similarities between germ-free mice and ketogenic humans

tracing a chain of ideas

Sometimes the assumptions that scientists start with about what is “good”, “healthy”, or “normal” can cause them to interpret results in a completely different way than someone starting with different assumptions would have. Then, the resulting conclusions become the assumptions in the next round of interpretation, leading to a chain of logic in which one questionable assumption leads to another.

We recently read a paper in which the authors made a series of logical steps, and it became almost comical to us how at each step we would have interpreted their results in an opposite way than they did.

When the results of their experiment are looked at from our perspective, it suggests an intriguing hypothesis:

Maybe some of the health benefits a ketogenic diet are due, not just to the diet being low in digestible carbohydrate and thus leading to ketosis, but also to being low in indigestible fiber and thus starving certain gut bacteria.

Or, to phrase the same hypothesis differently, maybe one mechanism by which a glycolytic or high-fiber diet causes health problems is that it feeds harmful gut bacteria, and the presence of those bacteria causes the health problems.

If that hypothesis were true, it would imply that if you are eating a low-carb diet, then including a lot of low-carb vegetables would feed these hypothesized harmful gut bacteria and reduce some of the potential health benefits of a low-carb diet.

in brief

The purpose of this article is two-fold:

• First, to compare the authors' interpretations of the observations to ours, given what we know about the metabolic effects of ketogenic diets.

  We draw attention to the fact that the metabolism of germ-free mice is strikingly similar to that of ketogenic dieters. This similarity holds at the whole-body level in terms of behaviour and physical characteristics, as well as the level of mitochondrial energetics. We show that these characteristics appear to be beneficial.

• Second, to raise the following questions:

  Are some of the benefits of a ketogenic diet mediated by starving gut bacteria, and if so, does eating fiber (i.e. low-carb vegetables) reduce some of the health benefits of a keto diet? Would eating a carbohydrate- and fiber- free diet confer some keto-like benefits even in the absence of ketosis?

the end of the chain

It all started when we saw the following statement on the Wikipedia page about butyrate :

"Butyrates are important as food for cells lining the mammalian colon (colonocytes). Without butyrates for energy, colon cells undergo autophagy (self digestion) and die.[1] Short-chain fatty acids, which include butyrate, are produced by beneficial colonic bacteria (probiotics) that feed on, or ferment prebiotics, which are plant products that contain adequate amounts of dietary fiber."

The topic of butyrate is exciting to some scientists, because they have the idea that eating indigestible fiber is good for human health. Epidemiological studies have found correlations between high fiber intake and relatively less disease. However, randomised controlled trials have repeatedly failed to confirm the hypotheses that the fiber intake was actually protective. [1]. Therefore when a new idea comes up that might explain how eating fiber would be good for human health, scientists still hoping for such evidence latch onto it. Butyrate is an example of such a candidate mechanism for how eating fiber would be good for human health.

To us, since we think that eating fiber is useless (at best) for health, the statement above poses a challenge and a mystery. Almost the only source of butyrate in the human body is, as the wikipedia page explains in the excerpt above, from gut bacteria digesting fiber that you ate. (There's also some butyrate in butter, presumably made the same way in cows.)

If butyrate is necessary for the health and even the survival of colon cells, wouldn't that mean that a low-fiber diet — such as an all-meat diet — would be very unhealthy? Amber hasn't eaten any significant amount of indigestible fiber in more than four years; does this mean that her colon cells have died off?

So we set out to investigate what led to that statement on wikipedia. Our investigation ultimately led to an intriguing hypothesis about a candidate mechanism to explain some of the health benefits of a keto diet.

The paper referenced as "[1]" on the wikipedia page is Donohoe-2011-“The Microbiome and Butyrate Regulate Energy Metabolism and Autophagy in the Mammalian Colon”. We read it with interest.

The authors of this paper performed a good experiment, made precise measurements, got interesting results, and clearly reported their results. But when it came to interpretation, they started with some assumptions we don't think are warranted, and therefore produced a chain of reasoning that eventually led them and their readers, such as the authors of the wikipedia page, to conclusions that are opposite from ours.

other links in the chain

The paper describes an experiment in which they compare the metabolic mechanisms of “germ free” mice — mice that have been raised from birth without ever being allowed to contact common gut bacteria — with “conventional” mice. “Conventional” mice are genetically identical to the germ-free mice, but the researchers allow the conventional mice to be colonized by the bacteria that colonize all lab mice that aren't specially isolated.

The authors cite previous experimenters who have raised germ-free mice and compared them to conventional mice. Those previous experiments compared body weight of the mice, how active they were, how much food they ate, and levels of different hormones and substances in their blood.

Here is their summary:

"[Germ-free] mice must consume 10–30% more food to maintain the same body weight as conventional controls. Despite this increased food intake, germ-free mice are leaner with a ~40% decrease in the size of their epididymal fat pads. They have a similar decrease in liver glycogen levels. [Germ-free] mice also have lower blood glucose and insulin levels and are resistant to obesity induced by a high-fat diet." — from Donohoe-2011

The authors view this as evidence of detrimental metabolic inefficiency. They also note differences in metabolic workings of the mitochondria in the colon cells of the mice that they believe account for the inefficiency. Therefore they construct an experiment to discover whether feeding butyrate to the colon cells of the germ-free mice would restore them to working the way conventional colon cells work (which it does).

To us, on the other hand, the germ-free mouse sounds like the healthier mouse!

similarities between germ-free mice and ketogenic dieters

If you have followed the debates about low-carb diets conferring a metabolic advantage (demonstrated by their superior performance as weight loss diets [2], [3]) then the above description of germ-free mice should sound familiar. Compared to low-fat dieters, ketogenic dieters tend to be leaner (i.e. have a higher ratio of muscle to body fat), and have lower insulin and blood glucose levels. This can happen despite similar caloric intake. Their liver glycogen levels are also lower; ketogenesis may depend on low glycogen levels [4].

However, the germ-free mice are not ketogenic, presumably because they are consuming regular, glucose-plentiful diets. In fact they are less ketogenic than the conventional mice, as measured by beta-hydroxybutyrate in the blood.

So what is the cause of the similarity in metabolism between germ-free mice and ketogenic humans?

There is one other important way in which the germ-free mice were different from the conventional mice. They had lower NADH/NAD+ ratios and ATP levels (per mitochondrion) in their colon cells, but not in the liver, heart, or kidneys. Note that the heart, liver and kidneys favour fat metabolism, even in glycolytic (non-ketogenic) dieters [5].

The authors took this to be further evidence that the mice were in a state of energy deprivation, even though by all accounts they appeared to be using substantially more energy. They even previously mentioned this in connection with the reduced fatness:

"[Germ-free] mice exhibit increased locomotor activity. Therefore, the increased food consumption and decreased body fat of germ-free mice may simply be due to increased energy expenditure." — from Donohoe-2011

However, it is a mistake to assume that lower NADH/NAD+ ratios and ATP levels per mitochondrion corresponds to less cellular energy. In fact, it is likely to be the opposite.

mitochondrial energetics is the commonality

There are three other conditions we know of that reduce the NADH/NAD+ ratio: calorie restriction [6], ketogenic diets [7], and the diabetes drug metformin [8]. Ketogenic diets share mechanisms with caloric restriction. Indeed, it seems likely that benefits of calorie restriction come from the activation of ketone bodies [9].

When you use fat and ketones for fuel instead of glucose, you produce fewer free radicals through reducing the NADH/NAD+ ratio [6]. This is probably the main mechanism by which it achieves the neuroprotection we mentioned in a recent post. Similarly, the reduction of the NADH/NAD+ ratio is probably one of the mechanisms by which calorie restriction can increase lifespan [5].

Calorie restriction also preserves ATP production, but it does this by increasing the number of mitochondria to match or exceed the lower ATP yield per mitochondrion [10], [11]. There is preliminary evidence that a ketogenic diet also increases mitochondrial number [12], [13]. So the idea of Donohoe et al. — that total ATP production is compromised because the NADH/NAD+ ratio in the individual mitochondrion has lowered ATP output per mitochondrion — seems unwarranted. Instead, there is likely to be a compensatory increase in mitochondrial number. That would be consistent with the fact that the mice appear to have more energy, not less. Ketogenic dieters have also been measured to have more energy expenditure than low-fat dieters [14].

So, a reduction of NADH/NAD+ ratio is associated with health benefits, and proposed longevity mechanisms. As you might now suspect, previous studies have shown that germ-free animals have increased lifespans [15]. (They also show decreased anxiety [16] and increased bone mass [17]. Once again, to us this sounds like a better kind of mouse to be!)

fiber-free for better health?

Given this observation — that some of the benefits of ketogenic diets are present in mice that don't have gut bacteria which process dietary fiber, even though the mice are not in ketosis — it raises the following questions:

1. Could the starvation of gut bacteria be a part of the mechanism of the benefits of ketogenic diets?
2. Since butyrate restores the mitochondrial working of the cells to be like the conventional controls (which, from our perspective, is a worse physiological state), could fiber be actually counter-productive to a ketogenic diet?
3. In analogy to the way the putative benefits of fiber may simply be that they displace refined carbohydrates in the diet, could the reason probiotics can lead to improved health be not because they are beneficial, but because they push out more harmful strains [18]?
4. Could a diet free of carb and fiber (i.e. one extremely low in plants) have benefits independent of its tendency to be ketogenic?

