Total Ketogenic Ratio (TKR)

The degree of ketosis produced by a particular diet has been long determined by the Ketogenic Ratio (KR), proposed by Woodyatt (1):

KR = K / AK

where, K represents the ketogenic factors and AK represents the anti-ketogenic factors. 

Experimentally determined values for each dietary component are expressed by the equation:

Where:

f = dietary fats in grams
p = dietary protein in grams
c = dietary carbohydrate in grams

Another easier way to determine the KR was proposed by Metcalf and Moriarty:

KR = f / p + c

The formula doesn't take into account the antiketogenic properties of some amino acids but gives an approximate result to the Woodyat equation. 

A paper published by I.A. Cohen (2) adressed the issue brought by these equations: they only apply to isocaloric conditions because they fail to take into account the utilization of energy stores in a hypocaloric diet, underestimating the degree of ketosis of a given diet. The Woodyatt equation then is referred as the Dietary Ketogenic Ratio (DKR), representing the effect of the dietary components on ketosis. 

The formula proposed by Cohen tries to model the impact of weight reduction dieting under ideal conditions, that is, the utilization of stored lipid without the catabolism of protein, scenario normally reached after a few days of fasting and/or low carbohydrate-high fat dieting. 

The Cohen formula is derived from the Woodyatt equation, in a series of simple, mathematical steps giving the new equation:

Where

e = total energy expenditure in calories
p = dietary protein in grams
c = dietary carbohydrate in grams

There are some limitations of this equation, as stated by the author:


1. It needs further evaluation for proving the hypothesis.
2. It describes an ideal situation in which any energy deficit is met entirely by stored lipids. 
3. Dietary energy established by bomb calorimetry may exceed from the energy obtainable by the organism consuming that diet.
4. Energy efficiencies brought about by dieting, as well as changes in the use of dietary nutrients for non-energy uses may alter the expected outcome. For instance, subjects who have not utilized particular metabolic pathways for an extended period may show changes in energy utilization over time.
5. Urinary loss of ketones is not accounted for in these formulae.
6. The nutrient mix itself, as well as the state of the organism may influence the ability to fully utilize the energy available in the consumed nutrients.
7. There is inter-individual variability for achieving the same degree of ketosis. Genetic factors and differences in intestinal flora may alter this parameter.
8. Woodyatt analysis of the ketogenic and anti-ketogenic properties of protein depends upon the proportions of specific amino acids, raising the possibility of differing properties depending upon protein sources. This may necessitate having to alter Woodyatt's constants depending upon protein mix.
9. Differences on both the absorption and utilization of fats resulting from the types of fat included in the diet might alter the response, ie. saturated vs. unsaturated, polyunsaturated vs. monounsaturated, inclusion of MCT or the presence of abnormalities in individual gall bladder functioning.
10. The antiketogenic effects of carbohydrates in food might be altered by the rapidity of their conversion to glucose, as expressed in their glycemic index. The timing of meals may alter the results, particularly if individual feedings varied greatly in their ketogenic to anti-ketogenic properties. For example, meals that contained an isolated bolus of carbohydrate might be expected to rapidly decrease ketosis, even when the total amount of carbohydrate would not be expected to do so when evaluated over a 24-h period. 
11. Ketosis is but one factor among many in weight-loss dieting. The behaviour of any particular factor, by itself, should not imply suitability nor superiority. 


Let's see some examples. 


Asume that an hypocaloric diet consisting of 100g of protein, 70g of fat and 20g of carbohydrate, for an energy expenditure of 2000kcal:


KR (Woodyatt) = 1.28
TKR (Cohen) = 2.09

The energy supplied by the diet in this example is 1110 kcal, which creates a 900kcal deficit. This additional fat increases the production of ketones, not accounted by the Woodyatt equation.

If we up the protein intake to 150g while maintaining calories, we get:

KR (Woodyatt) = 1.00

TKR (Cohen) = 1.65

So even if we consider a PSMF (Protein Sparing Modified Fast) type diet, 200g of protein with 20g of fat and 20g of carbohydrate, we get:

KR (Woodyatt) = 0.79

TKR (Cohen) = 1.37


While the Woodyatt formula might underestimate the level of ketosis, it is probable that the Cohen formula overestimates it, specially in obese and overweight subjects. 

