to produce and secrete adequate amounts of structurally normal collagen by collagen producing cells (Kivirikko and Myllyla, 1985; Prockop and Kivirikko, 1995). Precollagen, synthesized in the endoplasmic reticulum, consists of amino acid repeats rich in proline. Specific prolyl and lysyl residues are hydroxylated, proline is converted to either 3-hydroxyproline or 4-hydroxyproline, and lysine is converted to hydroxylysine. The reactions catalyzed by prolyl 3-hydroxylase, prolyl 4- hydroxylase, and lysyl hydroxylase (Peterkofsky, 1991; Prockop and Kivirikko, 1995; Pekkala et al, 2003) require vitamin C as a cofactor. Hydroxylation aids in the formation of the stable triple helical structure of collagen, which is transported to the Golgi apparatus and eventually secreted by secretory granules. In the absence of hydroxylation, secretion of procollagen decreases (Peterkofsky, 1991) and it probably undergoes faster degradation. However, some hydroxylation can occur even in the absence of vitamin C (Parsons et al, 2006). Secreted procollagen is enzymatically cleaved to form tropocollagen that spontaneously forms collagen fibrils in the extracellular space. These fibrils form intermolecular collagen cross-links, giving collagen its structural strength. Independent of its effects on hydroxylation, ascorbate may stimulate collagen synthesis (Geesin et al, 1988; Sullivan et al, 1994). Collagen synthesis may be decreased in scorbutic animals (Peterkofsky, 1991; Kipp et al, 1996; Tsuchiya and Bates, 2003). Reduced collagen cross-links may be a marker of vitamin C deficiency in the guinea pig (Tsuchiya and Bates, 2003) but this may not be specific to vitamin C deficiency. Although many features of human scurvy appear to be due to weakening of connective tissue, it has not been shown that these lesions are due to defective collagen synthesis."
Evidence type: non-human animal experiment
J Mårtensson, J Han, O W Griffith, and A Meister
Proc Natl Acad Sci U S A. 1993 Jan 1; 90(1): 317–321.
"Guinea pigs given an ascorbate-deficient diet gained weight through day 14, but gained at a slower rate than the control animals,and then lost weight(Table1,groupA).The animals givenGSH ester(groupB)gained more weight than those of group A, and the weight gain during days 10-14 was =70% of the control group. Animals in group A became obviously sick after about day 17. They could not walk and moved very little, apparently immobilized by fractures of the hind legs and by swelling of the joints of the extremities, which were tender and had periosteal hematomas. Radiography showed major fractures of the femur in two animals. Animals in group A died or were sacrificed on day 21 or 22. Animals in group B(GSHester)did not have fractures or hematomas; 75% of these animals were indistinguishable by general appearance from controls. Histological study showed significant loss of osteoid material from long bones in group A,whereas most animals in group B had no decrease of osteoid material (Fig.1)or only a moderate decrease. In a separate experiment, several animals comparable to those of group B were kept for 40 days and showed no significant signs of scurvy (tender swollen joints,fractures);they exhibited some weight loss."
Evidence type: in vitro experiment
Li X, Qu ZC, May JM.
Antioxid Redox Signal. 2001 Dec;3(6):1089-97.
"Liver is the site of ascorbic acid synthesis in most mammals. As human liver cannot synthesize ascorbate de novo, it may differ from liver of other species in the capacity or mechanism for ascorbate recycling from its oxidized forms. Therefore, we compared the ability of cultured liver-derived cells from humans (HepG2 cells) and rats (H4IIE cells) to take up and reduce dehydroascorbic acid (DHA) to ascorbate. Neither cell type contained appreciable amounts of ascorbate in culture, but both rapidly took up and reduced DHA to ascorbate. Intracellular ascorbate accumulated to concentrations of 10-20 mM following loading with DHA. The capacity of HepG2 cells to take up and reduce DHA to ascorbate was more than twice that of H4IIE cells. In both cell types, DHA reduction lowered glutathione (GSH) concentrations and was inhibited by prior depletion of GSH with diethyl maleate, buthionine sulfoximine, and phenylarsine oxide. NADPH-dependent DHA reduction due to thioredoxin reductase occurred in overnight-dialyzed extracts of both cell types. These results show that cells derived from rat liver synthesize little ascorbate in culture, that cultured human-derived liver cells have a greater capacity for DHA reduction than do rat-derived liver cells, but that both cell types rely largely on GSH- or NADPH-dependent mechanisms for ascorbate recycling from DHA."