We don't have enough evidence to settle these questions, but they are interesting hypotheses that come directly from the results of this study.

in sum

• Contrary to the conclusions of the authors and Wikipedia editors that butyrate is necessary for cell energy, we interpret the results as showing improved cellular energy in the absence of butyrate.
• We now have another source for making hypotheses about potentially important cellular metabolism. Before we learned about germ-free mice, we could already use mechanisms discovered from caloric restriction and compare them to mechanisms of ketogenic diets. Now we can compare and contrast mechanisms from caloric restriction, ketogenic diets, and germ-free animals.
• This raises some interesting (perhaps even provocative) questions about the health effects of dietary fiber and gut flora on human health.

notes

[1]

Evidence type: review The hot air and cold facts of dietary fibre. Carla S Coffin, MD FRCPC and Eldon A Shaffer, MD FRCPC Can J Gastroenterol. 2006 April; 20(4): 255–256. (emphasis ours) "A recent pooled analysis of 13 prospective cohort studies (6) found that dietary fibre was not associated with a reduced risk of colorectal cancer after adjusting for other dietary risk factors. The Cochrane collaboration (7) systematically reviewed five studies of over 4000 subjects for the effect of dietary fibre on the incidence or recurrence of colorectal adenomas and incidence of colorectal cancer over a two-to four-year period. The population included all subjects that had adenomatous polyps but no history of colorectal cancer or a documented ‘clean colon’ at baseline with follow-up colonoscopy. Study interventions included soluble and insoluble dietary fibre or a comprehensive dietary intervention with high fibre whole food sources. The combined data showed no outcome difference between the intervention and control groups in the number of subjects with at least one adenoma or a new diagnosis of colorectal cancer. The Cochrane reviewers (7) concluded that there was no evidence from randomized controlled trials to suggest that increased dietary fibre intake would reduce the incidence or recurrence of adenomatous polyps. "Widespread popular media advertisements have purported the benefits of soluble fibre in lowering the risk of atherosclerotic coronary artery disease, mainly by modifying the main coronary artery disease risk factors (ie, dyslipidemia, diabetes and obesity). As for diabetes, high fibre diets slow the postprandial rise in blood glucose and thus, improve glycemic control (8). In dyslipidemic patients, pundits have proposed that psyllium lowers serum cholesterol by binding bile acids in the intestinal lumen resulting in decreased absorption and increased fecal excretion. The ensuing bile acid depletion increases hepatic demand for the de novo synthesis of bile acids from cholesterol. Investigating this mechanism, Van Rosendaal et al (9) found that fibre administration had no effect and certainly did not lower serum cholesterol. Similarly, an earlier study (10) comparing the effect of wheat bran on serum cholesterol of hyperlipidemic and normolipidemic controls showed no change in total cholesterol or ratio of low density lipoprotein to high density lipoprotein cholesterol. Another trial (11) of intensive dietary advice regarding fat, cereal fibre and fish intake on diet and mortality of men with a recent history of myocardial infarction did not find any substantial long-term benefit. The authors admitted to limitations of dietary data in the study (ie, only short-term period of advice and limited number of questions), but there was no evidence to guide decisions about value of dietary advice to increase fish or cereal fibre by people with coronary disease. We await the results of three Cochrane protocols undertaken to review the evidence of dietary fibre in fruits and vegetables, wholegrain cereals or high-fat, low fibre dietary intervention in the prevention of coronary heart disease (12–14). Any conclusions regarding the effectiveness of fibre for the prevention of heart disease appear premature." […] "In one of the first randomized, placebo-controlled trials of the role of bran in patients with [diverticular disease] (17), the authors concluded that dietary fibre supplements do nothing more than relieve constipation, and the impression that fibre helps [diverticular disease] is “simply a manifestation of western civilization’s obsession with the need for frequent defecation”. Recent systematic reviews (18,19) of the role of dietary fibre and [diverticular disease] (both asymptomatic diverticulosis and symptomatic diverticulitis) conclude that most of the positive evidence of the effects of fibre supplementation in treating or preventing disease is from retrospective analyses with inherent limitations and high risk of bias." […] "Systematic reviews have shown that the treatment of IBS patients with fibre is controversial. One recent meta-analysis of 17 randomized controlled trials (20) quantified the effectiveness of different types of fibre. The reviewers found that fibre was only marginally effective in terms of global symptom improvement or constipation and there was no effect in IBS related abdominal pain. Fibre has a role in treating constipation but its value for IBS, pain and diarrhea is controversial. Any effectivenss of fibre in the long-term management of IBS remains questionable. Clinically, bran is no better than placebo in the relief of the overall symptoms of IBS, and is possibly worse than a normal diet for some symptoms."

[2]

Evidence type: review of randomised controlled trials in humans. Systematic review of randomized controlled trials of low-carbohydrate vs. low-fat/low-calorie diets in the management of obesity and its comorbidities. Hession M, Rolland C, Kulkarni U, Wise A, Broom J. Obes Rev. 2009 Jan;10(1):36-50. doi: 10.1111/j.1467-789X.2008.00518.x. Epub 2008 Aug 11. "Abstract There are few studies comparing the effects of low-carbohydrate/high-protein diets with low-fat/high-carbohydrate diets for obesity and cardiovascular disease risk. This systematic review focuses on randomized controlled trials of low-carbohydrate diets compared with low-fat/low-calorie diets. Studies conducted in adult populations with mean or median body mass index of > or =28 kg m(-2) were included. Thirteen electronic databases were searched and randomized controlled trials from January 2000 to March 2007 were evaluated. Trials were included if they lasted at least 6 months and assessed the weight-loss effects of low-carbohydrate diets against low-fat/low-calorie diets. For each study, data were abstracted and checked by two researchers prior to electronic data entry. The computer program Review Manager 4.2.2 was used for the data analysis. Thirteen articles met the inclusion criteria. There were significant differences between the groups for weight, high-density lipoprotein cholesterol, triacylglycerols and systolic blood pressure, favouring the low-carbohydrate diet. There was a higher attrition rate in the low-fat compared with the low-carbohydrate groups suggesting a patient preference for a low-carbohydrate/high-protein approach as opposed to the Public Health preference of a low-fat/high-carbohydrate diet. Evidence from this systematic review demonstrates that low-carbohydrate/high-protein diets are more effective at 6 months and are as effective, if not more, as low-fat diets in reducing weight and cardiovascular disease risk up to 1 year. More evidence and longer-term studies are needed to assess the long-term cardiovascular benefits from the weight loss achieved using these diets."

[3]

Evidence type: review of randomised controlled trials in humans. Although this analysis is not peer-reveiwed, it is thorough, appears to be accurate, and does not omit any counter-evidence as far as we are aware. 23 Studies on Low-Carb and Low-Fat Diets – Time to Retire The Fad Kris Gunnars October 15, 2013 Authority Nutrition (emphasis ours) "In this article, I have analyzed the data from 23 of these studies comparing low-carb and low-fat diets. "All of the studies are randomized controlled trials, the gold standard of science. All are published in respected, peer-reviewed journals. … "The majority of studies achieved statistically significant differences in weight loss (always in favor of low-carb). There are several other factors that are worth noting: The low-carb groups often lost 2-3 times as much weight as the low-fat groups. In a few instances there was no significant difference. In most cases, calories were restricted in the low-fat groups, while the low-carb groups could eat as much as they wanted. When both groups restricted calories, the low-carb dieters still lost more weight (7, 13, 19), although it was not always significant (8, 18, 20). There was only one study where the low-fat group lost more weight (23) although the difference was small (0.5 kg – 1.1 lb) and not statistically significant. In several of the studies, weight loss was greatest in the beginning. Then people start regaining the weight over time as they abandon the diet. When the researchers looked at abdominal fat (the unhealthy visceral fat) directly, low-carb diets had a clear advantage (5, 7, 19).

[4]

Evidence type: review of experiments Hormonal control of ketogenesis. Biochemical considerations. McGarry JD, Foster DW. Arch Intern Med. 1977 Apr;137(4):495-501. "Abstract A two-site, bihormonal concept for the control of ketone body production is proposed. Thus, ketosis is viewed as the result of increased mobilization of free fatty acids from adipose tissue (site 1) to the liver (site 2), coupled with simultaneous enhancement of the liver's capacity to convert these substrates into acetoacetic and beta-hydroxybutyric acids. The former event is believed to be triggered by a fall in plasma insulin levels while the latter is considered to be effected primarily by the concomitant glucagon excess characteristic of the ketotic state. Although the precise mechanism whereby elevation of the circulating [glucagon]:[insulin] ratio stimulates hepatic ketogenic potential is not known, activation of the carnitine acyltransferase reaction, the first step in the oxidation of fatty acids, is an essential feature. Two prerequisites for this metabolic adaptation in liver appear to be an elevation in its carnitine content and depletion of its glycogen stores. Despite present limitations the model (evolved mainly from rat studies) provides a framework for the description of various types of clinical ketosis in biochemical terms and may be useful for future studies."

[5]

Evidence type: authority Dynamic Adaptation of Nutrient Utilization in Humans. El Bacha, T., Luz, M. & Da Poian, A. (2010) Nature Education 3(9):8 "[M]any different cells do oxidize fatty acids for ATP production. Between meals, cardiac muscle cells meet 90% of their ATP demands by oxidizing fatty acids. Although these proportions may fall to about 60% depending on the nutritional status and the intensity of contractions, fatty acids may be considered the major fuel consumed by cardiac muscle. ... Other organs that use primarily fatty acid oxidation are the kidney and the liver."

[6]

Evidence type: controlled non-human animal experiments Calorie restriction extends yeast life span by lowering the level of NADH. Su-Ju Lin, Ethan Ford, Marcia Haigis, Greg Liszt, and Leonard Guarente. Genes Dev. 2004 January 1; 18(1): 12–16. "Our studies show that a switch to oxidative metabolism during CR increases the NAD/NADH ratio by decreasing NADH levels. NADH is a competitive inhibitor of Sir2, implying that a reduction in this dinucleotide activates Sir2 to extend the life span in CR. Indeed, overexpression of the NADH dehydrogenase specifically lowers NADH levels and extends the life span, providing strong support for this hypothesis. Regulation of the life span by NADH is also consistent with the earlier finding that electron transport is required for longevity during CR (Lin et al. 2002). The NAD/NADH ratio reflects the intracellular redox state and is a readout of metabolic activity. Our findings suggest that this ratio can serve a critical regulatory function, namely, the determination of the life span of yeast mother cells. It remains to be seen whether this ratio will serve related regulatory functions in higher organisms."

[7]

Evidence type: controlled non-human animal experiments Ketones inhibit mitochondrial production of reactive oxygen species production following glutamate excitotoxicity by increasing NADH oxidation. Marwan Maalouf, Patrick G. Sullivan, Laurie Davis, Do Young Kim, and Jong M. Rho Neuroscience. 2007 March 2; 145(1): 256–264. "[W]e demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD+/NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress."

[8]

Evidence type: controlled non-human animal experiment Metformin suppresses hepatic gluconeogenesis through induction of SIRT1 and GCN5 Paul W Caton, Nanda K Nayuni, Julius Kieswich, Noorafza Q Khan, Muhammed M Yaqoob and Roger Corder J Endocrinol April 1, 2010 205 97-106 (emphasis ours) "Results Metformin increases SIRT1 in db/db mice Systemic activation of SIRT1 with the activator SRT1720 is reported to lower blood glucose and improve insulin sensitivity in Zucker rats and diet-induced obese mice in part through inhibition of hepatic gluconeogenesis (Milne et al. 2007). Therefore, we investigated whether metformin inhibited gluconeogenesis through changes in hepatic SIRT1. Eight-week-old db/db or control (db/m) mice were administered metformin (250 mg/kg per day; 7 days). Levels of SIRT1 protein, activity and NAD+/NADH ratio were significantly increased in metformin-treated db/db mice compared with the controls and untreated db/db mice (Fig. 1A, C and D). Despite increased protein levels, Sirt1 mRNA levels were unchanged following metformin treatment (Fig. 1B). Levels of SIRT1 protein and activity as well as NAD+/NADH levels were unchanged between the control and untreated mice (Fig. 1A–C). Metformin had no effect on SIRT1 in control mice (data not shown). Furthermore, incubation of HepG2 cells with metformin (2 mM) also resulted in increased levels of SIRT1 protein and activity and NAD+/NADH ratio (Fig. 1E–G). This indicates that increasing SIRT1 protein and activity could be a key mechanism by which metformin inhibits gluconeogenic gene expression."