Cohen IA (2009). A model for determining total ketogenic ratio (TKR) for evaluating the ketogenic property of a weight-reduction diet. Medical hypotheses, 73 (3), 377-81 PMID: 19410378

Bioenergetics

Metabolic efficiency can be defined as the capacity for producing useful energy with the least amount of metabolic by-products and heat. In a machine, for example, efficiency can be measured as the ratio of the work done to the energy supplied to it. You can do more with less. There are other definitions of metabolic efficiency around the web such as the capacity for fat utilization for a given activity. 

For aerobic metabolism, the main pathway for ATP production in eukaryotes, there are two substrates which compete with each other: glucose and fatty acids. Amino acids can also be used but are not (or should not) be an important source of fuel. For knowing which fuel is more efficient (giving the higher amount of energy per mole, joule per mole) we must review the pathways for glucose and fatty acid catabolism before they converge into the Krebs Cycle.

Glucose oxidation, ie. glycolysis, is the pathway for oxidizing glucose to pyruvate. The preparatory phase of glycolysis requires the "investment" of two molecules of ATP. This is "payed off" in the following steps, producing 4 ATPs. So the net gain of the conversion glucose --> pyruvate is 2 ATPs. This process also produces 2 NADH+.  Pyruvate must be converted to acetyl CoA, producing one more NADH+ and CO2. So the total oxidation of one molecule of glucose to acetyl CoA yields:

1 glucose molecule to 2 pyruvates: 2 NADH+
2 pyruvates to 2 Acetyl CoA:  2NADH+, 2CO2

Total = 4 NADH+ plus 2CO2

On the other hand, fatty acid oxidation is relatively simpler. Once triglycerides are hydrolyzed, free fatty acids are taken up by cells. In the cytosol, they are esterified to conezyme A to form a fatty acyl CoA. The fatty acyl group is transferred to carnitine and moved across the inner mitochondrial membrane by an acylcarnitine transporter protein. Once in the matrix side, the fatty acyl group is released from carnitine and reattached to another CoA molecule. The mitochondrial fatty acyl CoA is oxidized in a series of reactions to produce Acetyl CoA. The conversion of a fatty acid to acetyl CoA yields (taking palmitate as an example):

1 palmitate to 1 palmitoyl CoA: 2 ATP
1 palmitoyl CoA to 8 Acetyl CoA: 7 FADH2 + 7 NADH+

Overall, in these preliminary reactions before entering the Krebs Cycle we have that 1 molecule of glucose can yield 2 Acetyl CoA while 1 molecule of palmitate can produce 8 Acetyl CoA. Oxidation of glucose until this step produces 2 molecules of CO2 and 4 NADH+, while oxidation of palmitate produces an equal number of FADH2 and NADH+, 7.

Once acetyl CoA reacts with oxaloacetate, forming citrate, it enters the Krebs Cycle. This pathway produces 3 NADH+, 1FADH2, 1 GTP and 2CO2. So, overall we have:

1 molecule of glucose produces: 

2 Acetyl CoA
6 CO2
10 NADH+
2 FADH2

Ratio NADH+:FADH2 = 5:1

ATPs produced from complete oxidation: 30-32 (assuming 2.5 ATP from NADH+ and 1.5 ATP from FADH2)

1 molecule of palmitate produces:

8 Acetyl CoA
16 CO2
31 NADH+
15 FADH2
Ratio NADH+:FADH2 = 2:1 (depending on carbon length)

ATPs produced from complete oxidation: 108 (assuming 2.5 ATP from NADH+ and 1.5 ATP from FADH2)

The final electron acceptors are the coenzymes NADH+ and FADH2 which transfer electrons to the mitochondrial respiratory chain. Understanding this process is of utmost importance for understanding the differences between glucose and fatty acid metabolism. For a basic tutorial in oxidative phosphorylation please read this. 

Electrons carried by NADH+ are transferred to Complex I, also called NADH dehydrogenase. Complex II, succinate dehydrogenase, is the same enzyme encountered in the Krebs Cycle, which oxidizes succinate and transfers electrons to FADH2. Studies have shown that Complex I and complex III are the main producers of ROS in the electron transport chain (1, 2, 3, 4)*. So, the ratio of NADH+:FADH2 is important for the potential ROS production in the mitochondria. Because glucose generates 5 times more NADH+ than FADH2, complex I activity is increased. Fatty acid oxidation produces only twice the amount of NADH+ than FADH2, shifting to a more balanced utilization between complex I and complex II. There are other ways to pass electrons into the respiratory chain. In the first step of the beta oxidation pathway, catalyzed by acyl-CoA dehydrogenase, electrons from the substrate are transferred to the FAD of the dehydrogenase, then to electron-transferring flavoprotein (ETF), which in turn passes its electrons to ETF:ubiquinone oxidoreductase. This enzyme reduces ubiquinone directly, without the need for other complexes. Glycerol-3-phosphate derived either from glycerol or from glycolysis is oxidized by glycerol-3-phosphate dehydrogenase and reduces ubiquinone directly. 