Evidence type: non-human animal experiment
Jarrett SG, Milder JB, Liang LP, Patel M.
J Neurochem. 2008 Aug;106(3):1044-51. doi: 10.1111/j.1471-4159.2008.05460.x. Epub 2008 May 5.
"The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague-Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of beta-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H2O2 production and H2O2-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H2O2 production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage."
Evidence type: non-human animal experiment
Milder JB, Liang LP, Patel M.
Neurobiol Dis. 2010 Oct;40(1):238-44. doi: 10.1016/j.nbd.2010.05.030. Epub 2010 May 31.
"The mechanisms underlying the efficacy of the ketogenic diet (KD) remain unknown. Recently, we showed that the KD increased glutathione (GSH) biosynthesis. Since the NF E2-related factor 2 (Nrf2) transcription factor is a primary responder to cellular stress and can upregulate GSH biosynthesis, we asked whether the KD activates the Nrf2 pathway. Here we report that rats consuming a KD show acute production of H(2)O(2) from hippocampal mitochondria, which decreases below control levels by 3 weeks, suggestive of an adaptive response. 4-Hydroxy-2-nonenal (4-HNE), an electrophilic lipid peroxidation end product known to activate the Nrf2 detoxification pathway, was also acutely increased by the KD. Nrf2 nuclear accumulation was evident in both the hippocampus and liver, and the Nrf2 target, NAD(P)H:quinone oxidoreductase (NQO1), exhibited increased activity in both the hippocampus and liver after 3 weeks. We also found chronic depletion of liver tissue GSH, while liver mitochondrial antioxidant capacity was preserved. These data suggest that the KD initially produces mild oxidative and electrophilic stress, which may systemically activate the Nrf2 pathway via redox signaling, leading to chronic cellular adaptation, induction of protective proteins, and improvement of the mitochondrial redox state."
Evidence type: review
Meister A
J Biol Chem. 1994 Apr 1;269(13):9397-400.
"Ascorbate and GSH have actions in common and can spare each other under appropriate experimental conditions; this redundancy reflects the metabolic importance of such antioxidant activity.
[Sorry, this paper is hard to quote. It's free. Go look. :-)]
Evidence type: review
Bruce N. Ames, Richard Cathcart, Elizabeth Schwiers, and Paul Hochstein
Proc. NatL Acad. Sci. USA Vol. 78, No. 11, pp. 6858-6862, November 1981 Biochemistry
"During primate evolution, a major factor in lengthening life-span and decreasing age-specific cancer rates may have been improved protective mechanisms against oxygen radicals. We propose that one of these protective systems is plasma uric acid, the level of which increased markedly during primate evolution as a consequence of a series of mutations. Uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. We show that, at physiological concentrations, urate reduces the oxo-heme oxidant formed by peroxide reaction with hemoglobin, protects erythrocyte ghosts against lipid peroxidation, and protects erythrocytes from peroxidative damage leading to lysis. Urate is about as effective an antioxidant as ascorbate in these experiments. Urate is much more easily oxidized than deoxynucleosides by singlet oxygen and is destroyed by hydroxyl radicals at a comparable rate. The plasma urate level in humans (about 300 ILM) is considerably higher than the ascorbate level, making it one of the major antioxidants in humans. Previous work on urate reported in the literature supports our experiments and interpretations, although the findings were not discussed in a physiological context."
Evidence type: review
Glantzounis GK, Tsimoyiannis EC, Kappas AM, Galaris DA.
Curr Pharm Des. 2005;11(32):4145-51.
"It has been proposed that UA may represent one of the most important low-molecular-mass antioxidants in the human biological fluids [23-26]. Ames et al. proposed in the early eighties that UA can have biological significance as an antioxidant and showed, by in vitro experiments, that it is a powerful scavenger of peroxyl radicals (RO2 . ), hydroxyl radicals (. OH) and singlet oxygen [23]. The authors speculated that UA may contribute to increased life-span in humans by providing protection against oxidative stress-provoked ageing and cancer. UA is an oxidizable substrate for haem protein/H2O2 systems and is able to protect against oxidative damage by acting as an electron donor [27]. Apart from its action as radical scavenger, UA can also chelate metal ions, like iron and copper, converting them to poorly reactive forms unable to catalyse free-radical reactions [28-30]."