[9]

Evidence type: review of experiments The neuroprotective properties of calorie restriction, the ketogenic diet, and ketone bodies. Marwan A. Maalouf, Jong M. Rho, and Mark P. Mattson Brain Res Rev. 2009 March; 59(2): 293–315. "Calorie restriction and the ketogenic diet share two characteristics: reduced carbohydrate intake and a compensatory rise in ketone bodies. The neuroprotective effects of reduced carbohydrate per se are being investigated by several research groups (Mattson et al. 2003; Ingram et al. 2006). We have evaluated the possibility that ketone bodies might mediate the neuroprotective effects of calorie restriction and of the ketogenic diet. An expanding body of evidence indicates that ketone bodies are indeed neuroprotective and that the underlying mechanisms are similar to those associated with calorie restriction - specifically at the mitochondrial level."

[10]

Evidence type: review of clinical reports Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency. G. López-Lluch, N. Hunt, B. Jones, M. Zhu, H. Jamieson, S. Hilmer, M. V. Cascajo, J. Allard, D. K. Ingram, P. Navas, and R. de Cabo Proc Natl Acad Sci U S A. 2006 February 7; 103(6): 1768–1773. "[M]itochondria under CR conditions show less oxygen consumption, reduce membrane potential, and generate less reactive oxygen species than controls, but remarkably they are able to maintain their critical ATP production. In effect, CR can induce a peroxisome proliferation-activated receptor coactivator 1α-dependent increase in mitochondria capable of efficient and balanced bioenergetics to reduce oxidative stress and attenuate age-dependent endogenous oxidative damage."

[11]

Evidence type: review of controlled experiments The neuroprotective properties of calorie restriction, the ketogenic diet, and ketone bodies Marwan A. Maalouf, Jong M. Rho, and Mark P. Mattson Brain Res Rev. 2009 March; 59(2): 293–315. (emphasis ours) "Slowing of brain aging in calorie-restricted animals was originally believed to result from reduced metabolic activity and, hence, decreased production of reactive oxygen species, a natural byproduct of oxidative metabolism (Wolf 2006). Several studies revealed that calorie restriction was associated with energy conservation (Gonzales-Pacheco et al. 1993; Santos-Pintos et al. 2001) and that mitochondria isolated from calorie-restricted animals produced less ATP than those from controls fed ad libitum, a finding compatible with increased UCP activity (Sreekumar et al. 2002; Drew et al. 2003). However, separate investigations in rodents have suggested that, when adjusted for body weight, metabolic rate does not decrease with calorie restriction (Masoro et al. 1982; McCarter et al. 1985; Masoro 1993). More importantly, calorie restriction prevents the age-related decline in oxidative metabolism in muscle (Hepple et al. 2005; Baker et al. 2006). These data are supported by recent studies indicating that, in contrast to isolated mitochondria, ATP synthesis in intact myocytes and in vivo does not decrease following calorie restriction (Lopez-Lluch et al. 2006; Zangarelli et al. 2006). Additional support is provided by the finding that, in yeast, oxidative metabolism increases with calorie restriction (Lin et al. 2002). […] Although the effects of calorie restriction on ATP generation might appear to contradict those on uncoupling proteins, this discrepancy can be explained by the fact that calorie restriction also promotes mitochondrial biogenesis, thereby enhancing total metabolic output per cell while decreasing mitochondrial production of reactive oxygen species (Diano et al. 2003; Nisoli et al. 2005; Civitarese et al. 2007)."

[12]

Evidence type: in vitro non-human animal experiment Mitochondrial biogenesis in the anticonvulsant mechanism of the ketogenic diet. Bough KJ, Wetherington J, Hassel B, Pare JF, Gawryluk JW, Greene JG, Shaw R, Smith Y, Geiger JD, Dingledine RJ. Ann Neurol. 2006 Aug;60(2):223-35. "OBJECTIVE The full anticonvulsant effect of the ketogenic diet (KD) can require weeks to develop in rats, suggesting that altered gene expression is involved. The KD typically is used in pediatric epilepsies, but is effective also in adolescents and adults. Our goal was to use microarray and complementary technologies in adolescent rats to understand its anticonvulsant effect. METHODS Microarrays were used to define patterns of gene expression in the hippocampus of rats fed a KD or control diet for 3 weeks. Hippocampi from control- and KD-fed rats were also compared for the number of mitochondrial profiles in electron micrographs, the levels of selected energy metabolites and enzyme activities, and the effect of low glucose on synaptic transmission. RESULTS Most striking was a coordinated upregulation of all (n = 34) differentially regulated transcripts encoding energy metabolism enzymes and 39 of 42 transcripts encoding mitochondrial proteins, which was accompanied by an increased number of mitochondrial profiles, a higher phosphocreatine/creatine ratio, elevated glutamate levels, and decreased glycogen levels. Consistent with increased energy reserves, synaptic transmission in hippocampal slices from KD-fed animals was resistant to low glucose. CONCLUSION These data show that a calorie-restricted KD enhances brain metabolism. We propose an anticonvulsant mechanism of the KD involving mitochondrial biogenesis leading to enhanced alternative energy stores."

[13]

Evidence type: controlled non-human animal experiments Mitochondrial biogenesis and increased uncoupling protein 1 in brown adipose tissue of mice fed a ketone ester diet. Srivastava S, Kashiwaya Y, King MT, Baxa U, Tam J, Niu G, Chen X, Clarke K, Veech RL. FASEB J. 2012 June; 26(6): 2351–2362. (Emphasis ours) Abstract "We measured the effects of a diet in which d-β-hydroxybutyrate-(R)-1,3 butanediol monoester [ketone ester (KE)] replaced equicaloric amounts of carbohydrate on 8-wk-old male C57BL/6J mice. Diets contained equal amounts of fat, protein, and micronutrients. The KE group was fed ad libitum, whereas the control (Ctrl) mice were pair-fed to the KE group. Blood d-β-hydroxybutyrate levels in the KE group were 3-5 times those reported with high-fat ketogenic diets. Voluntary food intake was reduced dose dependently with the KE diet. Feeding the KE diet for up to 1 mo increased the number of mitochondria and doubled the electron transport chain proteins, uncoupling protein 1, and mitochondrial biogenesis-regulating proteins in the interscapular brown adipose tissue (IBAT). [18F]-Fluorodeoxyglucose uptake in IBAT of the KE group was twice that in IBAT of the Ctrl group. Plasma leptin levels of the KE group were more than 2-fold those of the Ctrl group and were associated with increased sympathetic nervous system activity to IBAT. The KE group exhibited 14% greater resting energy expenditure, but the total energy expenditure measured over a 24-h period or body weights was not different. The quantitative insulin-sensitivity check index was 73% higher in the KE group. These results identify KE as a potential antiobesity supplement."

[14]

Evidence type: randomised controlled clinical trial Effects of Dietary Composition on Energy Expenditure During Weight-Loss Maintenance Cara B. Ebbeling, PhD; Janis F. Swain, MS, RD; Henry A. Feldman, PhD; William W. Wong, PhD; David L. Hachey, PhD; Erica Garcia-Lago, BA; David S. Ludwig, MD, PhD JAMA. 2012;307(24):2627-2634. doi:10.1001/jama.2012.6607. "The results of our study challenge the notion that a calorie is a calorie from a metabolic perspective. During isocaloric feeding following weight loss, REE was 67 kcal/d higher with the very low-carbohydrate diet compared with the low-fat diet. TEE differed by approximately 300 kcal/d between these 2 diets, an effect corresponding with the amount of energy typically expended in 1 hour of moderate-intensity physical activity."

[15]

Evidence type: review of non-human animal experiments The gnotobiotic animal as a tool in the study of host microbial relationships. H A Gordon and L Pesti. Bacteriol Rev. 1971 December; 35(4): 390–429. "Two attempts have been made to construct life tables and to determine lesions at natural death in germ-free and conventional animals. One study (105) was conducted in genetically closely linked Swiss Webster mice and included over 300 germ-free and the same number of conventional controls which were introduced into the colony at the age of 12 months (to eliminate the effect of early losses). At natural death, the ages of the mice were (means and standard errors in days 19; females, are given): germ-free males, 723 681 i 12; conventional males, 480 i 10; females, 516 i 10. This pattern of survival rates seemed to continue throughout the course of the experi- ment. In the second study (335), approximately 50 germ-free and the same number of conven- tional ICR mice were introduced into the colony after weaning. At natural death, the age of the mice was (using the same mode of expression): germ-free males, 556 i 43; females, 535 + 46; 41; females, 547 ± conventional males, 536 45. In the first trimester of life, the survival rate was essentially the same in the germ-free and conventional control groups. In the middle third, the germ-free mice displayed an increased survival rate (e.g., 40% cumulative mortality was reached for the combined group of germ-free males and females only at the age of approximately 580 days, whereas for the conventional controls this value was approximately 410 days). Increased mortality of the germ-free group at more advanced age resulted in the similarity of mean ages between the opposing animal groups when all animals participating in the study were considered."

[16]

Evidence type: controlled non-human animal experiment Normal gut microbiota modulates brain development and behavior. Rochellys Diaz Heijtz, Shugui Wang, Farhana Anuar, Yu Qian, Britta Björkholm, Annika Samuelsson, Martin L. Hibberd, Hans Forssberg, and Sven Pettersson Proc Natl Acad Sci U S A. 2011 February 15; 108(7): 3047–3052. "Microbial colonization of mammals is an evolution-driven process that modulate host physiology, many of which are associated with immunity and nutrient intake. Here, we report that colonization by gut microbiota impacts mammalian brain development and subsequent adult behavior. Using measures of motor activity and anxiety-like behavior, we demonstrate that germ free (GF) mice display increased motor activity and reduced anxiety, compared with specific pathogen free (SPF) mice with a normal gut microbiota. This behavioral phenotype is associated with altered expression of genes known to be involved in second messenger pathways and synaptic long-term potentiation in brain regions implicated in motor control and anxiety-like behavior. GF mice exposed to gut microbiota early in life display similar characteristics as SPF mice, including reduced expression of PSD-95 and synaptophysin in the striatum. Hence, our results suggest that the microbial colonization process initiates signaling mechanisms that affect neuronal circuits involved in motor control and anxiety behavior."