The overall message is that glucose's complete oxidation involves more complex I activity, ultimately promoting more ROS production by the mitochondria. A more in depth review of the implications of glucose and fatty acid metabolism, and mitochondrial complex utilization in aging and disease can be found in this paper by Mobbs et al. (yes, the same guy from the nephropathy reversal study):

"Therefore shifting away from glucose utilization toward lipid and amino acid utilization would be expected to substantially reduce the production of reactive oxygen species, without necessarily reducing ATP production. As described below, other beneficial effects also occur as a result of this altered pattern of glucose fuel use, including a shift toward producing antioxidizing NADPH and increased protein and lipid turnover, which reduces the accumulation of oxidized protein and lipids."

Ok, so we have seen why depending on fat as an energy source seems a better idea than depending on glucose. But what about ketones?

Kurt Harris thinks ketosis is metabolically stressful. Considering that the ketogenesis pathway only demands one molecule of NADH+ and one H2O, I dont see the "stressful" part.

Let's see how ketones alter mitochondrial energy metabolism**. Remember that the respiratory electron transport chain is the process which generates ATP by creating an electrochemical proton gradient. Electrons carried through redox couples are ultimately combined to H+ and O2 to form H2O. The total energy available from the movement of electrons up the respiratory chain is determined by the difference between the variable redox potential of the mitochondrial NAD couple and the O2 couple (this is because the first step in the chain is NADH dehydrogenase and the last step is production of H2O). We know experimentally the reduction potential of both couples (5): 

[NAD]/[NADH+] couple: -0.28V
[O2]/[H2O] couple: +1.2V
So we can calculate the total energy yield given by:

∆G' = -nF∆E

Where: n is the number of electrons, F is 96.485 kJ/mol/V (Faraday's constant) and ∆E the difference between potentials. Replacing in the equation with the values we have:

∆G' = -(2)(96.485)(0.92)

∆G' = -177.5324 or -178kJ/2mol

The energy of the proton gradient created by the electron transport chain is given by: 

∆G' [H+] cyto/[H+] mito = RTln[H+] cyto/[H+] mito + FEmito/cyto

Where  Emito/cyto is the electric potential of the mitochondrial relative to the cytosolic phase.

3 ATPs are generated by the transport of one electron pair through the respiratory chain. Since the maximum energy available from the redox reactions of the chain is -178kJ, the energy available for the synthesis of each ATP cannot be more than -59.2kJ/mol (178/3). Experiments done on different cells have shown that this energy is in the range of -54 to -58kJ/mol (very efficient). 

As the electrons liberated by complex I are carried by coenzyme Q to complex III, the difference between the redox potential of the [NAD]/[NADH+] mitochondrial couple and the coenzyme Q couple determines the energy of the proton gradient generated by the mitochondria. This difference determines the energy of hydrolysis of ATP generated by the mitochondrial F1 ATPase. 

bOHB and AcAc are in near-equilibrium with the free mitochondrial [NAD]/[NADH+] ratio. Veech and colleages showed that the metabolism of ketone bodies in the heart produced a reduction of the mitochondrial NAD couple and oxidation of the coenzyme Q couple, increasing the redox span between the two couples, making more energy available for ATP synthesis because: 

∆G = -nF∆EQ/NADH

In this classic study (6), the authors found that***:

"The Eh7 of the mitochondrial NAD couple, Eh7 NAD+/NADH (Eq. 16), was -280 mV and decreased to about - 300 mV on addition of insulin, ketones, and the combination.

The ratio [fumarate2-]/[succinate2-] increased about 1.5-fold on addition of insulin, about 2.5-fold on addition of ketones, and about 4-fold on addition of the combination (Fig. 1). Taking this change to indicate the redox state of the [Q]/[QH2] couple, which is the cofactor for the succinate dehydrogenase (EC 1.3.5.1) reaction, the observed increase in [fumarate2-]/[succinate2-] indicates a 1.5- to 4-fold oxidation of mitochondrial [Q]/[QH2] on addition of insulin, ketones, or the combination. The Eh[Q]/[QH2], calculated from the succinate dehydrogenase reaction (Eqs. 20-22, Fig. 2), increased progressively from -4 mV during perfusion with glucose alone to + 15 mV on the addition of insulin and ketones.