"A randomized placebo-controlled double-blind study has evaluated the effects of systemic administration of 100 mg UA, in healthy volunteers compared with vitamin C 1000 mg [45]. A significant increase in serum free radical scavenging capacity from baseline was observed during UA and vitamin C infusion – but the effect of UA was substantially greater. No adverse reactions to UA administration were reported. Another clinical study indicated a significant inverse relationship between serum UA concentrations and oxidative stress during acute aerobic exercise [46], while an increase in muscle allantoin levels was detected [32]. The authors concluded that ROS are formed in human skeletal muscle during intense sub-maximal exercise and urate is used as a local antioxidant. Another clinical trial involving healthy young men showed that 50 and 80 km marches led to 25 % and 37 % rises, respectively, in plasma levels of UA, probably due to increases in the metabolic rate and consequently pyrimidine nucleotide metabolism [47]. A randomized double-blind placebo controlled crossover study evaluated the free radical properties of UA in healthy volunteers [48]. UA (0.5 g in 250 ml of 0.1 % lithium carbonate / 4 % dextrose vehicle or vehicle alone as control) was given to subjects who then performed high intensity aerobic exercise for 20 min to induce oxidative stress. A single high-intensity exercise caused oxidative stress (as reflected by increased plasma F2- isoprostanes) immediately after exercise and recovery. Administration of UA increased circulating UA concentrations, which increased serum free radical scavenging capacity and reduced the exercise-induced increases in plasma F2-isoprostanes. The authors concluded that the antioxidant properties of UA are of physiological consequence and support the view that UA has potentially important free radical scavenging effects in vivo."
Evidence type: observational
"Urate has been shown to be a major antioxidant in human serum and was postulated to have a biological role in protecting tissues against the toxic effects of oxygen radicals and in determining the longevity of primates. This possibility has been tested by determining if the maximum lifespan potentials of 22 primate and 17 non-primate mammalian species are positively correlated with the concentration of urate in serum and brain per specific metabolic rate. This analysis is based on the concept that the degree of protection a tissue has against oxygen radicals is proportional to antioxidant concentration per rate of oxygen metabolism of that tissue. Ascorbate, another potentially important antioxidant in determining longevity of mammalian species, was also investigated using this method. The results show a highly significant positive correlation of maximum lifespan potential with the concentration of urate in serum and brain per specific metabolic rate. No significant correlation was found for ascorbate. These results support the hypothesis that urate is biologically active as an antioxidant and is involved in determining the longevity of primate species, particularly for humans and the great apes. Ascorbate appears to have played little or no role as a longevity determinant in mammalian species."
Evidence Type: review
Charles V. Mobbs, Jason Mastaitis, Minhua Zhang, Fumiko Isoda, Hui Cheng, and Kelvin Yen
Interdiscip Top Gerontol. 2007; 35: 39–68.
(emphasis mine)
"Glucose Oxidation Favors Complex I, Lipid/Amino Acid Oxidation Favors Complex II
"The significance of the shift in source of carbon atoms for oxidation produced by dietary restriction may be that the oxidation of lipids and amino acids depends much more on mitochondrial complex II than on (free-radical generating) complex I, whereas glucose oxidation depends much more on complex I than on complex II. When glucose is broken down by glycolysis, the only reducing equivalents it makes are in the form of NADH. When the final carbon product of glucose, pyruvate, is metabolized in the Krebs cycle, almost all the reducing equivalents are produced in the form of NADH, except for one step at complex II (succinate dehydrogenase) that makes (then oxidizes) FADH2. Ultimately the metabolism of one molecule of glucose produces an NADH: FADH2 ratio of 5:1 [53, p. 20]. In contrast, when lipids are broken down by β-oxidation (fatty acid counterpart to glycolysis), an equal number of NADH and FADH2 molecules are formed. When the lipid-derived carbons are metabolized in the Krebs cycle, reducing equivalents are produced in the ratio of 3 NADH molecules per FADH2 molecule. Therefore ultimately lipid metabolism yields an NADH:FADH2 ratio of about 2:1 [53, p. 38] or even more if the fatty acid contains enough carbon atoms. For example, when one molecule of palmitate is oxidized, it produces 15 molecules of FADH2 and 31 molecules of NADH, which are ultimately oxidized to produce a net total of 129 ATP molecules. In contrast, production of the same number of ATP molecules from glucose would entail producing then oxidizing 8.66 FADH2 and 43.3 NADH molecules. Amino acid oxidation also proceeds by a similar 2-step mechanism yielding an NADH:FADH2 ratio between that of lipids and that of glucose, the precise number depending on the specific amino acid. The significance of this shift in the NADH:FADH2 ratio is that NADH is oxidized only at mitochondrial complex I, whereas FADH2 is oxidized only at complex II [53, p. 17]. Thus palmitate oxidation entails utilizing complex II at roughly twice the (FADH2-dependent) rate as glucose oxidation entails. Therefore shifting away from glucose utilization toward lipid and amino acid utilization would be expected to substantially reduce the production of reactive oxygen species, without necessarily reducing ATP production. As described below, other beneficial effects also occur as a result of this altered pattern of glucose fuel use, including a shift toward producing antioxidizing NADPH and increased protein and lipid turnover, which reduces the accumulation of oxidized protein and lipids."