[17]

Evidence type: controlled non-human animal experiment The gut microbiota regulates bone mass in mice. Sjögren K, Engdahl C, Henning P, Lerner UH, Tremaroli V, Lagerquist MK, Bäckhed F, Ohlsson C. J Bone Miner Res. 2012 Jun;27(6):1357-67. doi: 10.1002/jbmr.1588. "Abstract The gut microbiota modulates host metabolism and development of immune status. Here we show that the gut microbiota is also a major regulator of bone mass in mice. Germ-free (GF) mice exhibit increased bone mass associated with reduced number of osteoclasts per bone surface compared with conventionally raised (CONV-R) mice. Colonization of GF mice with a normal gut microbiota normalizes bone mass. Furthermore, GF mice have decreased frequency of CD4(+) T cells and CD11b(+) /GR 1 osteoclast precursor cells in bone marrow, which could be normalized by colonization. GF mice exhibited reduced expression of inflammatory cytokines in bone and bone marrow compared with CONV-R mice. In summary, the gut microbiota regulates bone mass in mice, and we provide evidence for a mechanism involving altered immune status in bone and thereby affected osteoclast-mediated bone resorption. Further studies are required to evaluate the gut microbiota as a novel therapeutic target for osteoporosis."

[18]

Evidence type: review of experiments and hypotheses Probiotics and their fermented food products are beneficial for health. Parvez S, Malik KA, Ah Kang S, Kim HY. J Appl Microbiol. 2006 Jun;100(6):1171-85. (emphasis ours) "Abstract Probiotics are usually defined as microbial food supplements with beneficial effects on the consumers. Most probiotics fall into the group of organisms' known as lactic acid-producing bacteria and are normally consumed in the form of yogurt, fermented milks or other fermented foods. Some of the beneficial effect of lactic acid bacteria consumption include: (i) improving intestinal tract health; (ii) enhancing the immune system, synthesizing and enhancing the bioavailability of nutrients; (iii) reducing symptoms of lactose intolerance, decreasing the prevalence of allergy in susceptible individuals; and (iv) reducing risk of certain cancers. The mechanisms by which probiotics exert their effects are largely unknown, but may involve modifying gut pH, antagonizing pathogens through production of antimicrobial compounds, competing for pathogen binding and receptor sites as well as for available nutrients and growth factors, stimulating immunomodulatory cells, and producing lactase. Selection criteria, efficacy, food and supplement sources and safety issues around probiotics are reviewed. Recent scientific investigation has supported the important role of probiotics as a part of a healthy diet for human as well as for animals and may be an avenue to provide a safe, cost effective, and 'natural' approach that adds a barrier against microbial infection. This paper presents a review of probiotics in health maintenance and disease prevention."

The Ketogenic Diet Reverses Indicators of Heart Disease

Cardiovascular disease (CVD) is the leading cause of death worldwide ¹. Because of its prevalence and life-threatening nature, and because it appears that a keto diet is likely to reverse it, we consider it one of the most important conditions to discuss here.

In our last post, we argued that CVD, being a disease strongly associated with metabolic syndrome, is likely to be best treated with a ketogenic diet. In this post we will present more evidence that ketogenic diets do improve heart disease risk factors.

Unfortunately, there is much confusion and misinformation about the impact of nutrition on CVD among scientists and non-scientists alike. Not only does a high fat, keto diet not worsen heart disease risk — as would commonly be assumed — it actually improves it. This confusion about dietary fat is probably the reason that we do not yet have clinical trials directly testing the effects of ketogenic diets on CVD outcomes.

However, we already have many trials of ketogenic diets that measured known CVD risk factors, especially cholesterol profiles. It turns out that these trials show a powerful heart disease risk reduction in those following a ketogenic diet. It is powerful both in absolute terms, and in comparison with low-fat diets, which tend to improve some weakly predictive factors while worsening stronger predictors.

As such, a high-fat ketogenic diet is currently the best known non-drug intervention for heart disease, as defined by mainstream measures of risk. It is arguably better than drug interventions, too.

In brief:

• Total cholesterol and LDL cholesterol are only weak predictors of CVD.
• Triglycerides, HDL, LDL particle size, and the HDL-to-triglyceride ratio are much stronger predictors of CVD.
• Keto diets improve triglyceride levels, HDL, and LDL particle size — precisely those measures that strongly indicate risk.

Total cholesterol and LDL cholesterol are only weakly associated with CVD

The connection between blood cholesterol levels and the development of heart disease began to be explored in the last century. Over the last several decades, our understanding of the predictive power of various blood lipids has gone through many refinements as our ability to measure finer and finer detail has advanced.

In the early years, it appeared that high levels of total cholesterol carried some risk of heart disease in many cases. However, it is now well established that total cholesterol by itself is a weak predictor ^{2, 3, 4}.

The reason is quite simple. The different subtypes of cholesterol work together in an intricately balanced system. There is a wide range of total cholesterol levels that are perfectly healthy, so long as the proportions of the subtypes are healthy ones. By the same token, a given level of total cholesterol, even if it is perfectly normal, could be pathological when examined by subtype. Strong evidence from recent decades suggests that the best known blood lipid measures for predicting future risk of CVD are HDL, triglycerides, and related ratios (see below).

Similarly, while LDL cholesterol is probably important, it appears that it does not have good predictive power when looking at its magnitude alone ^{5, 6, 7, 8}.

One reason for this is that like total cholesterol, LDL is not uniform. Just as we distinguish between HDL and LDL, the so-called “good” and “bad” cholesterol, LDL itself is now known to have two important subtypes with opposite risk implications. Having more large, light LDL particles (also called Pattern A), does not indicate high CVD risk, but having more small, dense particles (Pattern B) does ^{9, 10, 11, 12, 13}. Therefore high LDL by itself is not necessarily indicative of CVD.

Low HDL cholesterol is strongly associated with CVD

Having high blood levels of HDL is now widely recognized as predicting lower levels of heart disease. The proportion of total cholesterol that is HDL cholesterol is a particularly strong predictor. In 2007, a meta-analysis was published in the Lancet that examined information from 61 prospective observational studies, consisting of almost 900,000 adults. Information about HDL was available for about 150,000 of them, among whom there were 5000 vascular deaths. According to the authors, "the ratio of total to HDL cholesterol is a substantially more informative predictor of IHD mortality than are total cholesterol, HDL cholesterol, or non-HDL cholesterol." ¹⁴

This is consistent with many other studies, for example this very recent analysis from the COURAGE trial ¹⁵.

High triglycerides are strongly associated with CVD

There has been drawn out controversy in the medical community as to the relationship of triglyceride levels to CVD. There are two parts to the controversy: whether or not triglycerides are an independent predictor of CVD, and whether or not triglycerides play a causative role in CVD.

In both cases, however, it doesn't matter in which way the controversy is resolved! Whether or not triglycerides independently predict CVD (and there is at least some evidence that they do), and whether or not they cause CVD, there is no controversy about whether they predict CVD. The association between triglyceride levels and CVD still holds and is strongly predictive ^{16, 17, 18}. In fact it is so predictive that those who argue that triglyceride levels are not an independent risk factor, call it instead a “biomarker” for CVD ¹⁹. In other words, seeing high triglycerides is tantamount to seeing the progression of heart disease.

HDL-to-Triglycerides Ratio: compounding evidence

Triglycerides and HDL levels statistically interact. That means it is a mistake to treat one as redundant with respect to the other. If you do, you will miss the fact that the effect of one on your outcome of interest changes depending on the value of the other. Despite the fact that most heart disease researchers who study risk factors have not used methods tuned to find interactions between triglycerides and HDL, many studies have at least measured both. This has allowed others to do the appropriate analysis. When triglycerides and HDL have been examined with respect to each other, that is, when the effect of triglycerides is measured under the condition of low HDL, or when the effect of HDL is measured under the condition of high triglycerides, this combination of factors turns out to be even more indicative of CVD ^{20, 21, 22, 23}.

One of the most interesting aspects of this finding from our perspective, is that the ratio of triglyceride levels to HDL is considered to be a surrogate marker of insulin resistance (See The Ketogenic Diet as a Treatment for Metabolic Syndrome.) In other words, the best lipid predictors of CVD are also those that indicate insulin resistance.

Ketogenic Diets improve risk factors for CVD

There is now ample evidence that a low carbohydrate, ketogenic diet improves lipid profiles, particularly with respect to the risk factors outlined above: triglycerides, HDL, and their ratio ^{24, 25, 26, 27, 28, 29, 30, 31}.

Although a ketogenic diet typically raises LDL levels, which has been traditionally seen as a risk factor, it has also been shown to improve LDL particle size. In other words, although the absolute amount of LDL goes up, it is the "good" LDL that goes up, whereas the "bad" LDL goes down ^{31, 32}. This is hardly surprising, since LDL particle size is also strongly predicted by triglycerides ^{33, 34, 35}.

Although there have not yet been intervention studies testing the effect of a ketogenic diet on the rate of actual CVD incidents (e.g. heart attacks), the evidence about lipid profiles is strong enough to make ketogenic diets more likely to reduce heart disease than any other known intervention.

Summary:

• Current medical practice uses blood lipid measurements to assess the risk of heart disease.
• Despite the continuing tradition of measuring total cholesterol and LDL, we have known for decades that triglycerides, HDL, and the ratio of the two, are much better predictors of heart disease. LDL particle size is also considered strongly predictive.
• A ketogenic diet has a very favourable impact on these risk factors, and thus should be considered the diet of choice for those at risk of CVD.

In their 2011 paper, "Low-carbohydrate diet review: shifting the paradigm", Hite et al. display the following graph (VLCKD stands for Very Low Carbohydrate Ketogenic Diet, and LFD for Low Fat Diet) ³⁶ based on data from ³¹:

It makes an excellent visualization of the factors at stake, and how powerful a ketogenic diet is. It also shows quite clearly that not only is restricting carbohydrate more effective for this purpose than a low fat diet, but that a low fat diet is detrimental for some important risk factors — apolipoprotein ratios, LDL particle size, and HDL — but a low carb diet is not. The ketogenic diet resulted in a significant improvement in every measure.

References:

¹ Evidence type: observational
World Health Organization Fact sheet N°317: Cardiovascular diseases (CVDs) September 2011

• CVDs are the number one cause of death globally: more people die annually from CVDs than from any other cause.
• An estimated 17.3 million people died from CVDs in 2008, representing 30% of all global deaths. Of these deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke.
• Low- and middle-income countries are disproportionally affected: over 80% of CVD deaths take place in low- and middle-income countries and occur almost equally in men and women.
• By 2030, almost 23.6 million people will die from CVDs, mainly from heart disease and stroke. These are projected to remain the single leading causes of death.

² Evidence type: observational
Role of lipid and lipoprotein profiles in risk assessment and therapy. Ballantyne CM, Hoogeveen RC. Am Heart J. 2003 Aug;146(2):227-33.

Despite a strong and consistent association within populations, elevated TC [(total cholesterol)] alone is not a useful test to discriminate between individuals who will have CHD [(coronary heart disease)] events and those who will not.