When the oxidation of the mitochondrial Q couple was combined with the reduction of the mitochondrial NAD couple resulting from these additions to the glucose perfused hearts, the estimated energy available in the transfer of 2 e from the mitochondrial NAD to the Q couple (Eq. 23 and Eq. 24) catalyzed by the NADH dehydrogenase multienzyme complex, ∆G QH2/NAD+, increased from -53 kJ/2 mol e during perfusion with glucose alone to -60 kJ/ 2 mol e with addition of insulin and ketones. This increase was paralleled by an increase in the cytosolic free energy of ATP hydrolysis, ∆GATP (Eq. 7 and Eq. 8), determined independently using NMR spectroscopy, which increased from -56 kJ/mol in control hearts to about -59 kJ/mol on addition of insulin or insulin plus ketones. 

The potential between mitochondrial and cytosolic phases, E mito/cyto (Eq. 9, Fig. 2, Table 3), was -143 mV in hearts perfused with glucose alone and unchanged on the addition of insulin but decreased to -120 mV on addition of ketones or -130 mV on addition of the combination. "

Summarizing (from the study data)****: 

For ketones (without insulin):
Eh7 NAD+/NADH = -299mV or -0.299V
Eh7 Q/QH2 = +7.8mV or 0.078V
Cytosolic pH = 7.05
Mitochondrial pH = 7.52

∆G = -nF∆E

∆G = -(2)*(96.485)(0.015-(-0.3))

∆G = -59.2 kJ/2mol

For glucose:

Eh7 NAD+/NADH = -280mV or -0.28V

Eh7 Q/QH2 = -0.4mV or -0.004V

Cytosolic pH = 7.05

Mitochondrial pH = 7.09

∆G = -nF∆E
∆G = -(2)*(96.485)(-0.004-(-0.28))
∆G = -53 kJ/2mol

Recall that the energy of the proton gradient is calculated by:

∆G' [H+] cyto/[H+] mito = RTln[H+] cyto/[H+] mito + FEmito/cyto

Expressing ∆G in terms of pH instead of concentration, we get:

∆G' [H+] cyto/[H+] mito = 2.3RT (∆pH) + FEmito/cyto

where, R is the gas constant (8.315 x 10^-3 kJ/mol.K) and T the absolute temperature (in this study, 311.15K).  

For ketones, Emito/cyto = -0.12V, so:

∆G = 2.3 (8.315*10^-3)(311.15) (∆pH) + (96.485)(-0.12)
∆G = (5.9 kJ/mol) (∆pH) + (-11.52)
∆G = (5.9 kJ/mol) (7.05-7.52) + (-11.52)
∆G = -14.2 kJ/mol

For glucose, Emito/cyto = -0.14V, so:

∆G = 2.3 (8.315*10^-3)(311.15) (∆pH) + (96.485)(-0.14)

∆G = (5.9 kJ/mol) (∆pH) + (-13.5)

∆G = (5.9 kJ/mol) (7.05-7.09) + (-13.5)

∆G = -13.7 kJ/mol

The mean calculated ∆G of cytosolic ATP hydrolysis was:

∆G ATP = -57.6 kJ/mol for ketones
∆G ATP = -56.6 kJ/mol for glucose

Concluding from this data we see that ketones cause a decrease in the potential between the mitochondria and cytosol (ie. Emito/cyto) while increasing the ∆G of ATP hydrolisis, paralleled by the increase in ∆G QH2/NAD+ and ∆G[H+]. Increased efficiency. 


But why bOHB is such a "superfuel"? This can be illustrated in the following table (7):

The table above shows the heats of combustion for different nutrients metabolized by the Krebs cycle. As shown, there is a wide variation in the inherent energy of different C2 units which enter the cycle, derived from different sources. For instance, as we have seen, one molecule of pyruvate produces 15 ATP. The ∆G' of ATP hydrolisis cannot exceed 59-60kJ/mol and the range of variation in cells is very narrow, less than 10% (-53 to -60kJ/mol). So, by simple maths, for producing 15 ATP, -795 to -900 kJ/15 moles are needed (15*-53; 15*-60). There are only -778kJ available from pyruvate, so the ∆G' of the ATP produced is of low energy (-778/15 = -51kJ/mol). C2 from fatty acids (ie. palmitate) have too much inherent energy to be accomodated in the 15 ATPs, metabolically implausible (-1247/15 = 83). If we look at the table, bOHB has -1021kJ/C2, only 13% excess energy (-1021/15=68). Compared to pyruvate, bOHB is more reduced (ie. more H+ per C). 