Evidence type: experiment
Greco T, Glenn TC, Hovda DA, Prins ML
J Cereb Blood Flow Metab. 2016 Sep;36(9):1603-13
"Mechanisms of ketogenic improvement
"As mentioned previously, it is thought that much of the KD’s improvement in cellular metabolism and neuroprotection is through its ability to act as an alternative substrate. Here, we show rather that it first acts in an antioxidant manner to reverse mitochondrial dysfunction. Both Complex I and II–III are inhibited in CCISTD mice at 6 h post-injury. Increased production of lactate is a reflection of impairment of oxidative phosphorylation as well as an attempt to maintain ATP concentrations and cellular membrane potential through increased glycolytic output.13 While Complex I activity returns to sham levels by 24 h, Complex II–III activity remains inhibited. ONOO has been shown to not only inhibit Complex II–III, but also Complex V40 and suggests that the observed decline in ATP production in PND35 animals13 is due in part to impaired Complex III and/or V activity. In addition to inhibition of mitochondrial complexes, decomposition products of ONOO increase the amount of lipid peroxidation leading to thiol linkages and pore formation in the inner membrane ultimately uncoupling the mitochondria. Although Complex I activity is inhibited in CCI-KD animals, Complex II–III activity is not. This will continue to allow electron flow through the respiratory chain and production of ATP. KD not only has antioxidant properties, but may provide substrates beyond Acetyl-CoA. The reaction of AcAc with Succinyl-CoA produces succinate, and animals either fed a KD or infused with ßOHB show a significant increase in succinate concentrations.41,42 Other groups have also shown that KD increases Complex II activity (succinate dehydrogenase activity).43 By increasing Complex II activity and its substrate, KD is able to maintain mitochondrial membrane potential and ATP production and prevent bioenergetic failure. At 24 h post-injury, KD is likely to exert its affects through three mechanisms: (1) continued direct and indirect ROS/RNS scavenging, (2) increased Complex II activity and (3) increased acetyl-CoA and succinate."

Evidence type: experiment
I cannot access the original experiment, but it is referred to here in the documents used by the RDA:
"Overall, while evidence suggests that vitamin C deficiency is linked to some aspects of periodontal disease, the relationship of vitamin C intake to periodontal health in the population at large is unclear. Beyond the amount needed to prevent scorbutic gingivitis (less than 10 mg/day) (Baker et al., 1971), the results from current studies are not sufficient to reliably estimate the vitamin C requirement for apparently healthy individuals based on oral health endpoints."
Baker EM, Hodges RE, Hood J, Sauberlich HE, March SC, Canham JE. 1971. Metabolism of 14C- and 3H-labeled L-ascorbic acid in human scurvy. Am J Clin Nutr 24:444–454.
Evidence type: observation
The current recommended dietary allowance (RDA) for vitamin C for adult nonsmoking men and women is 60 mg/d, which is based on a mean requirement of 46 mg/d to prevent the deficiency disease scurvy. However, recent scientific evidence indicates that an increased intake of vitamin C is associated with a reduced risk of chronic diseases such as cancer, cardiovascular disease, and cataract, probably through antioxidant mechanisms. It is likely that the amount of vitamin C required to prevent scurvy is not sufficient to optimally protect against these diseases. Because the RDA is defined as "the average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all healthy individuals in a group," it is appropriate to reevaluate the RDA for vitamin C. Therefore, we reviewed the biochemical, clinical, and epidemiologic evidence to date for a role of vitamin C in chronic disease prevention. The totality of the reviewed data suggests that an intake of 90-100 mg vitamin C/d is required for optimum reduction of chronic disease risk in nonsmoking men and women. This amount is about twice the amount on which the current RDA for vitamin C is based, suggesting a new RDA of 120 mg vitamin C/d.