³ Evidence type: observational
Relation of serum lipoprotein cholesterol levels to presence and severity of angiographic coronary artery disease. Philip A. Romm, MD, Curtis E. Green, MD, Kathleen Reagan, MD, Charles E. Rackley, MD. The American Journal of Cardiology Volume 67, Issue 6, 1 March 1991, Pages 479–483

Most CAD [(coronary artery disease)] occurs in persons who have only mild or moderate elevations in cholesterol levels. Total cholesterol level alone is a poor predictor of CAD, particularly in older patients in whom the major lipid risk factor is the HDL cholesterol level.

⁴ Evidence type: observational
Lipids, risk factors and ischaemic heart disease. Atherosclerosis. 1996 Jul;124 Suppl:S1-9. Castelli WP.

Those individuals who had TC [(total cholesterol)] levels of 150-300 mg/dl (3.9-7.8 mmol/1) fell into the overlapping area (Fig. 1), demonstrating that 90% of the TC levels measured were useless (by themselves) for predicting risk of CHD [(coronary heart disease)] in a general population. Indeed, twice as many individuals who had a lifetime TC level of less than 200 mg/dl (5.2 mmol/1) had CHD compared with those who had a TC level greater than 300 mg/dl (7.8 mmol/l) (Fig. 1).

⁵ Evidence type: observational
Range of Serum Cholesterol Values in the Population Developing Coronary Artery Disease. William B. Kannel, MD, MPH. The American Journal of Cardiology, Volume 76, Issue 9, Supplement 1, 28 September 1995, Pages 69C–77C

The ranges of serum cholesterol and LDL cholesterol levels varied widely both in the general population and in patients who had already manifested CAD (Figures 1 and 2). Because of the extensive overlap between levels, it was impossible to differentiate the patients with CAD from the control subjects.

⁶ Evidence type: observational
Lipoprotein cholesterol, apolipoprotein A-I and B and lipoprotein (a) abnormalities in men with premature coronary artery disease. Jacques Genest Jr., MD,FACC, Judith R. McNamara, MT, Jose M. Ordovas, PhD, Jennifer L. Jenner, BSc, Steven R. Silberman, PhD, Keaven M. Anderson, PhD, Peter W.F. Wilson, MD, Deeb N. Salem, MD, FACC, Ernst J. Schaefer, MD. Journal of the American College of Cardiology Volume 19, Issue 4, 15 March 1992, Pages 792–802.

Our data suggest that total and LDL cholesterol may not be the best discriminants for the presence of coronary artery disease despite the strong association between elevated cholesterol and the development of coronary artery disease in cross-sectional population studies and prospective epidemiologic studies.

⁷ Evidence type: observational
Apolipoprotein B and apolipoprotein A-I: risk indicators of coronary heart disease and targets for lipid-modifying therapy. Walldius, G. and Jungner, I. (2004), Journal of Internal Medicine, 255: 188–205. doi: 10.1046/j.1365-2796.2003.01276.x

(Emphasis ours.)

For over three decades it has been recognized that a high level of total blood cholesterol, particularly in the form of LDL cholesterol (LDL-C), is a major risk factor for developing coronary heart disease (CHD) [1–4]. However, as more recent research has expanded our understanding of lipoprotein function and metabolism, it has become apparent that LDL-C is not the only lipoprotein species involved in atherogenesis. A considerable proportion of patients with atherosclerotic disease have levels of LDL-C and total cholesterol (TC) within the recommended range [5, 6], and some patients who achieve significant LDL-C reduction with lipid-lowering therapy still develop CHD [7].

Other lipid parameters are also associated with elevated cardiovascular risk, and it has been suggested that LDL-C and TC may not be the best discriminants for the presence of coronary artery disease (CAD) [5].

⁸ Evidence type: observational
Plasma Lipoprotein Levels as Predictors of Cardiovascular Death in Women. Katherine Miller Bass, MD, MHS; Craig J. Newschaffer, MS; Michael J. Klag, MD, MPH; Trudy L. Bush, PhD, MHS. Arch Intern Med. 1993;153(19):2209-2216.

Using a sample of 1405 women aged 50 to 69 years from the Lipid Research Clinics' Follow-up Study, age-adjusted CVD death rates and summary relative risk (RR) estimates by categories of lipid and lipoprotein levels were calculated. Multivariate analysis was performed to provide RR estimates adjusted for other CVD risk factors.

RESULTS: Average follow-up was 14 years. High-density lipoprotein and triglyceride levels were strong predictors of CVD death in age-adjusted and multivariate analyses. Low-density lipoprotein and total cholesterol levels were poorer predictors of CVD mortality. After adjustment for other CVD risk factors, HDL levels less than 1.30 mmol/L (50 mg/dL) were strongly associated with cardiovascular mortality (RR = 1.74; 95% confidence interval [CI], 1.10 to 2.75). Triglyceride levels were associated with increased CVD mortality at levels of 2.25 to 4.49 mmol/L (200 to 399 mg/dL) (RR = 1.65; 95% CI, 0.99 to 2.77) and 4.50 mmol/L (400 mg/dL) or greater (RR = 3.44; 95% CI, 1.65 to 7.20). At total cholesterol levels of 5.20 mmol/L (200 mg/dL) or greater and at all levels of LDL and triglycerides, women with HDL levels of less than 1.30 mmol/L (< 50 mg/dL) had CVD death rates that were higher than those of women with HDL levels of 1.30 mmol/L (50 mg/dL) or greater.

⁹ Evidence type: plausible mechanism and observational review
Particle size: the key to the atherogenic lipoprotein? Rajman I, Maxwell S, Cramb R, Kendall M. QJM. 1994 Dec;87(12):709-20.

Using different analytical methods, up to 12 low-density lipoprotein (LDL) subfractions can be separated. LDL particle size decreases with increasing density. Smaller, denser LDL particles seem more atherogenic than the larger, lighter particles, based on the experimental findings that smaller LDL particles are more susceptible for oxidation in vitro, have lower binding affinity for the LDL receptors and lower catabolic rate, have a higher concentration of polyunsaturated fatty acids, and potentially interact more easily with proteoglycans of the arterial wall. Clinical studies have shown that a smaller LDL subfraction profile is associated with an increased risk of heart disease, even when total cholesterol level is only slightly raised. There is a strong inverse association between LDL particle size and triglyceride concentrations. Although LDL particle size is genetically determined, its phenotypic expression may also be affected by environmental factors such as drugs, diet, obesity, exercise or disease. Factors that shift the LDL subfractions profile towards larger particles may reduce the risk of heart disease.

¹⁰ Evidence type: nested case-control study
Association of Small Low-Density Lipoprotein Particles With the Incidence of Coronary Artery Disease in Men and Women. Christopher D. Gardner, PhD; Stephen P. Fortmann, MD; Ronald M. Krauss, MD JAMA. 1996;276(11):875-881. doi:10.1001/jama.1996.03540110029028.

Incident CAD cases were identified through FCP surveillance between 1979 and 1992. Controls were matched by sex, 5-year age groups, survey time point, ethnicity, and FCP treatment condition. The sample included 124 matched pairs: 90 pairs of men and 34 pairs of women.

...

LDL size was smaller among CAD cases than controls (mean ±SD) (26.17±1.00nm vs 26.68±0.90nm;P<.001).The association was graded across control quintiles of LDL size. The significant case-control difference in LDL size was independent of levels of high-density lipoprotein cholesterol (HDL-C), non—HDL cholesterol (non-HDL-C), triglyceride, smoking, systolic blood pressure, and body mass index, but was not significant after adjusting for the ratio of total cholesterol (TC) to HDL-C (TC:HDL-C). Among all the physiological risk factors, LDL size was the best differentiator of CAD status in conditional logistic regression. However, when added to the physiological parameters above, the TC:HDL-C ratio was found to be a stronger independent predictor of CAD status.

¹¹ Evidence type: review
The small, dense LDL phenotype and the risk of coronary heart disease: epidemiology, patho-physiology and therapeutic aspects. Lamarche B, Lemieux I, Després JP. Diabetes Metab. 1999 Sep;25(3):199-211.

More than decade ago, several cross-sectional studies have reported differences in LDL particle size, density and composition between coronary heart disease (CHD) patients and healthy controls. Three recent prospective, nested case-control studies have since confirmed that the presence of small, dense LDL particles was associated with more than a three-fold increase in the risk of CHD. The small, dense LDL phenotype rarely occurs as an isolated disorder. It is most frequently accompanied by hypertriglyceridemia, reduced HDL cholesterol levels, abdominal obesity, insulin resistance and by a series of other metabolic alterations predictive of an impaired endothelial function and increased susceptibility to thrombosis.

²⁸ Evidence type: prospective
A prospective, population-based study of low density lipoprotein particle size as a risk factor for ischemic heart disease in men. Lamarche B, St-Pierre AC, Ruel IL, Cantin B, Dagenais GR, Després JP. Can J Cardiol. 2001 Aug;17(8):859-65.

Analyses were conducted in a cohort of 2057 men who were all initially free of IHD, and who were followed up over a five-year period, during which 108 first IHD events (myocardial infarction, angina or coronary death) were recorded. LDL particle size was measured by nondenaturing gradient gel electrophoresis.

RESULTS: Cox proportional hazards analysis indicated that the relationship between LDL particle size and the risk of future IHD events was not linear. Men with an LDL particle size less than 256.0 A had a significant 2.2-fold increase in the five-year rate of IHD (P<0.001) compared with men having an LDL particle size greater than 256.0 A. Multivariate and subgroup analyses indicated that small, dense LDL particles predicted the rate of IHD independent of LDL cholesterol, triglycerides, high density lipoprotein (HDL) cholesterol, apolipoprotein B and the total cholesterol to HDL cholesterol ratio. Finally, the magnitude of the increase in IHD risk attributed to lipid risk factors was modulated to a significant extent by variations in LDL particle size.

¹³ Evidence type: review
Small, dense low-density-lipoproteins and the metabolic syndrome. Rizzo M, Berneis K. Diabetes Metab Res Rev. 2007 Jan;23(1):14-20.

Small, dense low-density-lipoproteins (LDL) are associated with increased risk for cardiovascular diseases and diabetes mellitus and a reduction in LDL size has been reported in patients with coronary and non-coronary forms of atherosclerosis. LDL size has been accepted as an important predictor of cardiovascular events and progression of coronary artery disease as well as an emerging cardiovascular risk factor by the National Cholesterol Education Program Adult Treatment Panel III. Small, dense LDL, with elevated triglyceride levels and low HDL-cholesterol concentrations, constitute the 'atherogenic lipoprotein phenotype (ALP)', a form of atherogenic dyslipidemia that is a feature of type 2 diabetes and the metabolic syndrome.