When interpreting this data, one obvious question arise. If the inherent energy of palmitic acid is greater than bOHB, why does palmitic acid is not a "superfuel"? The answer lies in the nature of beta oxidation. If we remember the sites for entering the respiratory chain, we can see that some reducing equivalents produced by the oxidation of fatty acids enter the chain via the ETF:ubiquinone oxidoreductase, resulting in a loss of approximately 1 of the 6 possible ATP produced, because of its redox potential (8). The remaining reducing equivalents are metabolized in the Krebs cycle normally, but in contrast with metabolizing bOHB, the Q couple is reduced and not oxidized, decreasing ∆E available for ATP synthesis. Last but not least, free fatty acids produce enzymatic/transcriptional changes which account for the difference in efficiency between palmitic acid and bOHB. Elevation of plasma FFA induce the expression of uncoupling proteins (9, 10) and peroxisomal beta-oxidation (11). 

Summing up

The response Dr. Harris gave me a couple of months ago included some statements like:

"It [bOHB] is only efficient if you think the steps it took to get it don't count."

""Glycolysis" is anaerobic and only happens by itself when speed is necessary - like weight lifting."

"Ketosis is an adaptive state that beats the alternative of glycogen depletion or muscle wasting that it is designed to counter. This makes it "good". In the same way, muscle wasting to keep your brain supplied with glucose is "good". Good in the sense of beating the alternatives. That does not mean we should seek to live continuously in either state, though does it?"

I hope that after this post (and after comparing the "alternatives"), my position on the subject is clear.

* This is a subject of much debate. Studying ROS production by mitochondria under physiological conditions is not an easy task. Ultimately, every complex is capable of producing ROS. 
** This explanation is summarized from the referenced article by Veech. A nice article on the subject can be found here.
*** Eh stands for the redox potential of a half reaction. Eh7 = Eh at pH 7.
****Im omitting some units for the sake of clarity. 

Sato K, Kashiwaya Y, Keon CA, Tsuchiya N, King MT, Radda GK, Chance B, Clarke K, & Veech RL (1995). Insulin, ketone bodies, and mitochondrial energy transduction. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 9 (8), 651-8 PMID: 7768357

Beta hydroxybutyrate does make you smarter

Someone tweeted this study which I have read before, but unfortunately lost. I find this paper rather exceptional because of the perspective of the authors regarding congition and mental health. Most researchers only agree that a KD is efficient for seizure control, not so much for neuroprotection and neuroregeneration. The notion that the brain only functions with glucose coupled with the idea that not eating glucose is metabolically stressful (next post is about this) has deviated attention from ketone bodies as memory enhancing agents. This, I think, is one of the reasons for the very little attention given to KD for Alzheimer and Parkinson, considering the very promising results of studies done on the subject. Maybe because it is not a drug...

Back to the study. 23 older adults with mild cognitive impairment (MCI) were assigned to either a very low carbohydrate diet (VLCD) or to a high carbohydrate diet (HCD) for 6 weeks. Carbohydrate calories were 5-10% on the VLCD and 50% in the HCD. They do not controlled calories, fat or protein*. Some interesting facts from the dietary intevention:

- "The high carbohydrate diet approximated the macronutrient profile consumed at the time of enrollment for most subjects, which included at least 50% of calories from carbohydrates."
< - "We advised the high carbohydrate subjects to consume fruits and vegetables as carbohydrate sources as much as possible.Those in the low carbohydrate diet were restricted from fruit and instructed to limit carbohydrate consumption to small portions of vegetables."

Let's look at the results. 

Memory performance was only increased significantly in the low carbohydrate group. Ok, but maybe the VLCD performed better because of the changes in weight or calorie intake and not necessarily because of the diet being ketogenic. 

(I'm omitting some statistical data on the text). 