¹⁴ Evidence type: meta-analysis of prospective studies
Blood cholesterol and vascular mortality by age, sex, and blood pressure: a meta-analysis of individual data from 61 prospective studies with 55,000 vascular deaths. Prospective Studies Collaboration, Lewington S, Whitlock G, Clarke R, Sherliker P, Emberson J, Halsey J, Qizilbash N, Peto R, Collins R. Lancet. 2007 Dec 1;370(9602):1829-39.

Of various simple indices involving HDL cholesterol, the ratio total/HDL cholesterol was the strongest predictor of IHD mortality (40% more informative than non-HDL cholesterol and more than twice as informative as total cholesterol). Total cholesterol was weakly positively related to ischaemic and total stroke mortality in early middle age (40-59 years), but this finding could be largely or wholly accounted for by the association of cholesterol with blood pressure. Moreover, a positive relation was seen only in middle age and only in those with below-average blood pressure; at older ages (70-89 years) and, particularly, for those with systolic blood pressure over about 145 mm Hg, total cholesterol was negatively related to haemorrhagic and total stroke mortality.

¹⁵ Evidence type: post-hoc analysis
Low Levels of High Density Lipoprotein Cholesterol and Increased Risk of Cardiovascular Events in Stable Ischemic Heart Disease Patients: A Post Hoc Analysis from the COURAGE Trial. Acharjee S, Boden WE, Hartigan PM, Teo KK, Maron DJ, Sedlis SP, Kostuk W, Spertus JA, Dada M, Chaitman BR, Mancini GB, Weintraub WS. J Am Coll Cardiol. 2013 Aug 8. pii: S0735-1097(13)03082-9. doi: 10.1016/j.jacc.2013.07.051. [Epub ahead of print]

Abstract:

OBJECTIVES: The aim of this study was to assess the independent effect of high-density lipoprotein cholesterol (HDL-C) level on cardiovascular risk in patients with stable ischemic heart disease (SIHD) while on optimal medical therapy (OMT).

BACKGROUND: While low HDL-C level is a powerful and independent predictor of cardiovascular risk, recent data suggest that this may not apply when low-density lipoprotein cholesterol (LDL-C) is reduced to optimal levels using intensive statin therapy.

METHODS: We performed a post hoc analysis in 2,193 men and women with stable ischemic heart disease (SIHD) from the COURAGE trial. The primary outcome measure was the composite of death from any cause or nonfatal myocardial infarction (MI). The independent association between HDL-C levels measured after 6 months on optimal medical therapy (OMT) and the rate of cardiovascular events after 4 years was assessed. Similar analyses were performed separately in subjects with LDL-C levels below 70 mg/dL (1.8 mmol/L).

RESULTS: In the overall population, the rate of death/MI was 33% lower in the highest HDL-C quartile as compared with the lowest quartile, with quartile of HDL-C being a significant, independent predictor of death/MI (P = 0.05), but with no interaction for LDL-C category (P=0.40). Among subjects with LDL-C levels < 70 mg/dL, those in the highest quintile of HDL-C had a 65% relative risk reduction in death or MI as compared to the lowest quintile, with HDL-C quintile demonstrating a significant, inverse predictive effect (P=0.02).

CONCLUSIONS: In this post hoc analysis, patients with SIHD continued to experience incremental cardiovascular risk associated with low HDL-C levels despite OMT during long-term follow-up. This relationship persisted and appeared more prominent even when LDL-C was reduced to optimal levels with intensive dyslipidemic therapy.

¹⁶ Evidence type: meta-analysis of prospective studies
Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies. Hokanson JE, Austin MA. J Cardiovasc Risk. 1996 Apr;3(2):213-9.

Seventeen studies were selected for the analysis based on published reports of population-based, prospective studies, including 46413 men and 10864 women. To insure comparability, only studies reporting the association between fasting triglyceride levels and incident cardiovascular endpoints were included. Using standard meta-analysis calculations, relative risks (RR) and 95% confidence intervals (CI) were calculated and standardized with respect to a 1 mmol/l increase in triglyceride. Multivariable-adjusted RRs were determined for the six studies in men and two studies in women that reported adjustments for HDL cholesterol.

RESULTS: For men and women, the univariate RRs for triglyceride were 1.32 (95% Cl 1.26-1.39) and 1.76 (95% Cl 1.50-2.07), respectively, indicating an approximately 30% increased risk in men and a 75% increase in women. Adjustment of HDL cholesterol and other risk factors attenuated these RRs to 1.14 (95% Cl 1.05-1.28) and 1.37 (95% Cl 1.13-1.66), respectively, which were still statistically significant values.

CONCLUSION: Based on combined data from prospective studies, triglyceride is a risk factor for cardiovascular disease for both men and women in the general population, independent of HDL cholesterol. These finding demonstrate the necessity for clinical trials to evaluate whether lowering plasma triglyceride decreases the risk of cardiovascular disease.

http://www.ncbi.nlm.nih.gov/pubmed/8782637

¹⁷ Evidence type: prospective cohort study
A prospective study of triglyceride level, low-density lipoprotein particle diameter, and risk of myocardial infarction. Stampfer MJ, Krauss RM, Ma J, Blanche PJ, Holl LG, Sacks FM, Hennekens CH. JAMA. 1996 Sep 18;276(11):882-8.

RESULTS: Cases (n=266) had a significantly smaller LDL diameter (mean [SD], 25.6 [0.9] nm) than did controls (n=308) matched on age and smoking (mean [SD], 25.9 [8] nm; P<.001). Cases also had higher median triglyceride levels (1.90 vs 1.49 mmol/L [168 vs 132 mg/dL]; P<.001). The LDL diameter had a high inverse correlation with triglyceride level (r=-0.71), and a high direct correlation with high-density lipoprotein cholesterol (HDL-C) level (r=0.60). We observed a significant multiplicative interaction between triglyceride and total cholesterol (TC) levels (P=.01). After simultaneous adjustment for lipids and a variety of coronary risk factors, LDL particle diameter was no longer a statistically significant risk indicator, with a relative risk (RR) of 1.09 (95% confidence interval [CI], 0.85-1.40) per 0.8-nm decrease. However, triglyceride level remained significant with an RR of 1.40 (95% CI, 1.10-1.77) per 1.13 mmol/L (100-mg/dL) increase. The association between triglyceride level and MI risk appeared linear across the distribution; men in the highest quintile had a risk about 2.5 times that of those in the lowest quintile. The TC level, but not HDL-C level, also remained significant, with an RR of 1.80 (95% CI, 1.44-2.26) per 1.03-mmol/L (40-mg/dL) increase.

CONCLUSIONS: These findings indicate that nonfasting triglyceride levels appear to be a strong and independent predictor of future risk of MI, particularly when the total cholesterol level is also elevated. In contrast, LDL particle diameter is associated with risk of MI, but not after adjustment for triglyceride level. Increased triglyceride level, small LDL particle diameter, and decreased HDL-C levels appear to reflect underlying metabolic perturbations with adverse consequences for risk of MI; elevated triglyceride levels may help identify high-risk individuals.

¹⁸ Evidence type: prospective study
The emergence of triglycerides as a significant independent risk factor in coronary artery disease. Assmann G, Schulte H, Funke H, von Eckardstein A. Eur Heart J. 1998 Oct;19 Suppl M:M8-14.

The Prospective Cardiovascular Münster (PROCAM) study involved 4849 middle-aged men who were followed up for 8 years to record the incidence of coronary heart disease (CHD) events according to the risk factors present at study entry. The study showed that fasting levels of triglycerides were an independent risk factor for CHD events, irrespective of serum levels of high density lipoprotein cholesterol (HDL-C) or low density lipoprotein cholesterol (LDL-C). Other independent predictors of CHD included serum levels of LDL-C and HDL-C, age, systolic blood pressure, cigarette smoking, diabetes mellitus, a family history of myocardial infarction and angina pectoris, but did not include total serum cholesterol levels. Individuals with an LDL-C/HDL-C ratio > 5 had a 19.2% chance of experiencing a CHD event in the next 8 years. Furthermore, if an LDL-C/HDL-C ratio > 5 was combined with hypertriglyceridaemia (> or = 2.3 mmol. l-1), the risk of CHD increased to 26.9%. The association between hypertriglyceridaemia and CHD events may be related to the presence of atherogenic, triglyceride-rich particles in plasma, such as LDL and very low density lipoproteins. High triglyceride levels may also predispose to thrombosis.

¹⁹ Evidence type: observation
The role of triglycerides in cardiovascular risk. Gandotra P, Miller M. Curr Cardiol Rep. 2008 Nov;10(6):505-11.

Triglycerides' role in coronary heart disease (CHD) risk assessment has long been debated. Although meta-analyses have suggested that triglycerides are an independent risk factor for CHD, a consensus has emerged that triglycerides more appropriately represent a biomarker of CHD risk rather than an independent risk factor. Ongoing studies will determine whether triglyceride lowering confers additional CHD benefit beyond that attained via low-density lipoprotein (LDL) cholesterol reduction. The American Diabetes Association presently recommends lowering elevated triglycerides as a secondary therapeutic target after LDL cholesterol, whereas other organizations, such as the National Cholesterol Education Program, recommend non-high-density lipoprotein cholesterol as the second priority after attaining the LDL cholesterol goal. However, reducing very high triglycerides (ie, > 500 mg/dL) remains a sufficiently high priority in affected individuals.

²⁰ Evidence type: analysis of observational results from a randomized controlled trial
The Triglyceride Issue RevisitedFindings From the Helsinki Heart Study Leena Tenkanen, PhD; Kati Pietilä; Vesa Manninen, MD; Matti Mänttäri, MD Arch Intern Med. 1994;154(23):2714-2720. doi:10.1001/archinte.1994.00420230107012.

Results: Triglycerides occupied a central role in the pattern of associations of the factors studied; in particular, the associations with HDL-C level, blood pressure, and blood glucose level were without threshold values. The prevalence of high triglyceride level plus low HDL-C level was strongly associated with blood pressure and blood glucose level, while the prevalence of low HDL-C level alone was not. Only the subgroup with both high triglyceride and low HDL-C levels showed a substantial CHD risk, while those with low HDL-C levels alone or high triglyceride levels alone showed a marginal risk.

Conclusions: Our results suggest that triglycerides play a central mediating role in the occurrence of several CHD risk factors, especially those related to the insulin resistance syndrome. Because of these interdependencies, the question of an independent effect of triglycerides is not relevant, and when assessing CHD risk, triglycerides should be considered jointly with HDL-C

²¹ Evidence type: prospective analysis
Relation of High TG–Low HDL Cholesterol and LDL Cholesterol to the Incidence of Ischemic Heart Disease: An 8-Year Follow-up in the Copenhagen Male Study. Jørgen Jeppesen, Hans Ole Hein, Poul Suadicani, Finn Gyntelberg. Arteriosclerosis, Thrombosis, and Vascular Biology. 1997; 17: 1114-1120

High triglyceride (TG) and low HDL cholesterol (HDL-C) is the characteristic dyslipidemia seen in insulin-resistant subjects. We examined the role of this dyslipidemia as a risk factor of ischemic heart disease (IHD) compared with that of high LDL cholesterol (LDL-C) in the Copenhagen Male Study. In total 2910 white men, aged 53 to 74 years, free of cardiovascular disease at baseline, were subdivided into four groups on the basis of fasting concentrations of serum TG, HDL-C, and LDL-C. “High TG–low HDL-C” was defined as belonging to both the highest third of TG and the lowest third of HDL-C; this group encompassed one fifth of the population. “High LDL-C” was defined as belonging to the highest fifth of LDL-C. A control group was defined as not belonging to either of these two groups. “Combined dyslipidemia” was defined as belonging to both dyslipidemic groups. Age-adjusted incidence of IHD during 8 years of follow-up was 11.4% in high TG–low HDL-C, 8.2% in high LDL-C, 6.6% in the control group, and 17.5% in combined dyslipidemia.

...

At both low and high levels of total cholesterol and LDL-C, the presence of high TG–low HDL-C approximately doubled the risk of IHD, and individuals with high TG–low HDL-C in the lowest fifth of LDL-C (≤3.6 mmol/L) had a similar risk of IHD to subjects without high TG–low HDL-C in the highest fifth of LDL-C (≥5.3 mmol/L). High TG–low HDL-C thus clearly identified a group at high risk of IHD, though they had LDL-C levels considered to be safe or borderline (<3.4 mmol/L).

²² Evidence type: observational
Fasting Triglycerides, High-Density Lipoprotein, and Risk of Myocardial Infarction. J. Michael Gaziano, MD, MPH; Charles H. Hennekens, MD, DrPH; Christopher J. O’Donnell, MD, MPH; Jan L. Breslow, MD; Julie E. Buring, ScD. Circulation. 1997; 96: 2520-2525 doi: 10.1161/​01.CIR.96.8.2520

We examined the interrelationships of fasting triglycerides, other lipid parameters, and nonlipid risk factors with risk of myocardial infarction among 340 cases and an equal number of age-, sex-, and community-matched control subjects. Cases were men or women of <76 years of age with no prior history of coronary disease who were discharged from one of six Boston area hospitals with the diagnosis of a confirmed myocardial infarction. In crude analyses, we observed a significant association of elevated fasting triglycerides with risk of myocardial infarction (relative risk [RR] in the highest compared with the lowest quartile=6.8; 95% confidence interval [CI]=3.8 to 12.1; P for trend <.001). Results were not materially altered after control for nonlipid coronary risk factors. As expected, the relationship was attenuated after adjustment for HDL but remained statistically significant (RR in the highest quartile=2.7; 95% confidence interval [CI]=1.4 to 5.5; P for trend=.016). Furthermore, the ratio of triglycerides to HDL was a strong predictor of myocardial infarction (RR in the highest compared with the lowest quartile=16.0; 95% CI=7.7 to 33.1; P for trend <.001).

²³ Evidence type: observational
High Ratio of Triglycerides to HDL-Cholesterol Predicts Extensive Coronary Disease. Protasio Lemos da Luz, Desiderio Favarato, Jose Rocha Faria-Neto Junior, Pedro Lemos, and Antonio Carlos Palandri Chagas. Clinics. 2008 August; 63(4): 427–432. doi: 10.1590/S1807-59322008000400003 PMCID: PMC2664115

High-risk patients (n = 374) submitted for coronary angiography had their lipid variables measured and coronary disease extent scored by the Friesinger index.

RESULTS: The subjects consisted of 220 males and 154 females, age 57.2 ± 11.1 years, with total cholesterol of 210± 50.3 mg/dL, triglycerides of 173.8 ± 169.8 mg/dL, HDL-cholesterol (HDL-c) of 40.1 ± 12.8 mg/dL, LDL-cholesterol (LDL-c) of 137.3 ± 46.2 mg/dL, TG/HDL-c of 5.1 ± 5.3, and a Friesinger index of 6.6 ± 4.7. The relationship between the extent of coronary disease (dichotomized by a Friesenger index of 5 and lipid levels (normal vs. abnormal) was statistically significant for the following: triglycerides, odds ratio of 2.02 (1.31-3.1; p = 0.0018); HDL-c, odds ratio of 2.21 (1.42-3.43; p = 0.0005); and TG/HDL-c, odds ratio of 2.01(1.30-3.09; p = 0.0018). However, the relationship was not significant between extent of coronary disease and total cholesterol [1.25 (0.82-1.91; p = 0.33)] or LDL-c [1.47 (0.96-2.25; p = 0.0842)]. The chi-square for linear trends for Friesinger > 4 and lipid quartiles was statistically significant for triglycerides (p = 0.0017), HDL-c (p = 0.0001), and TG/HDL-c (p = 0.0018), but not for total cholesterol (p = 0.393) or LDL-c (p = 0.0568). The multivariate analysis by logistic regression OR gave 1.3 ± 0.79 (p = .0001) for TG/HDL-c, 0.779 ± 0.074 (p = .0001) for HDL-c, and 1.234 ± 0.097 (p = 0.03) for LDL. Analysis of receiver operating characteristic curves showed that only TG/HDL-c and HDL-c were useful for detecting extensive coronary disease, with the former more strongly associated with disease.

CONCLUSIONS: Although some lipid variables were associated with the extent of coronary disease, the ratio of triglycerides to HDL-cholesterol showed the strongest association with extent.

²⁴ Evidence type: non-randomized experiment
A Ketogenic Diet Favorably Affects Serum Biomarkers for Cardiovascular Disease in Normal-Weight Men. Matthew J. Sharman, William J. Kraemer, Dawn M. Love, Neva G. Avery, Ana L. Gómez, Timothy P. Scheett, and Jeff S. Volek. J. Nutr. July 1, 2002 vol. 132 no. 7 1879-1885

The primary objective of this study was to examine how healthy normolipidemic, normal-weight men respond to a ketogenic diet in terms of fasting and postprandial CVD biomarkers. Ketogenic diets have been criticized on the grounds they jeopardize health (8); however, very few studies have directly evaluated the effects of a ketogenic diet on fasting and postprandial risk factors for CVD. Subjects consumed a diet that consisted of 8% carbohydrate (<50 g/d), 61% fat, and 30% protein. Adaptation to this ketogenic diet resulted in significant reductions in fasting TAG (−33%), postprandial lipemia after a fat-rich meal (−29%), and fasting insulin concentrations (−34%). There were significant increases in LDL particle size, and no change in the oxidative LDL concentrations. There was a significant increase in HDL cholesterol at wk 3 after the ketogenic diet. Collectively, the responses in serum lipids, insulin and lipid subclasses to the ketogenic diet were favorable in terms of overall CVD risk profile.

²⁵ Evidence type: review of experiments
Cardiovascular and hormonal aspects of very-low-carbohydrate ketogenic diets. Volek JS, Sharman MJ. Obes Res. 2004 Nov;12 Suppl 2:115S-23S.

Compared with low-fat diets, short-term VLCKDs [very low carb diets] consistently result in improvements in fat loss, fasting and postprandial triacylglycerols, high-density lipoprotein-cholesterol, the distribution of low-density lipoprotein-cholesterol subclasses, and insulin resistance.

²⁶ Evidence type: randomized controlled trial
Long-term effects of a ketogenic diet in obese patients. Dashti HM, Mathew TC, Hussein T, Asfar SK, Behbahani A, Khoursheed MA, Al-Sayer HM, Bo-Abbas YY, Al-Zaid NS. Exp Clin Cardiol. 2004 Fall;9(3):200-5.

The level of total cholesterol showed a significant decrease from week 1 to week 24 (Figure 3). The level of HDL cholesterol significantly increased (Figure 4), whereas LDL cholesterol levels significantly decreased with treatment (Figure 5). The level of triglycerides decreased significantly after 24 weeks of treatment. The initial level of triglycerides was 2.75±0.23 mmol/L, whereas at week 24, the level decreased to 1.09±0.08 mmol/L (Figure 6).

²⁷ Evidence type: randomized controlled trial
Very Low-Carbohydrate and Low-Fat Diets Affect Fasting Lipids and Postprandial Lipemia Differently in Overweight Men. Matthew J. Sharman, Ana L. Gómez, William J. Kraemer, and Jeff S. Volek. J. Nutr. April 1, 2004 vol. 134 no. 4 880-885

The primary purpose of this study was to compare the effects of a very low-carbohydrate and a low-fat diet on fasting blood lipids and postprandial lipemia in overweight men. In a balanced, randomized, crossover design, overweight men (n = 15; body fat >25%; BMI, 34 kg/m2) consumed 2 experimental diets for 2 consecutive 6-wk periods. One was a very low-carbohydrate (<10% energy as carbohydrate) diet and the other a low-fat (<30% energy as fat) diet. Blood was drawn from fasting subjects on separate days and an oral fat tolerance test was performed at baseline, after the very low-carbohydrate diet period, and after the low-fat diet period. Both diets had the same effect on serum total cholesterol, serum insulin, and homeostasis model analysis-insulin resistance (HOMA-IR). Neither diet affected serum HDL cholesterol (HDL-C) or oxidized LDL (oxLDL) concentrations. Serum LDL cholesterol (LDL-C) was reduced (P < 0.05) only by the low-fat diet (−18%). Fasting serum triacylglycerol (TAG), the TAG/HDL-C ratio, and glucose were significantly reduced only by the very low-carbohydrate diet (−44, −42, and −6%, respectively). Postprandial lipemia was significantly reduced when the men consumed both diets compared with baseline, but the reduction was significantly greater after intake of the very low-carbohydrate diet. Mean and peak LDL particle size increased only after the very low-carbohydrate diet. The short-term hypoenergetic low-fat diet was more effective at lowering serum LDL-C, but the very low-carbohydrate diet was more effective at improving characteristics of the metabolic syndrome as shown by a decrease in fasting serum TAG, the TAG/HDL-C ratio, postprandial lipemia, serum glucose, an increase in LDL particle size, and also greater weight loss (P < 0.05).

²⁸ Evidence type: uncontrolled trial
Long term effects of ketogenic diet in obese subjects with high cholesterol level. Dashti HM, Al-Zaid NS, Mathew TC, Al-Mousawi M, Talib H, Asfar SK, Behbahani AI. Mol Cell Biochem. 2006 Jun;286(1-2):1-9. Epub 2006 Apr 21.