"There were significant changes in anthropometric and metabolic values and in dietary parameters. After the intervention, weight (81 kg vs. 77 kg, adjusted means) and waist circumference (95 cm vs. 90 cm, adjusted means) were reduced for the low carbohydrate group. Likewise, fasting glucose (96 mg/dL vs. 86 mg/dL, adjusted means) and fasting insulin values (14.5 /mL vs. 11.9 /mL, adjusted means) were lower for the low carbohydrate but not high carbohydrate group. Urinary ketone bodies were not detected for the high carbohydrate subjects but were present for the low carbohydrate subjects (Table 2), and ketone body levels were related to memory performance."

Table 2:

The VLCD group reduced their calorie intake considerably compared to the HCD group. Note that pre intervention energy intake was 1697 kcal for the HCD and 1762 kcal for the VLCD. There was a slight increase in protein (and fat) intake, but not sufficient to be the responsible of the sudden drop of calories. What about insulin?

"(...) semipartial correlations indicated weak and nonsignificant relationships to memory performance for each of these factors: change in calories (rsp = -0.16, p = 0.46); change in insulin (rsp =-0.26, p = 0.24); and change in weight (rsp = 0.28, p = 0.20). However, within the low carbohydrate group, the relationship between change in insulin and change in memory performance was stronger although not statistically significant, r = 0.47, p = 0.11."

Ok so there was a weak relationship between insulin levels and memory performance, but only in the VLCD. In the end, the parameter that was best correlated to memory performance was ketone body levels. 

Interesting discussion excerpts:

"A number of mechanisms might be considered with respect to our memory finding. There are indications that central ketone metabolism may confer neurocognitive benefit and mitigate neurodegenerative processes in conjunction with, but also independent of, effects on insulin. Mean fasting insulin levels prior to the intervention indicated that, on average, subjects were hyperinsulinemic. We observed a significant reduction of insulin among the low carbohydrate subjects, suggesting that the memory improvement was related, in part, to increased insulin transport into the central nervous system (CNS) as a consequence of correction of peripheral hyperinsulinemia. The trend toward a moderate relationship between fasting insulin and memory performance within the low carbohydrate group would be expected to reach statistical significance in a larger sample.It is noteworthy that a recent trial involving 12 weeks’ calorie restriction in a sample of 50 middle-aged and older adults demonstrated improvement in memory function related to change in fasting insulin (Witte et al., 2009)."

"The absence of a strong relationship between insulin reduction and memory improvement suggests that neurocognitive benefit also might be associated with other aspects of the ketotic condition. Ketone metabolism has been shown to protect hippocampal neurons from Abeta toxicity (Kashiwaya et al., 2000), glutamate toxicity, and apoptosis (Noh et al., 2006), as well as other insults such as kainic acid (Noh et al., 2003) and hypoxia (Puchowizt et al., 2005). As compared with glucose metabolism, central ketone metabolism generates lower levels of oxidative stress (Prins, 2008) and has been shown to produce greater cellular energy output and antioxidant capacity, the latter by increasing glutathione peroxidase in hippocampal cells (Veech et al., 2001; Ziegler et al.. 2003). In addition, the presence of cerebral ketones is associated with decreased apoptosis and inflammation (Gasior et al., 2006; Malouf et al., 2009), which along with oxidative stress, have been identified as fundamental factors contributing to neurodegeneration (Cotman, 2000)."

Some final notes:

- Ketone body levels were measured with urinary strips, and BOHB was not measured directly.

- The KD was by not means a high fat diet (maybe if we consider the % of calories from fat). 

* As per the authors: "Very high levels of fat (90% of total calorie intake) have been prescribed traditionally to induce ketosis for seizure management (Vining, 1998). However, recent trials have indicated that protein restriction is not necessary to achieve ketosis (Boden et al., 2005; Cassady et al., 2007) or effective seizure control (Kossoff and Dorward, 2008; Kossoff et al.,2003), allowing for a less severe regimen." More on protein intake & Ketosis here.

Krikorian R, Shidler MD, Dangelo K, Couch SC, Benoit SC, & Clegg DJ (2010). Dietary ketosis enhances memory in mild cognitive impairment. Neurobiology of aging PMID: 21130529

Extra: Glycogen, de novo lipogenesis and carbohydrates

This is an unusual post as I dont like discussing about weight loss or dietary carbs vs. fat superiority in terms of dieting. 