In this study, 66 healthy obese subjects with body mass index (BMI) greater than 30, having high cholesterol level (Group I; n = 35) and those subjects with normal cholesterol level (Group II; n = 31) were selected. The body weight, body mass index, total cholesterol, LDL-cholesterol, HDL-cholesterol, urea, creatinine, glucose and triglycerides were determined before and after the administration of the ketogenic diet. Changes in these parameters were monitored at 8, 16, 24, 32, 40, 48 and 56 weeks of the treatment.

RESULTS: The body weight and body mass index of both groups decreased significantly (P < 0.0001). The level of total cholesterol, LDL cholesterol, triglycerides and blood glucose level decreased significantly (P < 0.0001), whereas HDL cholesterol increased significantly (P < 0.0001) after the treatment in both groups.

CONCLUSION: This study shows the beneficial effects of ketogenic diet following its long term administration in obese subjects with a high level of total cholesterol. Moreover, this study demonstrates that low carbohydrate diet is safe to use for a longer period of time in obese subjects with a high total cholesterol level and those with normocholesterolemia.

²⁹ Evidence type: randomized controlled trial
Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet. Shai I, Schwarzfuchs D, Henkin Y, Shahar DR, Witkow S, Greenberg I, Golan R, Fraser D, Bolotin A, Vardi H, Tangi-Rozental O, Zuk-Ramot R, Sarusi B, Brickner D, Schwartz Z, Sheiner E, Marko R, Katorza E, Thiery J, Fiedler GM, Blüher M, Stumvoll M, Stampfer MJ; Dietary Intervention Randomized Controlled Trial (DIRECT) Group. N Engl J Med. 2008 Jul 17;359(3):229-41. doi: 10.1056/NEJMoa0708681.

Changes in lipid profiles during the weight-loss and maintenance phases are shown in Figure 3. HDL cholesterol (Figure 3A) increased during the weight-loss and maintenance phases in all groups, with the greatest increase in the low-carbohydrate group (8.4 mg per deciliter [0.22 mmol per liter], P<0.01 for the interaction between diet group and time), as compared with the low-fat group (6.3 mg per deciliter [0.16 mmol per liter]). Triglyceride levels (Figure 3B) decreased significantly in the low-carbohydrate group (23.7 mg per deciliter [0.27 mmol per liter], P=0.03 for the interaction between diet group and time), as compared with the low-fat group (2.7 mg per deciliter [0.03 mmol per liter]). LDL cholesterol levels (Figure 3C) did not change significantly within groups, and there were no significant differences between the groups in the amount of change. Overall, the ratio of total cholesterol to HDL cholesterol (Figure 3D) decreased during both the weight-loss and the maintenance phases. The low-carbohydrate group had the greatest improvement, with a relative decrease of 20% (P=0.01 for the interaction between diet group and time), as compared with a decrease of 12% in the low-fat group.

³⁰ Evidence type: review
Influence of dietary carbohydrate and fat on LDL and HDL particle distributions. Siri PW, Krauss RM. Curr Atheroscler Rep. 2005 Nov;7(6):455-9.

Variations in the size and density distributions of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles have been related to risk for cardiovascular disease. In particular, increased levels of small, dense LDL particles, together with reduced levels of large HDL and increases in small HDL, are integral features of the atherogenic dyslipidemia found in patients with insulin resistance, obesity, and metabolic syndrome. Increased dietary carbohydrates, particularly simple sugars and starches with high glycemic index, can increase levels of small, dense LDL and HDL, primarily by mechanisms that involve increasing plasma triglyceride concentrations. Low-carbohydrate diets may have the opposite effects. Diets with differing fatty acid composition can also influence LDL and HDL particle distributions.

³¹ Evidence type: controlled experiment
Carbohydrate restriction has a more favorable impact on the metabolic syndrome than a low fat diet. Volek JS, Phinney SD, Forsythe CE, Quann EE, Wood RJ, Puglisi MJ, Kraemer WJ, Bibus DM, Fernandez ML, Feinman RD. Lipids. 2009 Apr;44(4):297-309. doi: 10.1007/s11745-008-3274-2. Epub 2008 Dec 12.

Abstract

We recently proposed that the biological markers improved by carbohydrate restriction were precisely those that define the metabolic syndrome (MetS), and that the common thread was regulation of insulin as a control element. We specifically tested the idea with a 12-week study comparing two hypocaloric diets (approximately 1,500 kcal): a carbohydrate-restricted diet (CRD) (%carbohydrate:fat:protein = 12:59:28) and a low-fat diet (LFD) (56:24:20) in 40 subjects with atherogenic dyslipidemia. Both interventions led to improvements in several metabolic markers, but subjects following the CRD had consistently reduced glucose (-12%) and insulin (-50%) concentrations, insulin sensitivity (-55%), weight loss (-10%), decreased adiposity (-14%), and more favorable triacylglycerol (TAG) (-51%), HDL-C (13%) and total cholesterol/HDL-C ratio (-14%) responses. In addition to these markers for MetS, the CRD subjects showed more favorable responses to alternative indicators of cardiovascular risk: postprandial lipemia (-47%), the Apo B/Apo A-1 ratio (-16%), and LDL particle distribution. Despite a threefold higher intake of dietary saturated fat during the CRD, saturated fatty acids in TAG and cholesteryl ester were significantly decreased, as was palmitoleic acid (16:1n-7), an endogenous marker of lipogenesis, compared to subjects consuming the LFD. Serum retinol binding protein 4 has been linked to insulin-resistant states, and only the CRD decreased this marker (-20%). The findings provide support for unifying the disparate markers of MetS and for the proposed intimate connection with dietary carbohydrate. The results support the use of dietary carbohydrate restriction as an effective approach to improve features of MetS and cardiovascular risk.

³² Evidence type: randomized controlled trial
Effect of a low-carbohydrate, ketogenic diet program compared to a low-fat diet on fasting lipoprotein subclasses. Westman EC, Yancy WS Jr, Olsen MK, Dudley T, Guyton JR. Int J Cardiol. 2006 Jun 16;110(2):212-6. Epub 2005 Nov 16.

Comparing baseline to 6 months, the LCKD [low carb ketogenic diet] group had significant changes in large VLDL (-78%), medium VLDL (-60%), small VLDL (-57%), LDL particle size (+2%), large LDL (+54%), medium LDL (-42%), small LDL (-78%), HDL particle size (+5%), large HDL (+21%), and LDL particle concentration (-11%).

...

CONCLUSIONS: The LCKD with nutritional supplementation led to beneficial changes in serum lipid subclasses during weight loss. While the LCKD did not lower total LDL cholesterol, it did result in a shift from small, dense LDL to large, buoyant LDL, which could lower cardiovascular disease risk.

³³ Evidence type: longitudinal analysis
Change in LDL particle size is associated with change in plasma triglyceride concentration. McNamara JR, Jenner JL, Li Z, Wilson PW, Schaefer EJ. Arterioscler Thromb. 1992 Nov;12(11):1284-90.

Low density lipoprotein (LDL) particle size is inversely associated with plasma triglyceride concentration in cross-sectional analyses. In the present study, changes in the LDL particle size of 227 participants of the Framingham Offspring Study were analyzed longitudinally by nondenaturing gradient gel electrophoresis at two examinations that were separated by 3-4 years. All subjects had triglyceride concentrations < 400 mg/dl at both exams. Using laser scanning densitometry to assess mean LDL particle size, 56% of samples displayed a change in size: 41% had a one-band size change, 13% had a two-band change, and 2% had a three-band change. These changes in size corresponded to a 15% change in pattern type, based on pattern A and B terminology. There was a significant inverse association between change in LDL size and change in triglyceride (p < 0.0001) and glucose (p < 0.004) concentrations, body weight (p < 0.02), and age (p < 0.03). There was also a significant positive association with change in high density lipoprotein (HDL) cholesterol concentration (p < 0.0001).

³⁴ Evidence type: observational
Ratio of triglycerides to HDL cholesterol is an indicator of LDL particle size in patients with type 2 diabetes and normal HDL cholesterol levels. Boizel R, Benhamou PY, Lardy B, Laporte F, Foulon T, Halimi S. Diabetes Care. 2000 Nov;23(11):1679-85.

LDL size correlated negatively with plasma triglycerides (TGs) (R2 = 0.52) and positively with HDL cholesterol (R2 = 0.14). However, an inverse correlation between the TG-to-HDL cholesterol molar ratio and LDL size was even stronger (R2 = 0.59). The ratio was > 1.33 in 90% of the patients with small LDL particles (95% CI 79.3-100) and 16.5% of those with larger LDL particles. A cutoff point of 1.33 for the TG-to-HDL cholesterol ratio distinguishes between patients having small LDL values better than TG cutoff of 1.70 and 1.45 mmol/l.

CONCLUSIONS: The TG-to-HDL cholesterol ratio may be related to the processes involved in LDL size pathophysiology and relevant with regard to the risk of clinical vascular disease. It may be suitable for the selection of patients needing an earlier and aggressive treatment of lipid abnormalities.

³⁵ Evidence type: observational
Assessment of LDL particle size by triglyceride/HDL-cholesterol ratio in non-diabetic, healthy subjects without prominent hyperlipidemia. Maruyama C, Imamura K, Teramoto T. J Atheroscler Thromb. 2003;10(3):186-91.

Abstract

Small, dense low-density lipoprotein (LDL) is an atherogenic lipoprotein because of its susceptibility to oxidative modification. However, evaluating LDL size requires highly sophisticated techniques. We investigated potentially convenient biochemical parameters for assessing the presence of small, dense LDL. Thirty-nine male subjects, who had been involved in a work-site health promotion program, were recruited. Subjects were divided into two groups: normal LDL size (> 25.5 nm, Normal LDL group) and small LDL (< /= 25.5 nm, Small LDL group). Significant negative correlations were observed between LDL size and both triglyceride (TG) (p <0.001) and remnant-like particle cholesterol concentrations (p < 0.01), while there was a significant positive correlation between LDL size and the high density lipoprotein cholesterol (HDL-C) concentration (p < 0.01). The TG concentration was a negative and the HDL-C concentration a positive independent variable predicting LDL size in multiple regression analysis (p < 0.0001). Seventy-five percent of the Small LDL group had TG/HDL-C ratios higher than 0.9 using mmol/L or 2.0 using mg/dL, while only 25% of the normal LDL group had ratios above the levels (p = 0.0013). A combined parameter, the TG/HDL-C ratio, is beneficial for assessing the presence of small LDL.

³⁶ Evidence type: review of experiments
Low-carbohydrate diet review: shifting the paradigm. Hite AH, Berkowitz VG, Berkowitz K. Nutr Clin Pract. 2011 Jun;26(3):300-8. doi: 10.1177/0884533611405791.

Note that there is an error in the text accompanying this figure (not visible in our image). The data is attributed to a study by Jakobsen et al., but it comes from the study in ³¹, by Volek et al.