The metabolic advantage is a never ending discussion in the blogosphere. Recently, a more "scientific" debate has started on Peter's blog. While some prefer to argue using scientific references and basic biochemistry concepts, others use their own experience mixed with their opinions. This happens in both who defend insulin's central role in weight loss/gain and in those who state that its calories in-calories out all the way. This last group of people, when trying to dismiss the carbohydrate drives insulin drives fat storage hypothesis, use an old study as their bible. It is not surprising that the one who "popularized" this study was Carb Sane, with her famous post Nutrient fates after absorption. I wrote a quick answer some months ago, which was posted but strangely has disappeared. 

The study which basically dismisses the carbohydrate-insulin obesity hypothesis is one by Eric Jequier, called "Nutrient effects: post-absorptive interactions". I will only focus on the "Metabolic Fate of Dietary Carbohydrate" part. 

The author states:

"Although  the  glycogen  stores  are  normally maintained  within  a  relatively  narrow  range, the capacity for storing large amounts of  dietary CHO by conversion to glycogen is relatively large (Acheson et al. 1982, 1984, 1985, 1988). Fig. 1 shows that a large load of CHO (500 g dextrin-maltose  given as three meals over 5 h) to healthy subjects induces a marked stimulation of CHO oxidation over the 14 h after the first meal (240 g oxidized,  260 g stored; Acheson et al. 1985). Net lipogenesis occurred at a low rate from 5 to 10 h  after  the  first  meal, but  this lipid accumulation was  offset  by  a greater rate  of  lipid  oxidation over the next 4 h. The fat balance calculated over 14 h was negative, indicating  that the large load of  CHO did not induce a gain in  fat."

Lets review the studies used to support his statements. 

1. Glycogen synthesis versus lipogenesis after a 500 gram carbohydrate meal in man

Subjects participating in the study were 6 healthy male volunteers. They tested the metabolic effect of consuming 480g of carbohydrates from bread, jam and fruit juice, with little butter added. The results were as expected, no net lipogenesis occured, just an increase in muscle glycogen capacity. Ok, one big high carbohydrate/low fat meal in healthy subjects does not induce fat gain in the short term (10h), rather increases energy expenditure, glucose storage and oxidation. Nothing new.

2. Glycogen storage capacity and de novo lipogenesis during massive carbohydrate overfeeding in man.
Lets look at the subjects and methods used in the study:

"Three healthy young men, one of whom was a competition swimmer at university level, (21-22 y, 62-72 kg, 174-180 cm, and 11-14% body fat) with no family history of diabetes or obesity and who were not taking any medication, participated in this study."

Ok, we are talking about healthy, young and lean men. Is everyone healthy, young and lean?  I think most of the regular blog readers are people with weight/metabolic issues. The subjects included a competitive swimmer, a fact that in my opinion is very relevant, besides the fact that all of the subjects were lean. This somehow escapes from the people referencing this study. 

"The experiment lasted 14 consecutive days. During the first 3 d the subjects consumed a restricted diet, high in fat and low in carbohydrate, and followed an exercise program. Halfway through this period the subjects were admitted into a respiration chamber in which respiratory exchange measurements were to be continued for 10 d. After 36 h in the chamber the diet was changed to a high-carbohydrate, low-fat diet that was ingested for the following 7 d. During the last 2 d while still in the chamber, the subjects received limited amounts of a high protein diet (protein-sparing modified-fast [PSMF], ‘ı2.5 MJ or 600 kcal) essentially devoid of carbohydrate. The subjects then left the respiration chamber but continued to consume the high-fat, low-carbohydrate diet in restricted amounts for a further 2 d."

I wonder, if a massive carbohydrate intake does not promotes fat storage as the previous study suggested, why using a glycogen depleting protocol before an extended massive carbohydrate intake? Hmm...

In short, the protocol lasted 14 days in which:

3 first days: HFLC + exercise.

7 days: HCLF

2 days: PSMF (LF, LC)

2 last days: HFLC, restricted amounts.

So we have a scenario in which glycogen is depleted by diet plus exercise and then there is a massive high carbohydrate low fat refeed for seven days. Glycogen storage increases until the 4th day, in which glycogen stores had become saturated and de novo lipogenesis starts to increase. This shows that you need 4 days of overfeeding carbohydrates to start getting fat? No. This shows that in LEAN HEALTHY YOUNG SUBJECTS, AFTER A GLYCOGEN DEPLETING PROTOCOL, a relatively well known phenomenon called GLYCOGEN SUPERCOMPENSATION occurs. 

I dont think we can extrapolate these results to everyone nor this study shows that you can eat 500g everyday and not get fat. Specially if you are not lean and/or healthy.

3. Nutritional influences on lipogenesis and thermogenesis after a carbohydrate meal.

I find odd the use of this study to support the author's hypothesis. Subjects consumed either a high fat, mixed or high carbohydrate diet during 3-6 days preceeding the test. From the abstract (unfortunately I dont have access to the full-text):

"CHO oxidation and conversion to fat was significantly less in the high-fat diet group (222 +/- 5 g) than in the mixed (300 +/- 13 g) or high-CHO diet (331 +/- 7 g) groups, resulting in a greater glycogen storage in the high-fat (278 +/- 6 g) than in the other two groups (197 +/- 11 and 170 +/- 2 g). Net lipogenesis occurred sooner and lasted longer in the high-CHO group, amounting to 0.8 +/- 0.5, 3.4 +/- 0.6, and 9 +/- 1 g of lipid synthesized in the high-fat, mixed, and high-CHO groups, respectively. The thermic effect of the CHO load was 5.2 +/- 0.5% on the high-fat, 6.5 +/- 0.4% on the mixed diet, and 8.6 +/- 0.4% on the high-CHO diet."

This study shows what is an already known fact about high fat diets and carbohydrate loading. HFLC reduce glucose oxidation and increase non-oxidative glucose disposal, ie. glycogen stores. Although net lipogenesis was small (9g on the high carbohydrate diet), measurements were done for 24 hours. While not significant for body composition in the short term, the metabolic effect might be significant, specially considering the effect a high carbohydrate meal has on gene expression (1, 2)*. This study shows the difference between eating a 500g carbohydrate meal after a HFLC and a LFHC diet. 

4. Contribution of 500 g naturally labeled 13C dextrin maltose to total carbohydrate utilization and the effect of the antecedent diet, in man

This study is a follow up on the previous study. Nothing new to add, really. From the study:

"After the load, carbohydrate oxidation increased in each group with a corresponding decrease in fat oxidation. However, in the high carbohydrate (Fig 3C) and mixed (Fig 3B) groups, fat oxidation decreased to zero at 2.5 h and 4 h respectively after which energy expenditure was principally due to carbohydrate oxidation and de novo lipogenesis for a further 9 h and 6.5 h respectively. The negative values of lipid oxidation represent an equivalent amount of carbohydrate energy converted into lipid, ie net de novo lipogenesis. During the evening fat oxidation increased progressively and became the major energy source throughout the night in each group."

The authors concluded:

"The results imply that the more the composition of the everyday diet is rich in carbohydrate, the more full the glycogen stores become, but at the same time more carbohydrate is oxidized to provide energy. When both storage and oxidation become saturated, de novo lipogenesis can occur but not at a rate sufficient to prevent increasing blood glucose and insulin concentrations."

Summing up

It is noteworthy to mention that the author of the review, Eric Jequier participated in all of the studies shown above. The conclusion he gets from his review is the following:

"The  practical consequence  which  results from  the  different fates  of  the  ingested 

nutrients  is  that  body-weight  regulation primarily  depends on fat intake. A chronic 

excess of  fat in the everyday diet is stored and contributes to increase the adipose tissue 

mass. (...) The fat content of the habitual diet could influence the steady-state of weight maintenance. When a high dietary fat intake is chronically ingested, body-weight gain occurs."

Talk about bias. Although he mentions that "The practical implication of these findings is  that the proportion of fat and CHO energy plays an important role in body-weight regulation" he goes on saying that "The ad  lib. consumption of high-CHO-low-fat  diets induces a slow rate of  weight loss, with a spontaneous decrease in energy intake" and "Prevention of obesity should benefit from this recent advance in our knowledge of the post-absorptive effects of nutrients".

I did a quick search on more studies done by Jequier and I found these gems:

Low fat diets are preferred

Is fat intake a risk factor for fat gain in children

Failure of dietary fat intake to promote fat oxidation: a factor favoring the development of obesity

Pathways to obesity

I encourage you to check the references used in the section "Metabolic Fate of Dietary Fat". 

Studies can show whatever the author wants to. The only way of knowing and interpreting the real data is reading the data source for yourself. Or reading trustful blogs. But hey, after all, fat is what makes us ugly, fat and ultimately kills us, doesnt it?

* Some further reading on ChREBP is recommended.

Jéquier E (1995). Nutrient effects: post-absorptive interactions. The Proceedings of the Nutrition Society, 54 (1), 253-65 PMID: 7568258